Methods for producing industrial products from plant lipids

Abstract

This invention relates to methods for producing industrial products from plant lipids, particularly from the nutrient components of plants. Specifically, the invention provides oil products such as biodiesel and synthetic diesel, methods for producing these, and plants having elevated levels of one or more nonpolar lipids such as triglycerides and increased total nonpolar lipid content.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580048054.8, entitled “Method for producing industrial products from plant lipids”, filed on July 7, 2015. Invention Field This invention relates to methods for producing industrial products from plant lipids, particularly from the nutrient parts of plants. Specifically, the invention provides oil products such as biodiesel and synthetic diesel, methods for producing these, and plants having elevated levels of one or more nonpolar lipids such as triglycerides and increased total nonpolar lipid content. In a particular embodiment, the invention relates to a combination of modifications of two or more of lipid-operating enzymes, oil body proteins, lipid-reducing enzymes, and / or transcription factors regulating lipid biosynthesis to increase the levels of one or more nonpolar lipids and / or total nonpolar lipids and / or monounsaturated fatty acid content in a plant or any part thereof. In one embodiment, the invention relates to a method for extracting lipids. In another embodiment, lipids are converted into one or more hydrocarbon products in the harvested nutrient parts of a plant to produce alkyl esters of fatty acids suitable for use as renewable biodiesel fuel. Background of the Invention The majority of the world's energy, especially that used for transportation, is provided by petroleum-derived fuels, and their supply is limited. There is a need for renewable alternative sources, such as biologically derived oils.

[0002] Triacylglycerol biosynthesis Triacylglycerols (TAGs) constitute the major form of lipids in seeds and consist of three acyl chains esterified into the glycerol backbone. Fatty acids are synthesized in plastids as intermediates of acyl-acyl carrier proteins (ACPs), where they undergo a first catalytic desaturation. This reaction is catalyzed by stearoyl-ACP desaturase to produce oleic acid (C18:1). Δ 9). Subsequently, the acyl chain, as an acyl-CoA thioester, is transferred to the cytoplasm and endoplasmic reticulum (ER). Prior to entering the main TAG biosynthetic pathway, also known as the Kennedy or glycerol-3-phosphate (G3P) pathway, the acyl chain is typically integrated into the phospholipids of the ER membrane, where it can undergo further desaturation. Two key enzymes in the production of polyunsaturated fatty acids are membrane-bound FAD2 and FAD3 desaturases, which produce linoleic acid (C18:2...). Δ9,12 ) and alpha-linolenic acid (C18: 3) Δ9,12,15 ).

[0003] TAG biosynthesis via the Kennedy pathway consists of a series of successive acylations, each using an acyl-CoA ester as an acyl donor. The first acylation step typically occurs on the G3P backbone.sn1 - This occurs at the location and is caused by glycerol-3-phosphoacyltransferase ( sn1 -GPAT) catalysis. Product sn1 -lysophosphatidic acid ( sn1 -LPA) serves as a substrate for lysophosphatidyl acyltransferase (LPAAT), which links the second acyl chain to... sn2 - The position is used to form phosphatidic acid. PA is further dephosphorylated to diacylglycerol (DAG) by phosphatidylphosphatase (PAP), thereby providing a substrate for the final acylation step. Finally, the third acyl chain is esterified to DAG in a reaction catalyzed by diacylglycerol acyltransferase (DGAT). sn3 The first step is to establish a site for the formation of TAGs that aggregate in the oil body. A second enzymatic reaction, phosphatidylglycerol acyltransferase (PDAT), also leads to the conversion of DAGs into TAGs. This reaction is independent of DGAT and uses phospholipids as acyl-donors.

[0004] To maximize the yield of lipids in commercial production, further measures are needed to increase lipid levels (especially nonpolar lipids such as DAG and TAG) in transgenic organisms or parts thereof, such as plants, seeds, leaves, algae, and fungi. Attempts to increase neutral lipid production in plants have primarily focused on individual key enzymatic steps involved in fatty acid biosynthesis or TAG assembly. However, these strategies have only resulted in general increases in oil content in seeds or leaves. Recent studies in the oil-producing yeast *Yersinia lipophila* have shown a significant improvement. Yarrowia lipolytica Metabolic engineering work in this field has confirmed the ability to increase glycerol-3-phosphate production and prevent TAG transmission. β - The combination of oxidative damage leads to an increase in total lipid content (Dulermo et al., 2011).

[0005] Plant lipids, such as seed oil triacylglycerols (TAGs), have numerous uses, including culinary applications (shortening, texture, flavoring), industrial applications (soap, candles, perfumes, cosmetics, suitable as desiccants, insulators, lubricants), and providing nutritional value. Interest in using plant lipids to produce biofuels is also growing.

[0006] To maximize the yield of lipid production in commercial organisms, further measures are needed to increase lipid levels, especially nonpolar lipids such as DAG and TAG, in genetically modified organisms or parts thereof, such as plants, seeds, leaves, algae, and fungi. Invention Overview The inventors have discovered a method for producing oil products from nutrient-rich plant parts.

[0007] In a first aspect, the present invention provides a method for producing oil products, the method comprising the following steps: (i) Processing the composition in a reactor, the composition comprising (a) The nutrient-rich plant portion having a dry weight of at least 2 g and a total nonpolar lipid content of at least 5% by weight on a dry weight basis. (b) A solvent comprising water, alcohol, or both, and (c) Any available catalyst, The treatment described herein includes exposure to approximately 50°C in an oxidizing, reducing, or inert environment. o C-approx. 450 o The composition was heated at a temperature of C and a pressure of 5-350 bar for 1-120 minutes. (ii) The oil product is recovered from the reactor at a yield of at least 35% by weight relative to the dry weight of the nutrient plant portion. Thus, the oil product is produced.

[0008] In one embodiment, the nutrient plant portion has a dry weight of at least 1 kg.

[0009] In one embodiment, the nutrient plant portion has a total nonpolar lipid content of at least 10%, at least 15%, at least 20%, about 25%, about 30%, about 35%, 10%-75%, 20%-75%, or preferably 30%-75% on a dry weight basis.

[0010] In one embodiment, the composition has a solids concentration of 5%-90%, preferably 15%-50% (dry weight / weight).

[0011] Any suitable catalyst can be used. In one embodiment, the catalyst is a base, acid, or noble metal catalyst. For example, in one embodiment, the catalyst comprises NaOH or KOH or both, preferably at a concentration of 0.1M-2M.

[0012] In one embodiment, the processing time is 1-60 minutes, preferably 10-60 minutes, more preferably 15-30 minutes. In one embodiment, when the pressure is less than 50 bar, the reaction time can be up to 24 hours or even up to 7 days. In a preferred embodiment, the temperature is 275°C. o C-360 o C, pressure of 100-200 bar, and the reaction occurs within 10-60 minutes.

[0013] In one embodiment, if the solvent is water, the method produces an oil product yield of at least 36%, 37%, 38%, 39%, or 40% by weight and at most 55% by weight or preferably 60% by weight (relative to the dry weight of the nutrient plant portion). In this embodiment, the oil product contains at least 2-fold, preferably at least 3-fold, more hydrocarbons than fatty acyl esters. Preferably, the oil product contains 35%, more preferably 40%, of C13-C22 hydrocarbons.

[0014] In another embodiment, if the solvent comprises an alcohol, preferably methanol, the method produces an oil product yield of at least 36%, 37%, 38%, 39%, or 40% by weight and at most 65% by weight or preferably 70% by weight (relative to the dry weight of the nutrient plant portion). In this embodiment, the oil product contains at least 1.5 times, preferably at least 2 times, more fatty acyl esters than hydrocarbons. Preferably, the oil product contains 40%, more preferably 50%, fatty acid methyl esters.

[0015] In another embodiment, if the solvent contains about 80% water, the oil product contains about 30% C13-C22 hydrocarbons, preferably about 35%, more preferably about 40% C13-C22 hydrocarbons.

[0016] In another embodiment, if the solvent contains about 50% methanol, then the oil product contains about 50% fatty acid methyl ester (FAME).

[0017] In another embodiment, the recovered oil product has a water content of less than about 15% by weight, preferably less than 5% by weight.

[0018] In another embodiment, the yield of the oil product is at least 2% by weight, preferably at least 4% by weight, relative to a corresponding method using the corresponding nutrient plant part (which has a nonpolar lipid content of less than 2% on a dry weight basis).

[0019] In one embodiment, the nutrient-rich plant portion has undergone one or more physical processing steps (i)(a) of drying, chopping, shredding, milling, rolling, pressing, crushing, or grinding. In an alternative embodiment, the nutrient-rich plant portion has not been dried to a moisture content of at least 10% prior to the preparation of the composition. For example, the nutrient-rich plant portion has a moisture content of at least 20% or at least 30%, or the nutrient-rich plant portion retains at least 50% of the moisture content it had at harvest.

[0020] In one embodiment, the method further includes one or more of the following: (i) Hydrodeoxygenation of recovered oil products, (ii) Treating the recovered oil products with hydrogen to reduce the ketone or sugar levels in the oil products. (iii) Producing syngas from recovered oil products, and (iv) The recovered oil products are graded and separated to produce one or more of fuel oil, diesel oil, kerosene, or gasoline. For example, the grading step is performed by fractionation.

[0021] In one embodiment, the nutrient plant portion comprises plant leaves, stems, or both.

[0022] In one embodiment, the nutrient plant portion comprises a combination of exogenous polynucleotides and / or genetic modifications as defined herein.

[0023] The inventors have also demonstrated that by manipulating fatty acid biosynthesis, lipid assembly, and lipid packaging pathways, as well as reducing lipid catabolism, the lipid content of organisms can be significantly increased, particularly in the nutrient parts and seeds of plants. Various gene combinations and reduced gene expression are used to achieve a substantial increase in oil content, which is of great significance for biofuels and other oil-derived industrial products.

[0024] In a second aspect, the present invention provides a recombinant eukaryotic cell comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and any one, two, or all three of the following c) A first genetic modification that, compared to corresponding cells lacking the genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triglycerides (TAG) in the cells. d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking a fourth exogenous polynucleotide, increases fatty acid efflux from the cytoplasm, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. Each exogenous polynucleotide is operatively linked to a promoter that can direct the expression of the polynucleotide in the cell.

[0025] In one embodiment, the cell comprises a), b), and c), and optionally d) or e).

[0026] In one embodiment, the cell comprises a), b), and d), and optionally c) or e).

[0027] In one embodiment, the cell comprises a), b), and e), and optionally c) or d).

[0028] In one embodiment, the cell further comprises one or more of the following: a) A fifth polynucleotide encoding an oil body coating (OBC) polypeptide, preferably a lipid droplet-associated protein (LDAP). b) A second genetic modification that, compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm, and c) A third genetic modification that, when compared with corresponding cells lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids.

[0029] In one embodiment, the recombinant eukaryotic cells comprise a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and c) A first genetic modification that, compared to corresponding cells lacking the genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triglycerides (TAG) in the cells. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the cell, and optionally the cell further comprises one or more of the following: d) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the fourth exogenous polynucleotide, increases fatty acid efflux from the cytoplasm. f) A second genetic modification that, when compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm. g) A third genetic modification that, when compared with corresponding cells lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids.

[0030] In one embodiment, the cells are plant cells derived from or within the vegetative part of a plant, and one or more promoters are expressed in the vegetative part at a higher level than in the plant seed.

[0031] In a preferred embodiment, the presence of c), d), or e), along with the first and second exogenous polynucleotides, increases the total nonpolar lipid content of the cells, preferably cells in vegetative plant parts such as leaves or stems, relative to the corresponding cells containing the first and second exogenous polynucleotides but lacking each of c), d), and e). More preferably, the increase is synergistic. Most preferably, at least the promoter directing the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0032] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT, and the polypeptide involved in the catabolic metabolism of TAG in the cells is SDP1 lipase.

[0033] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is the WRI1 polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT.

[0034] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT.

[0035] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in the catabolism of triglycerides (TAG) in the cells is an SDP1 lipase.

[0036] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the polypeptide involved in the catabolism of triacylglycerols (TAG) in the cells is an SDP1 lipase.

[0037] In one implementation, when present, the two transcription factors are WRI1 and LEC2, or WRI1 and LEC1.

[0038] In the above embodiments, preferably the cells are in the vegetative part of the plant, which grows in the soil or grows in the soil and is subsequently harvested from the plant part, and wherein, on a weight basis, the cells contain at least 8% TAG (% dry weight), for example 8%-75% or 8%-30%. More preferably, the TAG content is at least 10%, for example 10%-75% or 10%-30%. Preferably, these TAG levels are present in the vegetative part before or during flowering or before the fruiting stage of plant development. In these embodiments, preferably the ratio of TAG content in leaves to TAG content in plant stems is 1:1 to 10:1, and / or said ratio is increased relative to corresponding cells containing first and second exogenous polynucleotides and lacking the first genetic modification.

[0039] In the above embodiments, the cells preferably contain exogenous polynucleotides encoding DGAT and genetic modifications that downregulate the production of exogenous SDP1 lipase. More preferably, the cells do not contain exogenous polynucleotides encoding PDAT, and / or contain no substances other than *Nicotiana benthamiana* (Benjamin Butylcholine). Nicotiana benthamiana Cells other than ) cells, and / or WRI1 is except Arabidopsis thaliana ( Arabidopsis thaliana WRI1 other than WRI1 (SEQ ID NO: 21 or 22). Most preferably, at least one exogenous polynucleotide in the cell is expressed from a promoter that is not a constitutive promoter, such as a promoter that is preferentially expressed in the green tissue or stem of the plant or that is upregulated after flowering or during senescence.

[0040] In a third aspect, the present invention provides a recombinant eukaryotic cell comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and c) A third exogenous polynucleotide encoding an oil body coating (OBC) polypeptide, preferably a lipid droplet-associated polypeptide (LDAP). Each exogenous polynucleotide is operatively linked to a promoter that directs the expression of the polynucleotide in the cell, and the recombinant eukaryotic cell, relative to the corresponding cell containing a third exogenous polynucleotide (whose nucleotide sequence is complementary to the sequence provided in SEQ ID NO:176), has elevated levels of one or more nonpolar lipids and / or increased amounts of OBC peptides.

[0041] In one embodiment, the cells described above further comprise one or more of the following: d) A first genetic modification, which, compared to corresponding cells lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triglycerides (TAG) in said cells. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the fourth exogenous polynucleotide, increases fatty acid efflux from the cytoplasm. f) A second genetic modification that, when compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm. g) A third genetic modification that, when compared with corresponding cells lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids.

[0042] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT, and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0043] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is the WRI1 polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT.

[0044] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0045] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0046] In one embodiment, the cell contains two exogenous polynucleotides encoding two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

[0047] In a preferred embodiment, the presence of a third exogenous polynucleotide encoding an OBC polypeptide (preferably LDAP), relative to a corresponding plant cell containing the first and second exogenous polynucleotides but lacking the third exogenous polynucleotide, along with the first and second exogenous polynucleotides, increases the total nonpolar lipid content of the plant cell, preferably in cells of vegetative plant parts such as leaves or stems. More preferably, the increase is synergistic. Most preferably, at least the promoter directing the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0048] In a fourth aspect, the present invention provides a recombinant eukaryotic cell comprising a plastid and a first exogenous polynucleotide, the first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell, and one or more of the following; a) A second exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the second exogenous polynucleotide, increases fatty acid efflux from the cytoplasm. b) A first genetic modification that, when compared to corresponding cells lacking the first genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm. c) A second genetic modification that, compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. Each exogenous polynucleotide is operatively linked to a promoter that can direct the expression of the polynucleotide in the cell.

[0049] In one embodiment, the cell, preferably a plant cell, comprises a) and optionally b) or c).

[0050] In one embodiment, the cells described above further comprise one or more of the following: d) A third exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. e) A third genetic modification that, compared to corresponding cells lacking the third genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the cells, and f) A fourth exogenous polynucleotide encoding an oil-coated (OBC) polypeptide, preferably LDAP.

[0051] In a preferred embodiment, the cell, preferably a plant cell, contains a first, second, and third exogenous polynucleotide and optionally a third genetic modification or a fourth exogenous polynucleotide.

[0052] In a preferred embodiment, the presence of a second exogenous polynucleotide encoding a polypeptide (preferably a fatty acylthioesterase such as a FATA polypeptide) that increases fatty acid export from the cytoplasm, relative to a corresponding plant cell containing the first and third (if present) exogenous polynucleotides but lacking the second exogenous polynucleotide, together with the first and third (if present) exogenous polynucleotides, increases the total nonpolar lipid content of the plant cell, preferably in cells of vegetative plant parts such as leaves or stems. More preferably, the increase provided by the second exogenous polynucleotide is synergistic. Most preferably, at least the promoter directing the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0053] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, preferably a transcription factor other than Arabidopsis thaliana WRI1 (SEQ ID NO: 21 or 22), and the polypeptide that increases fatty acid export from the cytoplasm is a fatty acid thioesterase, preferably a FATA or FATB polypeptide, more preferably a FATA polypeptide or fatty acid thioesterase other than a medium-chain fatty acid thioesterase. The percentage of C12:0 and / or C14:0 fatty acids in the total fatty acid content of the cells is comparable to that of corresponding cells lacking exogenous polynucleotides encoding thioesterases, indicating the presence of thioesterases other than medium-chain thioesterases. Preferably, the cells further comprise an exogenous polynucleotide encoding DGAT and a genetic modification that downregulates the production of exogenous SDP1 lipase. In one embodiment, the reduced SDP1 lipase production synergizes with the transcription factor and fatty acid thioesterase to increase the total nonpolar lipid content in the cells. More preferably, the cells do not contain exogenous polynucleotides encoding PDAT, and / or are cells other than those of *Nicotiana benthamiana* cells. Most preferably, at least one exogenous polynucleotide in the cells is expressed from a promoter that is not a constitutive promoter, such as a promoter that is preferentially expressed in the green tissues or stems of the plant or that is upregulated during senescence.

[0054] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in introducing fatty acids into the cytoplasm is a TGD polypeptide.

[0055] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in diacylglycerol (DAG) production is plastid GPAT.

[0056] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide that increases fatty acid export from the cytoplasm is a fatty acid thioesterase, preferably a FATA or FATB polypeptide; and the polypeptide involved in the importation of fatty acids into the cytoplasm is a TGD polypeptide.

[0057] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide that increases fatty acid export from the cytoplasm is a fatty acid thioesterase, preferably a FATA or FATB polypeptide; and the polypeptide involved in diacylglycerol (DAG) production is plastid GPAT.

[0058] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in introducing fatty acids into the cytoplasm is a TGD polypeptide; and the polypeptide involved in the generation of diacylglycerol (DAG) is plastid GPAT.

[0059] In one embodiment, the cell contains two exogenous polynucleotides encoding two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

[0060] In embodiments of the second, third, and fourth aspects, when the cell contains an exogenous polynucleotide encoding a fatty acid thioesterase such as a FATA or FATB polypeptide, the thioesterase is preferably a FATA polypeptide or a fatty acid thioesterase other than a medium-chain fatty acid thioesterase.

[0061] In a fifth aspect, the present invention provides a recombinant eukaryotic cell comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells, preferably a WRI transcription factor. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, said polypeptide being LPAAT with preferential activity against fatty acids of medium chain length (C8-C14), and c) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the third exogenous polynucleotide, increases the export of C8-C14 fatty acids from the cytoplasm. Each exogenous polynucleotide is operatively linked to a promoter that can direct the expression of the polynucleotide in the cell.

[0062] In one embodiment, the third exogenous polynucleotide encodes a thioesterase, preferably a FATB thioesterase that has preferential activity for fatty acids with a medium chain length (C8-C14).

[0063] In a preferred embodiment, the presence of a third exogenous polynucleotide encoding a polypeptide that increases the export of C8-C14 fatty acids from the cytoplasm, relative to a corresponding plant cell containing the first and second exogenous polynucleotides but lacking the third exogenous polynucleotide, increases the total MCFA content of the cell, along with the first and second exogenous polynucleotides, preferably in cells of vegetative plant parts such as leaves, roots, or stems. More preferably, the increase provided by the third exogenous polynucleotide is synergistic. Most preferably, at least the promoter directing the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0064] In one embodiment, the exogenous polynucleotide encoding a FATB thioesterase with preferential activity for fatty acids of medium chain length (C8-C14) comprises an amino acid sequence as shown in any one of SEQ ID NO: 193-199, or a biologically active fragment of any one of them, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 193-199. More preferably, the exogenous polynucleotide encoding a FATB thioesterase with preferential activity for fatty acids of medium chain length (C8-C14) comprises an amino acid sequence as shown in SEQ ID NO: 193-199, or a biologically active fragment of any one of them, or a polypeptide whose amino acid sequence is at least 30% identical to any one or two of SEQ ID NO: 193-199.

[0065] In one embodiment of the fifth aspect, the transcription factor is not Arabidopsis thaliana WRI1 (SEQ ID NO:21 or 22).

[0066] In one embodiment of the fifth aspect, the exogenous polynucleotide encoding LPAAT comprises an amino acid sequence as shown in SEQ ID NO:200, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical thereto.

[0067] In one embodiment of the fifth aspect, the cell further comprises one or more of the following: d) A fourth exogenous polynucleotide encoding another polypeptide involved in the biosynthesis of one or more nonpolar lipids. e) A first genetic modification that, when compared to corresponding cells lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in said cells. f) A fifth exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. g) A second genetic modification that, when compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm. h) A third genetic modification that, compared to corresponding cells lacking the third genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. Each exogenous polynucleotide is operatively linked to a promoter that can direct the expression of the polynucleotide in the cell.

[0068] In one embodiment of the fifth aspect, the cell is a plant cell derived from or within a vegetative part of a plant, and one or more promoters are expressed in the vegetative part at a higher level than in the plant seed.

[0069] In one embodiment of the fifth aspect, the fatty acid having a medium chain length is at least myristic acid. In a preferred embodiment, the cell contains at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, 8%-25%, 8%-20%, 10%-25%, 11%-25%, about 15%-25%, or about 20%-25% (w / w dry weight) of myristic acid.

[0070] In embodiments of the third, fourth, and fifth aspects, preferably the cells are in the vegetative portion of the plant, which grows in soil or grows in soil and subsequently harvests the plant portion, and wherein, on a weight basis, the cells contain at least 8% TAG (% dry weight), for example 8%-75% or 8%-30%. More preferably, the TAG content is at least 10%, for example 10%-75% or 10%-30%. Preferably, these TAG levels are present in the vegetative portion before or during flowering or before the fruiting stage of plant development. In these embodiments, preferably, the ratio of TAG content in leaves to TAG content in plant stems is 1:1 to 10:1, and / or said ratio is increased relative to corresponding cells containing first and second exogenous polynucleotides and lacking the first genetic modification.

[0071] In embodiments of the second, third, fourth, and fifth aspects, the cells preferably contain exogenous polynucleotides encoding DGAT and genetic modifications that downregulate the production of exogenous SDP1 lipase. In a preferred embodiment, the cells do not contain exogenous polynucleotides encoding PDAT, and / or are cells other than those in *Nicotiana benthamiana* and / or cells other than those in *Raphanus pensilis* (European rapeseed). Brassica napus Cells other than cells. Most preferably, at least one exogenous polynucleotide in the cells is expressed from a promoter that is not a constitutive promoter, such as a promoter that is preferentially expressed in the green tissues or stems of plants or that is upregulated during senescence.

[0072] In one embodiment, the cell of the present invention (including the second, third, fourth and fifth aspects) has one or more of the following features (where applicable); i) The cells have increased total fatty acid synthesis relative to the corresponding cells lacking the first exogenous polynucleotide, or decreased total fatty acid catabolism relative to the corresponding cells lacking the first exogenous polynucleotide, or both, thus having elevated levels of total fatty acids relative to the corresponding cells lacking the first exogenous polynucleotide. ii) Relative to a cell having a first exogenous polynucleotide and lacking an exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, said cells have increased expression and / or activity of fatty acyltransferases that catalyze the synthesis of TAG, DAG, or MAG, preferably TAG. iii) Relative cells possessing a first exogenous polynucleotide and lacking genetic modifications that downregulate the endogenous generation and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in cytoplasms, said cells having reduced production of lysophosphatidic acid (LPA) from acyl-ACP and G3P in their plastids. iv) Compared to corresponding cells lacking exogenous polynucleotides and / or genetic modifications, said cells have an altered C16:3-C18:3 fatty acid ratio in their total fatty acid content and / or their galactolipid content, preferably a reduced ratio. v) The cells are located in the vegetative part of the plant and contain at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total nonpolar lipids. vi) The cells are located in the vegetative part of the plant and contain at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of TAG. vii) The transcription factor polypeptides are selected from the group consisting of Wrinkled 1 (WRI1), Leafy Cotyledon 1 (LEC1), LEC1-like, Leafy Cotyledon 2 (LEC2), BABY BOOM (BBM), FUS3, ABI3, ABI4, ABI5, Dof4, and Dof11, or the group consisting of MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2, and PHR1. viii) Oleic acid comprises at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%) of the total fatty acid content in the cells, preferably about 65% (mol%) or 20% to about 65%. (ix) The nonpolar lipids in the cells comprise fatty acids, which include hydroxyl, epoxy, cyclopropane, two-carbon, three-carbon, conjugated double bonds, branched chains such as methylated or hydroxylated branches, or combinations of two or more of these, or any two, three, four, five or six of the above groups, bonds or branches. x) The nonpolar lipids in the cells comprise one or more polyunsaturated fatty acids selected from eicosapentaenoic acid (EDA), arachidonic acid (ARA), octadecanoic acid (SDA), eicosatrienoic acid (ETE), eicosapentaenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or combinations of two or more of these. xi) The cells are in a plant or a part thereof, preferably a vegetative plant part, or the cells are algal cells such as diatoms, green algae, cyanophytes, chrysophytes, haptophytes, brown algae, and heteroflagellates, or the cells are derived from or are suitable for fermentation by organisms such as fungi. xii) One or more promoters are selected from promoters other than constitutive promoters, preferably tissue-specific promoters such as leaf and / or stem-specific promoters, developmental regulatory promoters such as senescence-specific promoters such as the SAG12 promoter, inducible promoters, or circadian rhythm regulatory promoters. Preferably, at least one promoter operatively linked to an exogenous polynucleotide encoding a transcription factor polypeptide is a promoter other than a constitutive promoter. (xiii) The cells contain a total fatty acid content (which includes medium-chain fatty acids, preferably C12:0, C14:0, or both, at a level of at least 5% of the total fatty acid content) and optionally present exogenous polynucleotides encoding LPAAT, wherein the LPAAT has preferential activity toward fatty acids having a medium chain length (C8-C14), preferably C12:0 or C14:0. xiv) The cells contain a total fatty acid content such that, relative to corresponding cells lacking exogenous polynucleotides and / or genetic modifications, their oleic acid and / or palmitic acid levels are increased by at least 2%, and / or their alpha-linolenic acid (ALA) and / or linoleic acid levels are decreased by at least 2% relative to corresponding cells lacking exogenous polynucleotides and / or genetic modifications. (xv) Nonpolar lipids in cells lacking exogenous polynucleotides and / or genetic modifications, wherein the nonpolar lipids in these cells comprise modified levels of total sterols, preferably free (non-esterified) sterols, stearoyl esters, and stearoyl glycosides. (xvi) The nonpolar lipids in the cells described contain waxes and / or wax esters. (xvii) The cell is a member of a group or collection of at least about 1000 such cells, preferably in the vegetative part of the plant or in the seed. (xviii) The cell contains an exogenous polynucleotide encoding a silencing repressor, wherein the exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the cell. xix) On a weight basis, the levels of one or more nonpolar lipids and / or total nonpolar lipids in the cells are at least 2% higher than those in corresponding cells containing exogenous polynucleotides encoding Arabidopsis thaliana WRI1 (SEQ ID NO:21) and Arabidopsis thaliana DGAT1 (SEQ ID NO:1), and xx) The total polyunsaturated fatty acid (PUFA) content is reduced relative to the total PUFA content of corresponding cells lacking exogenous polynucleotides and / or genetic modifications.

[0073] The following embodiments apply to the cells of the present invention (including the second, third, fourth, and fifth aspects), as well as methods for producing said cells and methods for using said cells. In these embodiments, when said cells are in the vegetative portion of a plant, the plant is preferably growing in soil.

[0074] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is a fatty acyl acyl transferase involved in the biosynthesis of TAG, DAG, or monoacylglycerol (MAG) in cells, preferably a TAG biosynthesis in cells, such as DGAT, PDAT, LPAAT, GPAT, or MGAT, with DGAT or PDAT being more preferred.

[0075] In one embodiment, the polypeptide involved in the catabolism of triacylglycerol (TAG) in cells is SDP1 lipase, Cgi58 polypeptide, acyl-CoA oxidase such as ACX1 or ACX2, or polypeptide involved in the β-oxidation of fatty acids in cells such as PXA1 peroxisome ATP-binding cassette transporter, preferably SDP1 lipase.

[0076] In one embodiment, the oil body coating (OBC) polypeptide is an oil protein, such as polyoleosin or caleosin, or preferably lipid droplet-associated protein (LDAP).

[0077] In one embodiment, the polypeptide that increases fatty acid export from the cytoplasm is a C16 or C18 fatty acid thioesterase such as FATA polypeptide or FATB polypeptide, a fatty acid transporter such as ABCA9 polypeptide, or a long-chain acyl-CoA synthase (LACS).

[0078] In one embodiment, the polypeptide involved in introducing fatty acids into the cytoplasm is a fatty acid transporter protein or its subunit, preferably a TGD polypeptide, such as TGD1 polypeptide, TGD2 polypeptide, TGD3 polypeptide or TGD4 polypeptide.

[0079] In one embodiment, the polypeptide involved in the generation of diacylglycerol (DAG) in the plasmid is plasmid GPAT, plasmid LPAAT, or plasmid PAP.

[0080] In one embodiment, the cells are derived from or in a 16:3 plant, or in its vegetative parts or seeds, and comprise one or more of the following: a) An exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the exogenous polynucleotide, increases fatty acid efflux from the cytoplasm. b) A first genetic modification that, when compared to corresponding cells lacking the first genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the importation of fatty acids into the cytoplasm. c) A second genetic modification that, compared to corresponding cells lacking the second genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. The exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the cell.

[0081] In an alternative embodiment, the cells originate from or are found in 18:3 plants, or in their vegetative parts or seeds.

[0082] In one embodiment, the cells are derived from or located in the leaves, stems, or roots of a plant before flowering, and the cells contain at least about 8%, at least about 10%, at least about 11%, 8%-15%, or 9%-12% (w / w dry weight) of total nonpolar lipids. In one embodiment, the total nonpolar lipid content of the cells is at least 3%, more preferably at least 5%, greater than that of corresponding cells transformed with genes encoding WRI1 and DGAT but lacking other exogenous polynucleotides and genetic modifications as described herein in aspects two, three, four, and five. More preferably, the increase is in cells located in the stems or roots of the plant.

[0083] In one embodiment, the addition of one or more exogenous polynucleotides or genetic modifications, preferably exogenous polynucleotides encoding OBC or fatty acylthioesterases or genetic modifications that downregulate the endogenous generation and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in cells, more preferably exogenous polynucleotides encoding FATA thioesterases or LDAP or reducing the expression of endogenous TAG lipases such as SDP1TAG lipases in cells, results in a synergistic increase in the total nonpolar lipid content of cells when added to the transgenic WRI1 and DGAT pair, particularly before flowering, and even more particularly in the stems and / or roots of the plant. See, for example, Examples 8, 11, and 15. In a preferred embodiment, the TAG content in cells of the stems or roots of the plant increases at least 2-fold, more preferably at least 3-fold, compared to corresponding cells transformed with genes encoding WRI1 and DGAT1 but lacking FATA thioesterase, LDAP, and genetic modifications that downregulate the endogenous generation and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in cells. Most preferably, the promoter that guides the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0084] The genetic modification can be any alteration of naturally occurring cells to achieve the desired effect. Methods for genetically modifying cells are well known in the art. In one embodiment, each of the one or more genetic modifications is an endogenous gene mutation that partially or completely inactivates a gene, preferably an introduced mutation such as a point mutation, insertion, or deletion (or a combination of one or more of these). A point mutation can be an early stop codon, splicing site mutation, frameshift mutation, or amino acid substitution that reduces the activity of a gene or its encoded polypeptide. A deletion can be the deletion of one or more nucleotides within a transcriptional exon or promoter of a gene, or an extension across or into more than one exon, or an extension to the entire gene. Preferably, the deletion is introduced using ZF, TALEN, or CRISPR technologies. In one embodiment, one or more genetic modifications are exogenous polynucleotides encoding RNA molecules that repress the expression of endogenous genes, wherein the exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the cell. Examples of exogenous polynucleotides that reduce the expression of endogenous genes are selected from antisense polynucleotides, sense polynucleotides, microRNAs, polynucleotides encoding polypeptides that bind to endogenous enzymes, double-stranded RNA molecules, and processed RNA molecules derived therefrom. In one embodiment, the cell comprises a genetic modification that introduces a mutation into an endogenous gene and an exogenous polynucleotide encoding an RNA molecule that reduces the expression of another endogenous gene.

[0085] In one embodiment, the exogenous polynucleotide encoding WRI1 comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 21-75 or 205-210, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 21-75 or 205-210. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridization to nucleotides i) and / or ii) under stringent conditions. Preferably, the WRI1 polypeptide is a WRI1 polypeptide other than Arabidopsis WRI1 (SEQ ID NO: 21 or 22). More preferably, the WRI1 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 208 or a bioactive fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical thereto.

[0086] In one embodiment of the second, third, fourth, or fifth aspect, the recombinant cells are potato ( Solanum tuberosum ) cells of tubers, beets ( Beta vulgaris ) cells of roots or leaves, sugarcane ( Saccharum sp.) or two-colored sorghum (sp.) Sorghum bicolor Cells of stems or leaves, endosperm cells of monocotyledonous plants, wherein the cells have an increased total fatty acid content relative to the corresponding wild-type endosperm cells, such as wheat ( Triticum aestivum Grains, rice (Oryza genus) Oryza sp.)) grains or corn (sp.)) Zea mays ) cells, with increased total fatty acid content in the genus Brassica ( Brassica The cells of seeds such as rapeseed, or legume seeds with increased total fatty acid content such as soybeans (sp.) Glycine max )The cells of the seed.

[0087] In a sixth aspect, the present invention provides a non-human object or a portion thereof comprising or composed of one or more cells of the present invention.

[0088] In one embodiment, the non-human part is a seed, fruit, or a nutrient part of a plant such as a surface plant part or a green part such as a leaf or stem.

[0089] In another embodiment, the non-human object is a photosynthetic organism such as a plant or algae, or an organism suitable for fermentation such as a fungus.

[0090] In a seventh aspect, the present invention provides a transgenic plant or a portion thereof, preferably a nutrient-rich plant portion, comprising... a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and any one, two, or all three of the following c) A genetic modification that, compared with a corresponding plant lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant. d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking a fourth exogenous polynucleotide, increases fatty acid efflux from the plant's cytoplasm, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant.

[0091] In one embodiment, the plant or a portion thereof comprises a), b), and c), and optionally d) or e).

[0092] In one embodiment, the plant or a portion thereof comprises a), b), and d), and optionally c) or e).

[0093] In one embodiment, the plant or a portion thereof comprises a), b), and e), and optionally c) or d).

[0094] In a preferred embodiment, the presence of c), d), or e), along with a), increases the total nonpolar lipid content of the plant or its portion, preferably a nutrient plant portion such as a leaf, root, or stem, relative to the corresponding plant or portion containing a) and b) but lacking each of c), d), and e). More preferably, the increase is synergistic. Most preferably, at least the promoter guiding the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0095] In one embodiment, the plant or a portion thereof further comprises one or more of the following: a) A fifth exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. b) A second genetic modification that, compared to a corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the input of fatty acids into the plastids of said plant, and c) A third genetic modification that, when compared with the corresponding plant lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in plastids.

[0096] In one embodiment, the genetically modified plant or a portion thereof contains a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant, preferably expressed from a promoter other than a constitutive promoter. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and c) A genetic modification that, compared with a corresponding plant lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant, and optionally the plant or a portion thereof further comprises one or more of the following: d) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the fourth exogenous polynucleotide, increases fatty acid efflux from the plant's plastids. f) A second genetic modification that, compared to a corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the input of fatty acids into the plastids of said plant, and g) A third genetic modification that, when compared with the corresponding plant lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids.

[0097] In one embodiment, the portion is a nutrient portion, and one or more promoters are expressed at a higher level in the nutrient portion relative to the seed of the plant.

[0098] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT, and the polypeptide involved in the catabolism of TAG in the plant is SDP1 lipase.

[0099] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is the WRI1 polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT.

[0100] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT.

[0101] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in the catabolism of triacylglycerol (TAG) in the plant is an SDP1 lipase.

[0102] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the polypeptide involved in the catabolism of triacylglycerol (TAG) in the plant is an SDP1 lipase.

[0103] In one implementation, when present, the two transcription factors are WRI1 and LEC2, or WRI1 and LEC1.

[0104] In the above embodiments, preferably the plant is growing in soil or growing in soil and subsequently harvesting a portion thereof. Preferably, on a weight basis, the nutrient portion of the plant contains at least 8% TAG (% dry weight), for example 8%-75% or 8%-30%. More preferably, the TAG content is at least 10%, for example 10%-75% or 10%-30%. Preferably, these TAG levels are present in the nutrient portion before or during flowering or before the fruiting stage of plant development. In these embodiments, preferably the ratio of TAG content in leaves to TAG content in plant stems is 1:1 to 10:1, and / or said ratio is increased relative to corresponding cells containing first and second exogenous polynucleotides and lacking the first genetic modification.

[0105] In the above embodiments, the total nonpolar lipid content of the plant or its parts is preferably at least 3% greater than that of the corresponding plant or its parts transformed with genes encoding WRI1 and DGAT but lacking other exogenous polynucleotides and genetic modifications as described herein. More preferably, this increase is in the stem or root tissue of the plant.

[0106] In the above embodiments, it is preferred to add one or more exogenous polynucleotides or genetic modifications, preferably exogenous polynucleotides encoding OBC or fatty acid thioesterases or genetic modifications that downregulate the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in cells, more preferably exogenous polynucleotides encoding LDAP or FATA thioesterases or reducing the expression of endogenous TAG lipases such as SDP1 TAG lipases in cells. When added to the transgenic WRI1 and DGAT pair, this results in a synergistic increase in the total nonpolar lipid content of the plant or parts thereof, particularly before flowering, and even more particularly in the stem and / or root tissues of the plant. See, for example, Examples 8, 11, and 15. In a preferred embodiment, relative to the portion transformed with genes encoding WRI1 and DGAT1 but lacking the corresponding portion of the exogenous polynucleotide encoding OBC or fatty acid thioesterases and the genetic modification that downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in cells, the TAG content in the leaves, stems, or roots of the plant, or all three, increases by at least 2-fold, more preferably at least 3-fold.

[0107] In the above embodiments, the plant or a portion thereof preferably contains a second exogenous polynucleotide encoding DGAT and a first genetic modification that downregulates the production of exogenous SDP1 lipase. More preferably, the plant or a portion thereof does not contain an exogenous polynucleotide encoding PDAT, and / or is a plant or a portion thereof other than Nicotiana benthamiana and / or Rapeseed, and / or WRI1 is a WRI1 other than Arabidopsis thaliana WRI1 (SEQ ID NOs: 21 or 22). In one embodiment, the plant is not sugarcane. More preferably, at least one exogenous polynucleotide in the plant is expressed from a promoter that is not a constitutive promoter, such as a promoter that is preferentially expressed in the green tissues or stems of the plant or upregulated after flowering or during senescence. Preferably, at least a first exogenous polynucleotide (encoding a transcription factor) is expressed from such a promoter.

[0108] In an eighth aspect, the present invention provides a transgenic plant or a portion thereof, preferably a nutrient-rich plant portion, comprising... a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in plants. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, and c) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. Each exogenous polynucleotide is operatively linked to a promoter that directs the expression of the polynucleotide in the plant, and the plant, relative to the corresponding plant containing a third exogenous polynucleotide (whose nucleotide sequence is complementary to the sequence provided in SEQ ID NO:176), has elevated levels of one or more nonpolar lipids and / or increased amounts of OBC polypeptides.

[0109] In a preferred embodiment, the presence of a third exogenous polynucleotide encoding an OBC polypeptide, relative to a corresponding plant part containing the first and second exogenous polynucleotides but lacking the third exogenous polynucleotide, together with the first and second exogenous polynucleotides, increases the total nonpolar lipid content of the plant or its parts, preferably nutrient plant parts such as leaves, roots, or stems. More preferably, the increase is synergistic. Most preferably, at least the promoter guiding the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0110] In one embodiment of the eighth aspect, the plant or a portion thereof further comprises one or more of the following: d) A first genetic modification that, compared to a corresponding plant lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in said plant. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the fourth exogenous polynucleotide, increases fatty acid efflux from the plant's plastids. f) A second genetic modification that, compared to a corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the input of fatty acids into the plastids of said plant, and g) A third genetic modification that, when compared with the corresponding plant lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids.

[0111] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT, and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0112] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is the WRI1 polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT.

[0113] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the OBC polypeptide is an oleogenous protein. Alternatively, the OBC polypeptide is LDAP.

[0114] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0115] In one embodiment, the cell contains two exogenous polynucleotides encoding two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant, such as WRI1 and LEC2, or WRI1 and LEC1.

[0116] In a ninth aspect, the present invention provides a transgenic plant or a portion thereof, preferably a nutrient plant portion, comprising a first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes for glycolysis and / or fatty acid biosynthesis in the plant, and one or more of the following; a) A second exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the second exogenous polynucleotide, increases fatty acid efflux from the plant's plastids. b) A first genetic modification that, compared to a corresponding plant lacking the first genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the input of fatty acids into the plant plastids. c) A second genetic modification that, compared to the corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant.

[0117] In one embodiment, the plant or a portion thereof, preferably a vegetative plant portion, comprises a) and optionally b) or c).

[0118] In one embodiment of the ninth aspect, the plant or a portion thereof further comprises one or more of the following: d) A third exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. e) A third genetic modification that, compared to a corresponding plant lacking the third genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the plant, and f) A fourth exogenous polynucleotide encoding an oil-coated (OBC) polypeptide, preferably LDAP.

[0119] In a preferred embodiment, the plant or a portion thereof, preferably a nutrient plant portion, comprises a first, second, and third exogenous polynucleotide, as well as an optional third genetic modification or a fourth exogenous polynucleotide.

[0120] In a preferred embodiment, the presence of a second exogenous polynucleotide encoding a polypeptide (preferably a fatty acylthioesterase such as a FATA polypeptide) that increases fatty acid output from the plant plastid, relative to a corresponding plant part containing the first and third (if present) exogenous polynucleotides but lacking the second exogenous polynucleotide, together with the first and third (if present) exogenous polynucleotides, increases the total nonpolar lipid content of the plant part, preferably a nutrient plant part such as a leaf, root, or stem. More preferably, the increase provided by the second exogenous polynucleotide is synergistic. Most preferably, at least the promoter guiding the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0121] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in introducing fatty acids into the cytoplasm is a TGD polypeptide.

[0122] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the polypeptide involved in diacylglycerol (DAG) production is plastid GPAT.

[0123] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide that increases fatty acid export from the plant plastid is a fatty acid thioesterase, preferably a FATA or FATB polypeptide; and the polypeptide involved in the importation of fatty acids into the plant plastid is a TGD polypeptide.

[0124] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide that increases fatty acid export from the plant plastid is a fatty acid thioesterase, preferably a FATA or FATB polypeptide; and the polypeptide involved in diacylglycerol (DAG) production is plastid GPAT.

[0125] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in introducing fatty acids into the plant plastids is a TGD polypeptide; and the polypeptide involved in the generation of diacylglycerol (DAG) is plastid GPAT.

[0126] In one embodiment, the plant contains two exogenous polynucleotides encoding two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

[0127] In embodiments of the seventh, eighth, and ninth aspects, when the plant contains an exogenous polynucleotide encoding a fatty acid thioesterase such as a FATA or FATB polypeptide, the thioesterase is preferably a FATA polypeptide or a fatty acid thioesterase other than a medium-chain fatty acid thioesterase.

[0128] In a tenth aspect, the present invention provides a transgenic plant or a portion thereof, preferably a nutrient portion, comprising... a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant, preferably a WRI transcription factor. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, said polypeptide being LPAAT with preferential activity against fatty acids of medium chain length (C8-C14), and c) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the third exogenous polynucleotide, increases the export of C8-C14 fatty acids from the plant's plastids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant.

[0129] In a preferred embodiment, the presence of a third exogenous polynucleotide encoding a polypeptide that increases the export of C8-C14 fatty acids from the plant plastid, relative to a corresponding plant part containing the first and second exogenous polynucleotides but lacking the third exogenous polynucleotide, increases the total MCFA content of the plant part, preferably a nutrient-rich plant part such as a leaf, root, or stem, in conjunction with the first and second exogenous polynucleotides. More preferably, the increase provided by the third exogenous polynucleotide is synergistic. Most preferably, at least the promoter guiding the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0130] In one embodiment of the tenth aspect, the transgenic plant or a portion thereof further comprises one or more of the following; d) A fourth exogenous polynucleotide encoding another polypeptide involved in the biosynthesis of one or more nonpolar lipids. e) A first genetic modification that, compared to a corresponding plant lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the plant. f) A fifth exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. g) A second genetic modification that, compared to a corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the input of fatty acids into the plastids of the plant, and h) A third genetic modification that, compared to the corresponding plant lacking the third genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant.

[0131] In one embodiment of the tenth aspect, the transcription factor is not Arabidopsis thaliana WRI1 (SEQ ID NO: 21 or 22), and / or the plant is not Nicotiana benthamiana (…). N. benthamiana ).

[0132] In one embodiment of the tenth aspect, the exogenous polynucleotide encoding LPAAT comprises an amino acid sequence as shown in SEQ ID NO:200, or a biologically active fragment thereof, or an LPAAT polypeptide whose amino acid sequence is at least 30% identical thereto.

[0133] In one embodiment of the tenth aspect, one or more promoters are expressed in the vegetative portion at a higher level than that of the seed of the plant, preferably including at least a promoter expressing a first exogenous polynucleotide.

[0134] In one embodiment of the tenth aspect, the fatty acid having a medium chain length is at least myristic acid (C14:0). In a preferred embodiment, the plant portion, preferably a nutrient plant portion, contains at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, 8%-25%, 8%-20%, 10%-25%, 11%-25%, about 15%-25%, or about 20%-25% (w / w dry weight) of myristic acid.

[0135] In embodiments of the sixth, seventh, eighth, ninth, and tenth aspects, the plant is preferably grown in soil or grown in soil and subsequently harvested, preferably a vegetative plant portion, and wherein, on a weight basis, the plant portion contains at least 8% TAG (% dry weight), for example 8%-75% or 8%-30%. More preferably, the TAG content is at least 10%, for example 10%-75% or 10%-30%. Preferably, these TAG levels are present in the vegetative portion before or during flowering or before the fruiting stage of plant development. In these embodiments, the ratio of TAG content in leaves to TAG content in plant stems is preferably 1:1 to 10:1, and / or said ratio is increased relative to corresponding cells containing first and second exogenous polynucleotides and lacking the first genetic modification.

[0136] In embodiments of the sixth, seventh, eighth, ninth, and tenth aspects, the plant or a portion thereof preferably contains an exogenous polynucleotide encoding DGAT and genetic modifications that downregulate the production of exogenous SDP1 lipase. In a preferred embodiment, the plant or a portion thereof does not contain an exogenous polynucleotide encoding PDAT, and / or is a plant other than *Nicotiana benthamiana*. Most preferably, at least one exogenous polynucleotide in the plant or a portion thereof is expressed from a promoter that is not a constitutive promoter, such as a promoter preferentially expressed in the green tissues or stems of the plant or upregulated during senescence.

[0137] In an eleventh aspect, the present invention provides a plant comprising a nutrient portion or the nutrient portion thereof, wherein the nutrient portion has a total nonpolar lipid content of at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein the nonpolar lipids comprise at least 90% triglycerides (TAG).

[0138] In a preferred embodiment, the nutrient plant portion is characterized by the properties described in aspects seven, eight, nine, and ten. The plant is preferably an 18:3 plant.

[0139] In one embodiment of the above aspects, the plant cells or plant parts have been treated so that they are no longer capable of propagating or producing living plants, i.e., they are dead. For example, the plant cells or plant parts have been dried and / or ground.

[0140] In a twelfth aspect, the present invention provides a plant comprising a nutrient portion or the nutrient portion thereof, wherein the nutrient portion has a TAG content of at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein the nonpolar lipid comprises at least 90% triglycerides (TAG). The plant is preferably an 18:3 plant.

[0141] In a thirteenth aspect, the present invention provides a plant comprising a nutrient portion or a nutrient portion thereof, wherein the nutrient portion has a total nonpolar lipid content of at least 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein the nonpolar lipids comprise at least 90% triglycerides (TAG), and wherein the plant is a 16:3 plant or a nutrient portion thereof.

[0142] In a fourteenth aspect, the present invention provides a plant comprising a nutrient portion or a nutrient portion thereof, wherein the nutrient portion has a TAG content of at least 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein the nonpolar lipid comprises at least 90% triglycerides (TAG), and wherein the plant is a 16:3 plant or a nutrient portion thereof.

[0143] In one embodiment, the cells of the present invention (including the second, third, fourth, and fifth aspects) are cells of the following species or genera, or the plant or part thereof of the present invention (including the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, and fourteenth aspects) is *Cocos nucifera* (coconut palm). Acrocomia aculeata (macauba palm)), Arabidopsis thaliana, peanut ( Aracinis hypogaea Peanuts, Mulu Star Palm ( Astrocaryum murumuru (murumuru)) Astrocaryum vulgare (tucuma) Attalea geraensis (Indaia-rateiro) Attalea humilis (American oil palm) Attalea oleifera (andaia) Attalea phalerata (uricuri) Attalea speciosa (babassu), oats Avena sativa Oats, sugar beets, and brassica species, etc. Brassica carinata , Brassica juncea , Brassica napobrassica European rapeseed (rapeseed), flaxseed ( Camelina sativa flax (false flax), cannabis ( Cannabis sativa Marijuana (hemp) and safflower ( Carthamus tinctorius safflower) Caryocar brasiliense (pequi), coconut ( Cocos nucifera Coconut, sea cabbage Crambe abyssinica sea ​​cabbage (Abyssinian kale), melon (Cucumis melo Melon, oil palm Elaeis guineensis African palm, soybean, upland cotton Gossypium hirsutum cotton), sunflower ( Helianthus sp.) such as sunflower ( Helianthus annuus Sunflower, barley Hordeum vulgare Barley, Jatropha curcas Jatropha curcas jatropha (physic nut) Joannesia princeps (arara nut-tree), duckweed (genus) Lemna sp., duckweed (like green duckweed) Lemna aequinoctialis ), Lemna disperma , Lemna ecuadoriensis Floating duckweed ( swollen duckweed), Japanese duckweed ( Lemna gibba ),duckweed( Lemna japonica ), Lemna minuta Lemna minor Rare vein duckweed ( Lemna obscura ), rare vein duckweed ( Lemna paucicostata ), Lemna perpusilla Lü Zao ( Lemna tenera ), duckweed ( Lemna trisulca Lemna ), trisulca , Lemna valdiviana Oticia rigida (Licania rigida, Oticica), flax ( Lemna yungensis Flax, lupins Linum usitatissimum Lupinus Lupin (Lupin) and Curved-leaved Palm (Lupinus cuspidata) angustifolius Buriti palm), Moti coconut ( Mauritia flexuosa Palm (Inaja palm), Miscanthus ( Maximiliana maripa sp.) such as allotriploid Miscanthus gypsum (sp.) Miscanthus ) and Chinese mango ( Miscanthus x giganteus Miscanthus sinensis ), the genus *Nicotiana* (tobacco), such as *Nicotiana safflower* (…). Nicotiana tabacum ) or Ben's tobacco, Bakkapa wine palm ( Oenocarpus berry Bakaba do-azeite, Bataoa do-azeite ( Oenocarpus bataua ,pataua) Oenocarpus distichus (bacaba-de-leque), genus *Rice* (rice), such as rice ( Rice green ) and Guangye rice ( Rice ), willow branch millet ( Panicum virgatum Switchgrass Paraqueiba parensis (mari), avocado Persea amencana Avocado, water citrus ( Pongamia pinnata Indian beech, hairy poplar People three-leafed ), castor bean ( Castor oil Castor, sugarcane, sesame Indian sesame Sesame, potato, sorghum ( Sorghum sp.) such as two-colored sorghum ( Sorghum bicolor ), sorghum ( Common sorghum ), Big Flower Cocoa ( Theobroma grandiflorum ,cupuassu, clover ( Clover sp.), Brazilian needle palm ( Trithrinax brasiliensis Brazilian needle palm, wheat ( Wheat sp., wheat (wheat) e.g., wheat ( Wheat summer ) and corn (corn).

[0144] In a fifteenth aspect, the present invention provides a potato plant or a portion thereof, preferably a tuber, having a diameter of at least 2 cm and having a TAG content of at least 0.5% on a dry weight basis and / or a fatty acid content of at least 1%, preferably at least 1.5% or at least 2.0% on a dry weight basis. When compared to a corresponding potato tuber lacking the genetic modification and / or exogenous polynucleotide, the potato tuber preferably has elevated levels of monounsaturated fatty acids (MUFA) and / or lower levels of polyunsaturated fatty acids (PUFA), such as elevated levels of oleic acid and decreased levels of ALA, in the total fatty acid content and the TAG portion of the total fatty acid content. Preferably, when compared to a corresponding potato tuber lacking the genetic modification and / or exogenous polynucleotide, the ALA level in the total fatty acid content of the tuber is reduced by at least 10% and / or the oleic acid level in the total fatty acid content is increased to at least 5%, preferably at least 10% or more preferably at least 15%. Furthermore, in one embodiment, when compared to corresponding potato tubers lacking the genetic modification and / or exogenous polynucleotides, the palmitic acid level in the total fatty acid content of the tubers is increased and / or the stearic acid (18:0) level in the total fatty acid content of the tubers is decreased. In one embodiment, when they are ground under the same conditions, the starch content of the tubers is about 90%-100% by weight relative to wild-type tubers.

[0145] In one embodiment, the potato plant or a portion thereof, preferably a tuber, of the present invention comprises a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the tuber, and b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the tuber during the growth of the potato plant.

[0146] In a preferred embodiment, the potato tubers further comprise one or more of the following: c) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. d) A first genetic modification that, compared to a corresponding tuber lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triglycerides (TAG) in the tuber, for example, when the peptide is SDP1. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding tuber lacking the fourth exogenous polynucleotide, increases fatty acid efflux from the plastid of the tuber. f) A second genetic modification that, compared to a corresponding tuber lacking the second genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the input of fatty acids into the plastids of the tuber, and g) A third genetic modification that, when compared with a corresponding tuber lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in the plastids of the tuber.

[0147] In other embodiments, additional genetic modifications in the tuber are as defined in the context of the cell or plant of the present invention.

[0148] In a sixteenth aspect, the present invention provides a sorghum or sugarcane plant, or a portion thereof, preferably stems or leaves, having a total fatty acid content of at least 6% or at least 8% on a dry weight basis and / or a TAG content of at least 2% or at least 3% on a dry weight basis in the stem and / or a TAG content in the stem increased by at least 50-fold on a weight basis and / or an increase of at least 100-fold in the leaves. In an embodiment, the sorghum or sugarcane plant, or a portion thereof, preferably stems or leaves, is characterized by characteristics as defined in the context of cells or plants or portions thereof according to the present invention.

[0149] In a seventeenth aspect, the present invention provides a sorghum or sugarcane plant, or a portion thereof, preferably stems or leaves, comprising a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts, and b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, wherein each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or a portion thereof during plant growth.

[0150] Preferably, the promoter guiding the expression of at least the first exogenous polynucleotide is a promoter other than the rice ubiquitin promoter (Rubi3). More preferably, the promoter is not a ubiquitin promoter or any other constitutive promoter. Preferably, the first and second exogenous polynucleotides and their respective promoters are linked to a genetic construct integrated into the plant genome.

[0151] In one embodiment, when they are ground under the same conditions, the sugar content of the sugarcane stalk is about 70%-100% by weight, relative to wild-type sugarcane stalks. Alternatively, the sugar content is 50%-70%.

[0152] In one embodiment, the sorghum or sugarcane plant of the present invention, or a portion thereof, preferably the stem or leaves, comprises a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the stem of the plant, and b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. At least one of the exogenous polynucleotides, preferably at least the first exogenous polynucleotide, is operatively linked to a promoter that is preferentially expressed in the stem relative to the leaf during the growth of the plant.

[0153] In one embodiment, the sorghum or sugarcane plant or a portion thereof of the present invention further comprises one or more of the following: c) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. d) A first genetic modification that, when compared to a corresponding plant or part thereof lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in said plant or part thereof. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking the fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof. f) A second genetic modification that, when compared to a corresponding plant or part thereof lacking the second genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the input of fatty acids into plastids of said plant or part thereof, and g) A third genetic modification that, when compared with a corresponding plant or part thereof lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids of the plant or part thereof.

[0154] When compared with the corresponding plants or portions thereof lacking the genetic modifications and / or exogenous polynucleotides, the sorghum or sugarcane plants or portions thereof of the present invention preferably have elevated levels of monounsaturated fatty acids (MUFA) and / or lower levels of polyunsaturated fatty acids (PUFA), such as elevated levels of oleic acid and reduced levels of ALA, in the total fatty acid content and the TAG portion of the total fatty acid content.

[0155] Preferably, when compared with the corresponding plant or part thereof lacking the genetic modification and / or exogenous polynucleotide, the ALA level in the total fatty acid content is less than 10% and / or the oleic acid level in the total fatty acid content is at least 5%, preferably at least 10% or more preferably at least 15%.

[0156] In other embodiments, additional genetic modifications in the sorghum or sugarcane plant or portions thereof are as defined in the context of the cell or plant of the present invention.

[0157] In an eighteenth aspect, the present invention provides a transgenic monocotyledonous plant or a portion thereof, preferably leaves, grains, stems, roots, or endosperm, having a total fatty acid content or TAG content that is at least 5-fold higher by weight compared to a corresponding non-transgenic monocotyledonous plant or a portion thereof. Alternatively, the present invention provides a transgenic monocotyledonous plant whose endosperm has a TAG content of at least 2.0%, preferably at least 3%, more preferably at least 4% or at least 5% by weight, or a portion of said plant, preferably leaves, stems, roots, grains, or endosperm. In one embodiment, the endosperm has a TAG content of at least 2%, which is at least 5-fold higher than that of the corresponding non-transgenic endosperm. Preferably, the plant is exclusively male- or female-reproducing, its pollen is substantially 100% viable, and its grains have a germination rate of 70%-100% compared to the corresponding wild-type grains. In one embodiment, the transgenic plant is a progeny of at least two generations of an initial transgenic wheat plant, and the transgene is preferably homozygous. In an embodiment, the monocotyledonous plant or a portion thereof, preferably a leaf, stem, grain, or endosperm, is further characterized by one or more properties as defined in the context of a cell or plant as described in this invention.

[0158] In a nineteenth aspect, the present invention provides a monocotyledonous plant or a portion thereof, preferably a leaf, grain, stem or endosperm, comprising a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts, and b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts during plant growth.

[0159] Preferably, at least the promoter that guides the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0160] In one embodiment, when the plants from which the grains are obtained are grown under the same conditions, the starch content of the monocotyledonous grains of the present invention is about 70%-100% by weight, relative to wild-type grains. Preferred monocotyledonous plants in both of the above aspects are wheat, rice, sorghum, and corn (maize).

[0161] In one embodiment, the monocotyledonous plant or a portion thereof, preferably a leaf, grain, or endosperm, of the present invention comprises... a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the endosperm of the plant, and b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. At least one of the exogenous polynucleotides, preferably at least the first exogenous polynucleotide, is operatively linked to a promoter that is expressed in the endosperm at a higher level than that in the leaves during the growth of the plant.

[0162] In a preferred embodiment, the monocotyledonous plant or a portion thereof further comprises one or more of the following: c) A third exogenous polynucleotide encoding an oil-body-coated (OBC) polypeptide, preferably LDAP. d) A first genetic modification that, when compared to a corresponding plant or part thereof lacking the first genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in said plant or part thereof. e) A fourth exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking the fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof. f) A second genetic modification that, when compared to a corresponding plant or part thereof lacking the second genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the input of fatty acids into plastids of said plant or part thereof, and g) A third genetic modification that, when compared with a corresponding plant or part thereof lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the generation of diacylglycerol (DAG) in plastids of the plant or part thereof.

[0163] In one embodiment, the monocotyledonous plant comprises one or two of the characteristics a), b), d), and e), and optionally one of c), f), and g).

[0164] When compared with the corresponding plant or part thereof lacking the genetic modification and / or exogenous polynucleotide, the monocotyledonous plant or part thereof of the present invention, preferably leaves, grains, stems or endosperm, preferably has elevated levels of monounsaturated fatty acids (MUFA) and / or lower levels of polyunsaturated fatty acids (PUFA) in the total fatty acid content and the TAG portion of the total fatty acid content, such as elevated levels of oleic acid and lower levels of LA (18:2).

[0165] Preferably, when compared with the corresponding plant or part thereof lacking the genetic modification and / or exogenous polynucleotide, the linoleic acid (LA, 18:2) level in the total fatty acid content of the grain or endosperm is reduced by at least 5% and / or the oleic acid level in the total fatty acid content is increased by at least 5%, preferably at least 10% or more preferably at least 15%, relative to the corresponding wild-type plant or part thereof.

[0166] The following implementation schemes apply to the plants or parts thereof in aspects 15, 16, 17, 18 and 19.

[0167] In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT, and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0168] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant or its parts is the WRI1 polypeptide, and the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT.

[0169] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant or its parts is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide, and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0170] In one embodiment, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the plant or its parts is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the OBC polypeptide is an oleogen. Alternatively, the OBC polypeptide is LDAP.

[0171] In one embodiment, the plant or a portion thereof contains two exogenous polynucleotides encoding two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

[0172] In each embodiment of the cell, plant, or part thereof of the present invention (including aspects two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth), preferably, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell is WRI1 polypeptide, LEC2 polypeptide, LEC1 polypeptide, or LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the polypeptide involved in the catabolism of triacylglycerol (TAG) in the cell is SDP1 lipase.

[0173] In each embodiment of the cell, plant, or part thereof of the present invention (including aspects two, three, five, six, seven, eight, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth, but excluding aspects five and ten), preferably, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1 polypeptide, or a LEC1-like polypeptide; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT or PDAT; and the polypeptide that increases fatty acid efflux cytoplasm is a fatty acid thioesterase, preferably a FATA or FATB polypeptide, more preferably a FATA polypeptide or a fatty acid thioesterase other than a medium-chain fatty acid thioesterase.

[0174] In each of the above embodiments of the cell, plant, or part thereof of the present invention (including aspects two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth), it is preferable that the transcription factor polypeptides expressing one or more glycolysis and / or fatty acid biosynthesis genes in the plant or part thereof are combinations of at least two polypeptides, preferably WRI1 polypeptide and LEC2 polypeptide. More preferably, the at least two transcription factor polypeptides are expressed from different promoters. Most preferably, the exogenous polynucleotides encoding the at least two polypeptides are linked to a single genetic construct integrated into the genome of the cell or plant.

[0175] In each of the above embodiments, when the plant is a dicotyledonous plant, the transcription factor may be a monocotyledonous transcription factor. Conversely, when the plant is a monocotyledonous plant, the transcription factor may be a dicotyledonous transcription factor. The transcription factor is preferably an anticotyledonous plant other than Arabidopsis thaliana. A. thaliana Transcription factors other than WRI1 (SEQ ID NO: 21 or 22).

[0176] In each of the above embodiments, the plant is preferably a transgenic progeny plant derived from the initial transgenic plant at least two generations prior, and the transgene is preferably homozygous.

[0177] In other embodiments, additional genetic modifications to the plant or its parts are as defined in the context of the cells of this invention.

[0178] In one embodiment, the plant or part thereof of the present invention (including aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth) has one or more or all of the following characteristics (where applicable); i) The plant comprises a portion, preferably a nutrient portion, which, relative to the corresponding portion lacking the first exogenous polynucleotide, has increased total fatty acid synthesis, or, relative to the corresponding portion lacking the first exogenous polynucleotide, has decreased total fatty acid catabolism, or both, thereby having an elevated level of total fatty acids relative to the corresponding portion lacking the first exogenous polynucleotide. ii) The plant contains a portion, preferably a nutrient portion, which, relative to the corresponding portion of an exogenous polynucleotide having a first exogenous polynucleotide and lacking a polypeptide encoding a peptide involved in the biosynthesis of one or more nonpolar lipids, has increased expression and / or activity of a fatty acyltransferase catalyzing the synthesis of TAG, DAG, or MAG, preferably TAG. iii) The plant comprises a portion, preferably a nutrient portion, which, relative to a corresponding portion having a first exogenous polynucleotide and lacking genetic modifications that downregulate the endogenous generation and / or activity of polypeptides involved in the formation of diacylglycerol (DAG) in the plastids of the plant portion, has reduced lysophosphatidic acid (LPA) formation from acyl-ACP and G3P in its plastids. iv) The plant comprises a portion, preferably a nutrient portion, which, relative to a corresponding portion lacking exogenous polynucleotides and / or genetic modifications, has an altered C16:3-C18:3 fatty acid ratio in its total fatty acid content and / or its galactolipid content, preferably a reduced ratio. v) The nutrient portion of the plant contains at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total nonpolar lipids, preferably before flowering. vi) The nutrient portion of the plant contains at least about 8%, at least about 10%, at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of TAG, preferably before flowering. vii) The transcription factor polypeptide is selected from the group consisting of WRI1, LEC1, LEC1-like, LEC2, BBM, FUS3, ABI3, ABI4, ABI5, Dof4, and Dof11, preferably WRI1, LEC1, or LEC2, or the group consisting of MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2, and PHR1. viii) Oleic acid comprises at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%) of the total fatty acids in the plant or its parts, preferably about 65% (mol%) or 20% to about 65%. (ix) The nonpolar lipids in the plant or its parts, preferably the nutrient parts, comprise elevated levels of one or more fatty acids, said fatty acids comprising hydroxyl, epoxy, cyclopropane, two-carbon, three-carbon, conjugated double bonds, branched chains such as methylated or hydroxylated branches, or combinations of two or more of these, or any two, three, four, five or six of the aforementioned groups, bonds or branches. x) The nonpolar lipids in the plant or its parts, preferably the nutrient parts, comprise one or more polyunsaturated fatty acids selected from eicosapentaenoic acid (EDA), arachidonic acid (ARA), octadecanoic acid (SDA), eicosatrienoic acid (ETE), eicosapentaenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or combinations of two or more of these. xi) The part mentioned is a vegetative plant part, such as a leaf or stem or part thereof. xii) One or more promoters are selected from promoters other than constitutive promoters, preferably tissue-specific promoters such as leaf and / or stem-specific promoters, developmental regulatory promoters such as senescence-specific promoters such as the SAG12 promoter, inducible promoters, or circadian rhythm regulatory promoters. Preferably, at least one promoter operatively linked to an exogenous polynucleotide encoding a transcription factor polypeptide is a promoter other than a constitutive promoter. (xiii) The plant or its parts, preferably the nutrient portion, comprises a total fatty acid content (containing medium-chain fatty acids, preferably C12:0, C14:0, or both, at a level of at least 5% of the total fatty acid content) and optionally present exogenous polynucleotides encoding LPAAT, said LPAAT having preferential activity toward fatty acids having a medium chain length (C8-C14), preferably C12:0 or C14:0. xiv) The plant or its parts, preferably the nutrient-rich parts, contain a total fatty acid content in which the oleic acid and / or palmitic acid levels are increased by at least 2% relative to the corresponding plant or its parts lacking exogenous polynucleotides and / or genetic modifications, and / or the α-linolenic acid (ALA) and / or linoleic acid levels are decreased by at least 2% relative to the corresponding plant or its parts lacking exogenous polynucleotides and / or genetic modifications. (xv) Nonpolar lipids in the corresponding plant or its parts, relative to those lacking exogenous polynucleotides and / or genetic modifications, wherein the nonpolar lipids in the plant or its parts, preferably in the vegetative parts, comprise modified levels of total sterols, preferably free (non-esterified) sterols, stearoyl esters, stearoyl glycosides, (xvi) The nonpolar lipids in the plant or its parts comprise waxes and / or wax esters. (xvii) The plant or part thereof, preferably the vegetative part, is one of the members of a group or collection of at least about 1000 such plants or parts thereof. (xviii) The plant contains an exogenous polynucleotide encoding a silencing repressor, wherein the exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant. (xix) On a weight basis, the levels of one or more nonpolar lipids and / or total nonpolar lipids in the plant or its parts, preferably nutrient parts, are at least 2% higher than those in the corresponding plants or parts containing exogenous polynucleotides encoding Arabidopsis thaliana WRI1 (SEQ ID NO:21) and Arabidopsis thaliana DGAT1 (SEQ ID NO:1). xx) The reduction in total polyunsaturated fatty acid (PUFA) content compared to the corresponding plants lacking exogenous polynucleotides and / or genetic modifications. The plant part mentioned in xxi is potato ( Solanum tuberosum ) tubers, beets ( Beetroot ) roots, sugarcane (Saccharum) or sorghum (bicolor sorghum) stems, and monocotyledonous seeds such as wheat that have increased total fatty acid content in their endosperm. Summer wheat Grains or corn Corn ) grains, tobacco leaves, or legume seeds with increased total fatty acid content such as brassica seeds or soybeans ( Glycine max )seed, xxii) If the plant part is a seed, the seed germinates at substantially the same rate as the corresponding wild-type seed, or when sown in soil, produces a plant whose seeds germinate at substantially the same rate as the corresponding wild-type seed. xxiii) The plants mentioned are algae, such as those from diatoms, green algae, cyanophyte, chrysophyte, haptophyte, brown algae, and heteroflagellates.

[0179] In the above implementation scheme, the preferred plant part is at least 1 cm in diameter. 2 The surface area of ​​the leaves or the stem segment with a length of at least 1 cm.

[0180] In one embodiment of the above aspects, the plant or plant part has been treated so that it can no longer reproduce or produce living plants, i.e., it is dead. For example, the plant or plant part has been dried and / or ground.

[0181] In the above embodiments, preferably, the total nonpolar lipid content of the plant portion is at least 3%, more preferably at least 5%, greater than that of the corresponding plant transformed with genes encoding WRI1 and DGAT but lacking other exogenous polynucleotides and genetic modifications as described herein. More preferably, the increase is in the stem or root of the plant.

[0182] In one embodiment, the addition of one or more exogenous polynucleotides or genetic modifications, preferably exogenous polynucleotides encoding OBC or fatty acylthioesterases, or genetic modifications that downregulate the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the plant, more preferably exogenous polynucleotides encoding FATA thioesterases or LDAP, or reducing the expression of endogenous TAG lipases such as SDP1 TAG lipases in the plant, results in a synergistic increase in the total nonpolar lipid content of the plant portion when added to the transgenic WRI1 and DGAT pair, particularly before flowering, and even more particularly in the stems and / or roots of the plant. See, for example, Examples 8, 11, and 15. In a preferred embodiment, the TAG content in the stems or roots of the plant increases at least 2-fold, more preferably at least 3-fold, relative to the corresponding portion transformed with genes encoding WRI1 and DGAT1 but lacking FATA thioesterase, LDAP, and genetic modifications that downregulate the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the plant. Most preferably, the promoter that guides the expression of the first exogenous polynucleotide is a promoter other than a constitutive promoter.

[0183] In the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, sixteenth, eighteenth, and nineteenth aspects, it is preferred that the plant or parts thereof are phenotypically normal because their growth and reproductive capacity is not significantly reduced when compared with unmodified plants or parts thereof. Preferably, when grown under the same conditions, the biomass produced, growth rate, germination rate, storage organ size, seed size, and / or the number of viable seeds is not less than 90% of that of the corresponding wild-type plant. In one embodiment, the plant reproduces male or female in the same manner as the corresponding wild-type plant, and its pollen (if produced) is viable as that of the corresponding wild-type plant pollen, preferably about 100% viable. In one embodiment, when the plant species produces seeds, the plant produces seeds with a germination rate of at least 90% relative to the germination rate of the corresponding seeds of the wild-type plant. In one embodiment, the plant of the present invention has a plant height of at least 90% relative to the height of the corresponding wild-type plant grown under the same conditions. Combinations of each of these characteristics are envisioned. In an alternative embodiment, the plant of the present invention has a height that is 60%-90% of the height of a corresponding wild-type plant grown under the same conditions. In one embodiment, the plant or a portion thereof of the present invention preferably does not exhibit increased necrosis in its leaves; that is, if present, the degree of necrosis is the same as that exhibited by a corresponding wild-type plant or a portion thereof grown under the same conditions and at the same stage of plant development. This characteristic is particularly applicable to plants or portions thereof containing exogenous polynucleotides encoding fatty acid thioesterases such as FATB thioesterase.

[0184] The following embodiments apply to the plants or parts thereof of the present invention (including aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, sixteenth, eighteenth, and nineteenth), and to methods of producing said plants or parts thereof, or methods of using said plants or parts thereof. In one embodiment, the polypeptide involved in the biosynthesis of one or more nonpolar lipids is a fatty acyl acyltransferase involved in the biosynthesis of TAG, DAG, or monoacylglycerol (MAG) in said plants or parts thereof, preferably TAG biosynthesis in said plants or parts thereof, such as DGAT, PDAT, LPAAT, GPAT, or MGAT, preferably DGAT or PDAT.

[0185] In another embodiment, the polypeptide involved in the catabolism of triacylglycerol (TAG) in the plant or its parts is SDP1 lipase, Cgi58 polypeptide, acyl-CoA oxidase such as ACX1 or ACX2, or polypeptide involved in the β-oxidation of fatty acids in the plant such as PXA1 peroxisome ATP-binding cassette transporter, preferably SDP1 lipase.

[0186] In one embodiment, the oil body coating (OBC) polypeptide is an oil protein, such as polyolein or caleosin, or preferably lipid droplet-associated protein (LDAP).

[0187] In one embodiment, the polypeptide that increases fatty acid export from the plant plastid is a C16 or C18 fatty acid thioesterase such as FATA polypeptide or FATB polypeptide, a fatty acid transporter such as ABCA9 polypeptide, or a long-chain acyl-CoA synthase (LACS).

[0188] In one embodiment, the polypeptide involved in introducing fatty acids into the plastids of the plant is a fatty acid transporter or its subunit, preferably a TGD polypeptide, such as TGD1 polypeptide, TGD2 polypeptide, TGD3 polypeptide or TGD4 polypeptide.

[0189] In one embodiment, the polypeptide involved in the generation of diacylglycerol (DAG) in the plasmid is plasmid GPAT, plasmid LPAAT, or plasmid PAP.

[0190] In one embodiment, the plant or a portion thereof of the present invention is a 16:3 plant or a portion thereof, and it comprises one or more of the following: a) An exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the exogenous polynucleotide, increases fatty acid efflux from the plant's plastids. b) A first genetic modification that, compared to a corresponding plant lacking the first genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the input of fatty acids into the plastids of the plant. c) A second genetic modification that, compared to the corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. The exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts.

[0191] In an alternative embodiment, the plant or a portion thereof of the present invention is an 18:3 plant or a portion thereof.

[0192] In one embodiment, prior to flowering, the nutrient portion of the plant contains at least about 8%, at least about 10%, about 11%, 8%-15%, or 9%-12% (w / w dry weight) of total nonpolar lipids.

[0193] In one embodiment, one or more genetic modifications are endogenous gene mutations that partially or completely inactivate a gene, such as point mutations, insertions, or deletions (or combinations thereof), preferably introduced mutations. Point mutations can be early stop codons, splicing site mutations, frameshift mutations, or amino acid substitutions that reduce the activity of the gene or the encoded polypeptide. Deletions can be the deletion of one or more nucleotides within a transcriptional exon or promoter of a gene, or extensions across or into more than one exon, or deletions extending to the entire gene. Preferably, deletions are introduced using ZF, TALEN, or CRISPR technologies. In an alternative embodiment, one or more genetic modifications are exogenous polynucleotides encoding RNA molecules that repress the expression of endogenous genes, wherein the exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts.

[0194] In one embodiment, the exogenous polynucleotide encoding WRI1 comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 21-75 or 205-210, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 21-75 or 205-210. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridization to nucleotides i) and / or ii) under stringent conditions. Preferably, the WRI1 polypeptide is a WRI1 polypeptide other than Arabidopsis WRI1 (SEQ ID NO: 21 or 22). More preferably, the WRI1 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 208 or a bioactive fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical thereto.

[0195] In one embodiment, the total nonpolar lipid content, or one or more nonpolar lipids, and / or the level of oleic acid or PUFA in the plant or its parts can be determined by gas chromatography analysis using fatty acid methyl esters obtained from the plant or its nutrient parts.

[0196] In another embodiment, the plant part is a leaf, and the total nonpolar lipid content of the leaf can be determined by nuclear magnetic resonance (NMR) analysis.

[0197] In one embodiment, the plant or part thereof is one of the members of a group or collection of at least about 1,000 such plants or parts.

[0198] On the other hand, the present invention provides a population of at least about 1,000 plants, each of which is a plant of the present invention, growing in the field.

[0199] On the other hand, the present invention provides a collection of at least about 1,000 nutrient plant parts, each of which is a nutrient plant part of the present invention, wherein said nutrient plant part has been harvested from plants growing in the field.

[0200] In embodiments of the present invention, including cells, non-human organisms, plants, or parts thereof, the transcription factor polypeptide that increases the expression of one or more glycolysis and / or fatty acid biosynthesis genes in the cells is the WRI1 transcription factor; the polypeptide involved in the biosynthesis of one or more nonpolar lipids is DGAT such as DGAT1 or DGAT2, or PDAT; and the polypeptide involved in the catabolism of triacylglycerols (TAGs) in the cells is the SDP1 lipase. In a preferred embodiment, the oil body coating (OBC) polypeptide is an olein protein; the polypeptide that increases fatty acid export from the cytoplasm is a fatty acid thioesterase such as FATA or FATB thioesterase; the polypeptide involved in the importation of fatty acids into the cytoplasm is a TGD polypeptide, preferably the TGD1 polypeptide; and the polypeptide involved in the generation of diacylglycerols (DAGs) in the plastids is plastid GPAT. In a more preferred embodiment, the cells are in a vegetative plant portion, and the TAG content of the vegetative plant portion is at least 8% (% dry weight) before the plant flowers.

[0201] In one embodiment, the plant, vegetative plant part, non-human object or part thereof, seed, or potato tuber comprises a first exogenous polynucleotide encoding WRI1, a second exogenous polynucleotide encoding DGAT or PDAT (preferably DGAT1), a third exogenous polynucleotide encoding RNA (which reduces gene expression encoding the SDP1 polypeptide), and a fourth exogenous polynucleotide encoding an oleic protein. In a preferred embodiment, the vegetative plant part, non-human object or part thereof, seed, or potato tuber has one or more of the following characteristics: i) Total lipid content of at least 8%, at least 10%, at least 12%, at least 14%, or at least 15.5% (by weight). ii) The total lipid content in the corresponding nutrient plant part or non-human organism that lacks the exogenous polynucleotide is at least 3 times, at least 5 times, at least 7 times, at least 8 times, or at least 10 times higher. iii) Total TAG content of at least 5%, at least 6%, at least 6.5%, or at least 7% (by weight of dry weight or seed weight). iv) Total TAG content that is at least 40, 50, 60, 70, 100, or 120 times higher than that of the corresponding nutrient plant part or non-human organism lacking the said exogenous polynucleotide. v) Oleic acid contains at least 15%, at least 19%, or at least 22% (by weight of dry weight or seed weight) of fatty acids in TAG. vi) TAG contains at least 10, 15, or 17 times higher levels of oleic acid compared to the corresponding nutrient plant parts or non-human organisms lacking the aforementioned exogenous polynucleotide. vii) Palmitic acid contains at least 20%, at least 25%, at least 30%, or at least 33% (by weight) of fatty acids in TAG. viii) Compared to the corresponding nutrient plant parts or non-human organisms lacking the aforementioned exogenous polynucleotide, TAG contains at least 1.5 times higher levels of palmitic acid. ix) Linoleic acid contains at least 22%, at least 25%, at least 30%, or at least 34% (by weight) of fatty acids in TAG. x) Alpha-linolenic acid contains less than 20%, less than 15%, less than 11%, or less than 8% (by weight) of fatty acids in TAG. xi) Compared to the corresponding nutrient plant parts or non-human organisms lacking the aforementioned exogenous polynucleotides, TAG contains at least 5 to 8 times lower levels of α-linolenic acid, and xii) For potato tubers, the total fatty acid content shall be at least 0.5% on a dry weight basis and / or at least 1%, preferably at least 1.5% or at least 2.0% on a dry weight basis.

[0202] Seeds of the plant of the present invention or seeds obtained from the plant of the present invention are also provided.

[0203] In another aspect, the present invention provides a transgenic plant stem, or a portion of a stem weighing at least 1 g dry weight, having a TAG content of at least 5%, preferably at least 6%, and more preferably at least 7% on a dry weight basis. In one embodiment, the transgenic plant stem or stem portion is of a dicotyledonous plant, or preferably harvested from a dicotyledonous plant. Alternatively, the transgenic plant stem or stem portion is of a monocotyledonous plant, or preferably harvested from a monocotyledonous plant. In one embodiment, the plant stem or stem portion is of a plant other than sugarcane, or derived from a plant other than sugarcane. In embodiments, a further feature of the plant stem or stem portion is one or more characteristics as defined in the context of cells or plants according to the present invention.

[0204] In another aspect, the present invention provides plant cells comprising... a) The first exogenous polynucleotide encoding PDAT, b) A first genetic modification that, compared to corresponding cells lacking the first genetic modification, downregulates the endogenous production and / or activity of a polypeptide (preferably a TGD polypeptide) involved in the importation of fatty acids into the cytoplasm, and one or more of the following c) A second genetic modification that, compared to corresponding cells lacking the genetic modification, downregulates the endogenous production and / or activity of peptides (preferably SDP1 peptides) involved in the catabolism of triacylglycerols (TAGs) in the cells. d) A second exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking the second exogenous polynucleotide, increases fatty acid efflux from the cytoplasm, preferably a fatty acylthioesterase, and e) A third genetic modification that, compared to corresponding cells lacking the third genetic modification, downregulates the endogenous production and / or activity of peptides involved in the generation of diacylglycerol (DAG) in plastids. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the cell. In a preferred embodiment, the presence of a first, second, or third genetic modification or a second exogenous polynucleotide in the cell synergistically increases the total nonpolar lipid content of the cell compared to a corresponding cell having PDAT but lacking additional genetic modifications or exogenous polynucleotides. More preferably, at least one exogenous polynucleotide is expressed from a promoter other than a constitutive promoter.

[0205] In another aspect, the present invention provides a method for obtaining the recombinant eukaryotic cells of the present invention, the method comprising the following steps: i) Introducing at least one exogenous polynucleotide and / or at least one genetic modification as defined herein into eukaryotic cells to produce eukaryotic cells containing a set of exogenous polynucleotides and / or genetic modifications as defined herein. ii) Express the exogenous polynucleotide in the cell or its progeny cells. iii) Analyze the lipid content of the cells or daughter cells, and iv) Select the cells of the present invention.

[0206] In one embodiment, the one or more exogenous polynucleotides are stably integrated into the genome of the cell or daughter cells.

[0207] In one embodiment, the method further includes the step of regenerating a transgenic plant from a cell or progeny cell containing the one or more exogenous polynucleotides.

[0208] In another embodiment, the step of regenerating the transgenic plant is performed before the step of expressing the one or more exogenous polynucleotides in the cells or their progeny cells, and / or before the step of analyzing the lipid content of the cells or progeny cells, and / or before the step of selecting cells or progeny cells with elevated levels of one or more nonpolar lipids.

[0209] In another embodiment, the method further includes the step of obtaining seeds or progeny plants from the transgenic plant, wherein the seeds or progeny plants contain one or more of the exogenous polynucleotides.

[0210] In another embodiment, the selected cell or the plant regenerated therefrom, or the vegetative plant part or seed of the regenerated plant, has one or more characteristics as defined herein.

[0211] In another aspect, the present invention provides a method for producing plants having a set of exogenous polynucleotides and / or genetic modifications as defined herein integrated into their genome, the method comprising the following steps: i) A hybridization of two parent plants, wherein one plant contains at least one exogenous polynucleotide and / or at least one genetic modification as defined herein, and the other plant contains at least one exogenous polynucleotide and / or at least one genetic modification as defined herein, and wherein, between them, the two parent plants contain a set of exogenous polynucleotides and / or genetic modifications as defined herein. ii) Screening for groups from one or more progeny plants of the hybrid that have or do not contain exogenous polynucleotides and / or genetic modifications as defined herein, and iii) Select progeny plants containing groups of exogenous polynucleotides and / or genetic modifications as defined herein. This produces the plant.

[0212] Also provided is a transgenic cell or transgenic plant obtained using the method of the present invention, or a portion thereof, comprising a group of exogenous polynucleotides and / or genetic modifications as defined herein.

[0213] A set of exogenous polynucleotides and / or genetic modifications as defined herein are also provided for use in the preparation of transgenic cells, transgenic non-human organisms or portions thereof or seeds, which, relative to corresponding cells, non-human organisms or portions thereof or seeds lacking the set of exogenous polynucleotides and / or genetic modifications, have an enhanced ability to produce one or more nonpolar lipids, wherein each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the exogenous polynucleotide in the transgenic cells, transgenic non-human organisms or portions thereof or seeds.

[0214] Preferably, at least one promoter operatively linked to an exogenous polynucleotide encoding a transcription factor polypeptide is a promoter other than a constitutive promoter.

[0215] In one embodiment, the transgenic cell, non-human object or part thereof, or seed contains one or more of the characteristics defined herein.

[0216] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining the recombinant eukaryotic cells of the present invention, the transgenic non-human organism or part thereof of the present invention, the transgenic plant or part thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plants or part thereof of the present invention, and ii) Converting at least some lipids in said cells, non-human objects or their parts, plants or their parts, or seeds into industrial products by in-situ application of heat, chemicals, or enzymatic methods, or any combination thereof, to said non-human objects or their parts in situ, and iii) Recycle the industrial products. Thus, the industrial products are produced.

[0217] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining the recombinant eukaryotic cells of the present invention, the transgenic non-human organism or part thereof of the present invention, the transgenic plant or part thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plants or part thereof of the present invention, and ii) The cells, non-human objects or parts thereof, plants or parts thereof, or seeds of the physical processing step i) iii) Converting at least some of the lipids in processed cells, non-human objects or parts thereof, plants or parts thereof, or seeds into industrial products by applying heat, chemical or enzymatic methods, or any combination thereof, to lipids in processed cells, non-human objects or parts thereof, plants or parts thereof, or seeds simultaneously or subsequently, and iv) Recycle the industrial products. Thus, the industrial products are produced.

[0218] In one embodiment of the above two aspects, the plant part is a nutrient plant part.

[0219] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining a nutrient-rich plant portion having a total nonpolar lipid content of at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight). ii) Converting at least some lipids in the nutrient plant portion into an industrial product by in-situ application of heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the nutrient plant portion, and iii) Recycle the industrial products. Thus, the industrial products are produced.

[0220] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining a nutrient-rich plant portion having a total nonpolar lipid content of at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight). ii) The nutrient-rich plant parts of the physical processing step i), iii) Converting at least some of the lipids in a processed nutrient plant part into an industrial product by applying heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the processed nutrient plant part, and iv) Recycle the industrial products. Thus, the industrial products are produced.

[0221] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining a nutrient plant portion having a total nonpolar lipid content of at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein said plant is a 16:3 plant or its nutrient portion. ii) Converting at least some lipids in the nutrient plant portion into an industrial product by in-situ application of heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the nutrient plant portion, and iii) Recycle the industrial products. Thus, the industrial products are produced.

[0222] In another aspect, the present invention provides a method for producing industrial products, the method comprising the following steps: i) Obtaining a nutrient plant portion having a total nonpolar lipid content of at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein said plant is a 16:3 plant or its nutrient portion. ii) The nutrient-rich plant parts of the physical processing step i), iii) Converting at least some of the lipids in a processed nutrient plant part into an industrial product by applying heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the processed nutrient plant part, and iv) Recycle the industrial products. Thus, the industrial products are produced.

[0223] In one embodiment, the steps of physically processing the cells, non-human objects or parts thereof, plants or parts thereof, or seeds include crushing, pressing, breaking, or grinding the cells, non-human objects or parts thereof, plants or parts thereof, or seeds. In one embodiment, the method includes the following steps: (a) Extracting at least some of the nonpolar lipids from the cells, non-human organisms or parts thereof, plants or parts thereof, or seeds into a nonpolar lipid content, and (b) Recovery of extracted nonpolar lipids, Steps (a) and (b) are performed prior to the step of converting at least some lipids in the cells, non-human objects or parts thereof, plants or parts thereof, or seeds into an industrial product.

[0224] In one embodiment, the extracted nonpolar lipids comprise triglycerides, wherein the triglycerides comprise at least 90%, preferably at least 95%, of the extracted lipids.

[0225] In one embodiment, the industrial product is a hydrocarbon product such as fatty acid esters, preferably methyl fatty acid esters and / or ethyl fatty acid esters, alkane such as methane, ethane, or longer-chain alkane, mixtures of longer-chain alkane, alkene, biofuel, carbon monoxide and / or hydrogen, bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen, and biochar. In a preferred embodiment, the total fatty acid content of the nutrient plant portion comprises at least 5% C12:O, C14:O, or the sum of C12:O and C14:O is at least 5% of the total fatty acid content, and the industrial product derived from the lipids in the nutrient plant portion is a component of aviation fuel.

[0226] In another aspect, the present invention provides a method for producing extracted lipids, the method comprising the following steps: i) Obtaining the recombinant eukaryotic cells of the present invention, the transgenic non-human organism or a portion thereof of the present invention, the transgenic plant or a portion thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plants or a portion thereof of the present invention. ii) Extracting lipids from said cells, non-human objects or parts thereof, plants or parts thereof, or seeds, and iii) Recover the extracted lipids. This process produces extracted lipids.

[0227] In another aspect, the present invention provides a method for producing extracted lipids, the method comprising the following steps: i) Obtaining a nutrient-rich plant portion having a total nonpolar lipid content of at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight). ii) Extracting lipids from the said nutrient plant parts, and iii) Recover the extracted lipids. This process produces extracted lipids.

[0228] In another aspect, the present invention provides a method for producing extracted lipids, the method comprising the following steps: i) Obtaining a nutrient plant portion having a total nonpolar lipid content of at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight), wherein said plant is a 16:3 plant or its nutrient portion. ii) Extracting lipids from the said nutrient plant parts, and iii) Recover the extracted lipids. This process produces extracted lipids.

[0229] In one embodiment, the extraction method includes drying, crushing, pressing, breaking or grinding one or more of the cells, non-human objects or parts thereof, plants or parts thereof, or seeds, and / or purifying the extracted lipids or seed oils.

[0230] In one embodiment, the method uses an organic solvent in the extraction process to extract the oil.

[0231] In another embodiment, the method includes recovering extracted lipids or oils by collecting them in a container and / or degumming, deodorizing, decolorizing, drying, grading, separating one or more of the extracted lipids or oils, removing at least some waxes and / or wax esters from the extracted lipids or oils, or analyzing the fatty acid composition of the extracted lipids or oils.

[0232] In one embodiment, the volume of the extracted lipids or oil is at least 1 liter.

[0233] In another implementation, one or more of the following characteristics are applied: (i) The extracted lipids or oils contain triglycerides, wherein the triglycerides comprise at least 90%, preferably at least 95% or at least 96% of the extracted lipids or oils. (ii) The extracted lipids or oils contain free sterols, stearoyl esters, stearoyl glycosides, waxes or wax esters, or any combination thereof, and (iii) The total sterol content and / or composition of the extracted lipids or oils are significantly different from the sterol content and / or composition of extracted lipids or oils derived from the corresponding cells, non-human objects or parts thereof, plants or parts thereof, or seeds.

[0234] In one embodiment, the method further includes converting the extracted lipids or oils into industrial products.

[0235] In one embodiment, the industrial product is a hydrocarbon product such as fatty acid esters, preferably fatty acid methyl esters and / or fatty acid ethyl esters, alkane such as methane, ethane, or longer-chain alkane, mixtures of longer-chain alkane, alkene, biofuel, carbon monoxide and / or hydrogen, bio-alcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen, and biochar. In a preferred embodiment, the total fatty acid content of the nutrient plant portion comprises at least 5% C12:O, C14:O, or the sum of C12:O and C14:O is at least 5% of the total fatty acid content, and the industrial product derived from the lipids in the nutrient plant portion is a component of aviation fuel.

[0236] In another embodiment, the plant part is a surface plant part or a green plant part, preferably a nutrient-rich plant part such as plant leaves or stems. In an alternative embodiment, the plant part is a tuber or root, such as a potato ( Solanum tuberosum ) tubers or beet roots.

[0237] In another embodiment, the method further includes the step of harvesting the cells, non-human objects or parts thereof, plants or parts thereof such as tubers or roots or seeds, preferably using a mechanical harvester, or by means of filtration, centrifugation, sedimentation, flotation or flocculation involving algae or fungal organisms.

[0238] In another embodiment, the lipid level in the cells, non-human objects or parts thereof, plants or parts thereof, or seeds and / or extracted lipids or oils can be determined by gas chromatography analysis using fatty acid methyl esters prepared from the self-extracted lipids or oils.

[0239] In another embodiment, the method further includes harvesting the portion from the plant.

[0240] In one embodiment, the plant portion is a nutrient plant portion containing at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total nonpolar lipids.

[0241] In another embodiment, the plant portion is a nutrient plant portion containing at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 18%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total TAG content.

[0242] In another embodiment, the plant portion is a nutrient plant portion containing at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total nonpolar lipids, and wherein the nutrient plant portion is derived from a 16:3 plant.

[0243] In another embodiment, the plant portion is a nutrient plant portion containing at least about 11%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 8%-75%, 10%-75%, 11%-75%, about 15%-75%, about 20%-75%, about 30%-75%, about 40%-75%, about 50%-75%, about 60%-75%, or about 25%-50% (w / w dry weight) of total TAG content, and wherein the nutrient plant portion is derived from a 16:3 plant.

[0244] A method for producing seeds is also provided, the method comprising: i) To enable the plant of the present invention to grow, and ii) Harvest seeds from the plant.

[0245] In one embodiment, the method includes growing a population of at least about 1,000 plants, each of which is a plant of the present invention, and harvesting seeds from the plant population.

[0246] In another aspect, the present invention provides a fermentation method comprising the following steps: i) A container comprising a lipid composition comprising the recombinant eukaryotic cells of the present invention or the transgenic non-human organism of the present invention, wherein the cells or non-human organism is suitable for fermentation, and components required for fermentation and fatty acid biosynthesis, and ii) Provide conditions conducive to the fermentation of the lipid composition contained in the container.

[0247] It also provides lipids that can be obtained from recombinant eukaryotic cells of the present invention, transgenic non-human organisms or parts thereof of the present invention, transgenic plants or parts thereof of the present invention, seeds of the present invention, or transgenic cells or transgenic plants or parts thereof of the present invention, or lipids that can be recovered or extracted by the methods of the present invention.

[0248] In another aspect, the present invention provides industrial products produced by the method of the present invention, which are hydrocarbon products such as fatty acid esters, preferably fatty acid methyl esters and / or fatty acid ethyl esters, alkane such as methane, ethane or longer chain alkane, mixtures of longer chain alkane, alkenes, biofuels, carbon monoxide and / or hydrogen, bio-alcohols such as ethanol, propanol or butanol, biochar, or combinations of carbon monoxide, hydrogen and biochar.

[0249] It also provides the use of the recombinant eukaryotic cells of the present invention, the transgenic non-human organisms or portions thereof of the present invention, the transgenic plants or portions thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plants or portions thereof of the present invention, or the use of the recovered or extracted lipids of the present invention in the manufacture of industrial products.

[0250] Examples of industrial products of the present invention include, but are not limited to, hydrocarbon products such as fatty acid esters, preferably fatty acid methyl esters and / or fatty acid ethyl esters, alkanes such as methane, ethane or longer-chain alkanes, mixtures of longer-chain alkanes, alkenes, biofuels, carbon monoxide and / or hydrogen, bio-alcohols such as ethanol, propanol or butanol, biochar, or combinations of carbon monoxide, hydrogen and biochar.

[0251] In another aspect, the present invention provides a method for producing fuel, the method comprising: i) The lipids of the present invention are optionally reacted with an alcohol in the presence of a catalyst to produce an alkyl ester, and ii) Optionally, the alkyl ester is mixed with a petroleum-based fuel.

[0252] In one embodiment of the above method, the alkyl ester is a methyl ester.

[0253] In another aspect, the present invention provides a method for producing synthetic diesel fuel, the method comprising: i) Converting the recombinant eukaryotic cells of the present invention, the transgenic non-human organisms of the present invention or parts thereof, the transgenic plants of the present invention or parts thereof, the seeds of the present invention, or the transgenic cells or transgenic plants of the present invention or parts thereof into bio-oil by means including pyrolysis or hydrothermal treatment, or converting them into syngas by gasification, and ii) Converting the bio-oil into synthetic diesel fuel by a method including staged separation, preferably at about 150 o C - Approximately 200 o C or approximately 200 o C - Approximately 300 o Hydrocarbons condensed under C2C, or syngas converted into biofuels using metal catalysts or microbial catalysts.

[0254] In another aspect, the present invention provides a method for producing biofuels, the method comprising converting lipids in the recombinant eukaryotic cells of the present invention, the transgenic non-human organisms of the present invention or parts thereof, the transgenic plants of the present invention or parts thereof, the seeds of the present invention, or the transgenic cells or transgenic plants of the present invention or parts thereof into biooils by pyrolysis, converting them into bioethanols by fermentation, or converting them into biogas by gasification or anaerobic digestion.

[0255] In one embodiment of the above method, the portion is a nutrient plant part.

[0256] A method for producing feed is also provided, the method comprising mixing the recombinant eukaryotic cells of the present invention, the transgenic non-human organism or a portion thereof of the present invention, the transgenic plant or a portion thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plant or a portion thereof of the present invention, or extracts or portions thereof obtained by the method of the present invention, with at least one other food ingredient.

[0257] In another aspect, the present invention provides feed, cosmetics, or chemicals comprising the recombinant eukaryotic cells of the present invention, the transgenic non-human organism or a portion thereof of the present invention, the transgenic plant or a portion thereof of the present invention, the seeds of the present invention, or the transgenic cells or transgenic plants or a portion thereof of the present invention, or which may be obtained by the methods of the present invention, or extracts or portions thereof.

[0258] In another aspect, the present invention provides a method for feeding animals, the method comprising providing the animals with the transgenic plant or a portion thereof of the present invention, the seeds of the present invention, or the transgenic plant or a portion thereof of the present invention, or the recovered or extracted lipids of the present invention.

[0259] Unless otherwise specified, any implementation method described herein shall be applicable to any other implementation method.

[0260] The scope of this invention is not limited to the specific embodiments described herein, which are merely illustrative. Clearly, functionally equivalent products, compositions, and methods are within the scope of the invention as described herein.

[0261] In this specification, unless otherwise specified or required by the context, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall cover one or more (i.e., one or more) of those steps, compositions of substances, groups of steps, or groups of compositions of substances.

[0262] The present invention will now be described with reference to the following non-limiting embodiments and the accompanying drawings. Brief description of the attached diagram Figure 1 A diagram illustrating lipid synthesis in eukaryotic cells, showing some fatty acids synthesized in plastids being exported to the endoplasmic reticulum (ER) via the plastid-associated membrane (PLAM), and some fatty acids being imported from the ER into the plastids for eukaryotic galactopyroslipase synthesis. (Abbreviations:) Acetyl-CoA and malonyl-CoA: Acetyl-CoA and malonyl-CoA; ACCase: Acetyl-CoA carboxylase; FAS: Fatty acid synthase complex; 16:0-ACP, 18:0-ACP and 18:1-ACP: C16:0-acyl carrier protein (ACP), C18:0-acyl carrier protein, C18:1-acyl carrier protein; KAS II: Ketoacyl-ACP synthase II (EC 2.3.1.41); PLPAAT: plastid LPAAT; PGPAT: Plastid GPAT; PAP: PA phosphorylase (EC 3.1.3.4); G3P: Glyceryl-3-phosphate; LPA: Lysophosphatidylcholine; PA: Phosphatidic acid; DAG: diacylglycerol; TAG: Triglycerides; Acyl-CoA and acyl-PC: acyl-CoA and acyl-phosphatidylcholine; PC: Phosphatidylcholine; GPAT: Glycerol-3-phosphoacyltransferase; LPAAT: Lysophosphatidyl acyltransferase (EC 2.3.1.51); LPCAT: Acyl-CoA: Lysophosphatidylcholine acyltransferase; or synonym 1-acylglycerol phosphocholine O -Acyltransferase; Acyl-CoA: 1-Acyl- sn -glycerol-3-phosphate choline O - Acyltransferase (EC 2.3.1.23); CPT: CDP-choline: diacylglycerol choline phosphotransferase; or synonyms: 1-alkyl-2-acetylglycerol choline phosphotransferase; alkyl acylglycerol choline phosphotransferase; choline phosphotransferase; phosphorylcholine-glycerol ester transferase (EC2.7.8.2); PDCT: Phosphatidylcholine diglyceride phosphotransferase; PLC: Phospholipase C (EC 3.1.4.3); PLD: Phospholipase D; Choline phosphatase; Lecithinase D; Phospholipase II (EC 3.1.4.4); PDAT: Phospholipid: Diacylglycerol acyltransferase; or synonym phospholipid: 1,2-diacyl- sn - Glycerol O-acyltransferase (EC 2.3.1.158); FAD2: Fatty acid Δ12-desaturase; FAD3: Fatty acid Δ15-desaturase; UDP-Gal: uridine diphosphate galactose; MGDS: Monogalactosyldiglycerol synthase; MGDG: Monogalactosyldiglycerol; DGDG: Digalactosyldiglycerol FAD6, 7, 8: plassomatic fatty acid Δ12-desaturase, plassomatic ω3-desaturase, and plassomatic ω3-desaturase induced at low temperature, respectively.

[0263] Figure 2 Genetic diagram of a construct that increases seed oil content in dicotyledonous plants. Abbreviation: PRO Pissa (pea globulin), pea ( Pea Pea globulin promoter and 5' UTR; TMV leader region, 5' UTR of tobacco mosaic virus; Arath-DGAT1, protein-coding region of Arabidopsis thaliana DGAT1; TER Glyma-lectin, cultivated soybean ( G. max ) The 3' terminator / polyadenylation region of the lectin gene; PRO Phavu-bean protein, derived from bean ( Common bean The promoter of the bean protein gene; Arath-WRI1, encoding the protein-coding region of Arabidopsis thaliana WRI1; TER Agrtu-NOS, the promoter of Agrobacterium tumefaciens (… Agrobacterium tumefaciens The 3' terminator / polyadenylation region of the Nos gene; PRO Phavu-PHA, the promoter of the bean protein gene in common bean; Sesin-oil protein, encoding sesame ( Sesame seeds The protein coding region of the oleic protein gene; TER Phavu-PHA, the 3' terminator / polyadenylation region of the bean protein gene in common bean.

[0264] Figure 3 Schematic diagram of vector pOIL122. Abbreviations: TER Agrtu-Nos, Agrobacterium tumefaciens carmine synthase terminator; NPTII, neomycin phosphotransferase protein coding region; PRO CaMV35S-Ex2, cauliflower mosaic virus 35S promoter with dual enhancer regions; Arath-DGAT1, Arabidopsis thaliana DGAT1 acyltransferase protein coding region; PRO Arath-RubiscoSSU, Arabidopsis thaliana Rubisco small subunit promoter; Arath-FATA2, Arabidopsis thaliana FATA2 thioesterase protein coding region; Arath-WRI, Arabidopsis thaliana WRI1 transcription factor protein coding region; TER Glyma-lectin, soybean lectin terminator; enTCUP2 promoter, cryptic constitutive promoter of Nicotiana scabra; attB1 and attB2, Gateway recombination sites; NB SDP1 fragment, hpRNAi silencing targeted Nicotiana benthamiana SDP1 region; OCS terminator, Agrobacterium tumefaciens ( A. tumefaciensOctopus alkaloid synthase terminator. The backbone features outside the T-DNA region originate from pORE04 (Coutu). and others (., 2007).

[0265] Figure 4 The total fatty acid methyl ester (FAME) spectrum (weight %) illustrates the effect of WRI1+DGAT1-mediated high oil background on MCFA formation in *Nicotiana benthamiana* leaves. The highest MCFA formation was observed after the addition of Arath-WRI1.

[0266] Figure 5 Total FAME spectrum (weight %) (n=4) illustrating the effect of WRI1 on MCFA accumulation. Compared to the previous addition of Cocnu-LPAAT alone, the addition of Arath-WRI1 significantly increased the formation of related fatty acids (C12:0, C14:0, or C16:0).

[0267] Figure 6 The levels of TFA (weight %), TAG (total fatty acids %), and MCFA (C16:0 and C14:0, %) in TFA and MCFA (fatty acid content of TAG) in TAG were measured in plant cells after expression of three oil palm DGATs in combination with FATB, LPAAT, and WRI1. Numbers 1-10 are as listed in the text (Example 9).

[0268] Figure 7 TAG levels (% leaf dry weight) in tobacco leaf tissue were determined by infiltration of genes encoding different WRI1 polypeptides, with or without (right bar) DGAT1 co-expression (n=3). All samples were also infiltrated using the P19 construct.

[0269] Figure 8 A schematic diagram of the Tobacco Bengal SDP1 hairpin construct. The genetic segments shown are as described in Example 11. Abbreviations are as follows. Figure 3 The attB site represents the recombination site from the pHELLSGATE12 vector.

[0270] Figure 9 TAG content was measured in green leaf samples of tobacco plants transformed with T-DNA from pOIL51, lines #61 and #69, harvested before flowering. Control (parental) samples were obtained from plants transformed with T-DNA from pJP3502.

[0271] Figure 10 Wild-type (wt) and transgenic red tobacco containing only T-DNA from pJP3502 or additionally having T-DNA from pOIL051 ( N. tobaccoTAG levels (% dry weight) in the root and stem tissues of plants.

[0272] Figure 11 TAG levels (% dry weight) in root and stem tissues of wild-type (wt) and transgenic red tobacco plants containing only T-DNA from pJP3502 or additionally T-DNA from pOIL049.

[0273] Figure 12 TAG content in leaf samples of transformed tobacco plants in the fruiting stage, transformed with T-DNA from pOIL049, lines #23c and #32b. Control (parental) samples were from plants transformed with T-DNA from pJP3502. The upper line indicates 18:2 percentage in TAG, while the lower line indicates 18:3 percentage in fatty acid content (ALA).

[0274] Figure 13 A. Starch content in leaf tissue of wild-type plants (WT) and transgenic plants containing T-DNA from pJP3502 (HO control), or from pJP3502 and pOIL051 (pOIL51.61 and pOIL51.69), or pJP3502 and pOIL049 (pOIL49.32b). Data represent pooled results from at least three individual plants. B. Correlation between starch and TAG content in leaf tissue of wild-type plants (WT) and transgenic plants containing T-DNA from pJP3502 (HO control), or from pJP3502 and pOIL051 (pOIL51.61 and pOIL51.69), or pJP3502 and pOIL049 (pOIL49.32b). Data represent pooled results from at least three individual plants.

[0275] Figure 14 Schematic diagram of the .pTV55 binary vector. Abbreviations: PRO, promoter; TER, 3' terminator / polyadenylation region; Arath, Arabidopsis thaliana; Linus, flax; Nicta, red tobacco; Glyma, cultivated soybean; Cnl1, conlinin 1 from flax; Cnl2, conlinin 2 from flax; MAR Nicat-RB7, matrix-binding region of RB7 from tobacco, or as... Figure 3 Gene abbreviations MGAT2, DGAT1, GPAT4, and WRI1 are as described in the text.

[0276] Figure 15 For example, flaxseed transformed with pTV55, pTV56, and pTV57, as determined by NMR ( C. sativaOil content (%) of T2 seeds. Each data point represents the average oil content of 50 mg seeds from three independent batches for each transgenic line. Negative control seeds were wild-type (untransformed) flaxseeds grown under identical conditions in a greenhouse. N represents the number of independent transgenic events for each construct.

[0277] Figure 16 .LDAP peptide phylogenetic tree (Example 15).

[0278] Figure 17 A schematic diagram of the genetic construct pJP3506, which includes the T-DNA region between its left and right boundaries. (Abbreviations are as follows) Figure 3 And: Sesin-oil protein, the protein coding region of sesame oil protein.

[0279] Figure 18 Changes in yield and calorific value of bio-oil production via HTP using wild-type and genetically modified high-oil tobacco nutrient plant materials as raw materials.

[0280] sequence SEQ ID NO:1 Arabidopsis thaliana DGAT1 polypeptide (CAB44774.1) SEQ ID NO:2 Arabidopsis thaliana DGAT2 peptide (NP_566952.1) SEQ ID NO:3 Castor bean DGAT2 polypeptide (AAY16324.1) SEQ ID NO:4 Tung oil ( Fordyce varnish DGAT2 polypeptide (ABC94474.1) SEQ ID NO:5 Raman spores ( Mortierella ramanniana DGAT2 peptide (AAK84179.1) SEQ ID NO:6 Homo sapiens ( Homo sapiens DGAT2 peptide (Q96PD7.2) SEQ ID NO:7 Homo sapiens DGAT2 peptide (Q58HT5.1) SEQ ID NO:8 Domestic cattle ( Bull DGAT2 peptide (Q70VZ8.1) SEQ ID NO:9 House mouse ( Mus musculus DGAT2 peptide (AAK84175.1) SEQ ID NO:10 YFP tripeptide – conserved DGAT2 and / or MGAT1 / 2 sequence motif SEQ ID NO:11 HPHG tetrapeptide – conserved DGAT2 and / or MGAT1 / 2 sequence motif SEQ ID NO:12 EPHS tetrapeptide – Conserved plant DGAT2 sequence motif SEQ ID NO:13 RXGFX(K / R)XAXXXGXXX(L / V)VPXXXFG(E / Q) – DGAT2 long conserved sequence motif, which is part of the putative glycerophospholipid domain. SEQ ID NO:14 FLXLXXXN – Conserved sequence motif of mouse DGAT2 and MGAT1 / 2, which is the putative neutral lipid-binding domain. SEQ ID NO:15 GPAT's plsC acyltransferase domain (PF01553) SEQ ID NO:16 HAD-like hydrolase (PF12710) superfamily domain of GPAT SEQ ID NO:17 Phosphoserine phosphatase domain (PF00702). GPAT4-8 contains an N-terminal region homologous to this domain. SEQ ID NO:18 Conserved GPAT amino acid sequence GDLVICPEGTTCREP SEQ ID NO:19 Conserved GPAT / phosphatase amino acid sequence (motif I) SEQ ID NO:20 Conserved GPAT / phosphatase amino acid sequence (motif III) SEQ ID NO:21 Arabidopsis thaliana WRI1 peptide (A8MS57) SEQ ID NO:22 Arabidopsis thaliana WRI1 polypeptide (Q6X5Y6) SEQ ID NO:23 Arabidopsis thaliana 'Fiddle Leaf' subspecies ( Arabidopsis lyrata subsp lyre WRI1 peptide (XP_002876251.1) SEQ ID NO:24 Rapeseed WRI1 peptide (ABD16282.1) SEQ ID NO:25 Rapeseed WRI1 peptide (ADO16346.1) SEQ ID NO:26 Soybean WRI1 peptide (XP_003530370.1) SEQ ID NO:27 Jatropha curcas WRI1 polypeptide (AEO22131.1) SEQ ID NO:28 Castor WRI1 polypeptide (XP_002525305.1) SEQ ID NO:29 Populus tomentosa WRI1 polypeptide (XP_002316459.1) SEQ ID NO:30 Grapes ( Wine grapevine WRI1 peptide (CBI29147.3) SEQ ID NO:31 Two-spike short-stalked grass ( Brachypodium distachyon WRI1 peptide (XP_003578997.1) SEQ ID NO:32 Barley ( Barley subsp Commonly WRI1 polypeptide (BAJ86627.1) SEQ ID NO:33 Rice WRI1 polypeptide (EAY79792.1) SEQ ID NO:34 Two-color sorghum WRI1 polypeptide (XP_002450194.1) SEQ ID NO:35 Maize WRI1 peptide (ACG32367.1) SEQ ID NO:36 *Brachys bipinnatus* WRI1 polypeptide (XP_003561189.1) SEQ ID NO:37 *Short-stalked Grass* ( Brachypodium sylvaticum WRI1 peptide (ABL85061.1) SEQ ID NO:38 Rice WRI1 polypeptide (BAD68417.1) SEQ ID NO:39 Two-color sorghum WRI1 polypeptide (XP_002437819.1) SEQ ID NO:40 Two-color sorghum WRI1 polypeptide (XP_002441444.1) SEQ ID NO:41 Soybean WRI1 peptide (XP_003530686.1) SEQ ID NO:42 Soybean WRI1 peptide (XP_003553203.1) SEQ ID NO:43 Populus pilosa WRI1 polypeptide (XP_002315794.1) SEQ ID NO:44 Grape WRI1 polypeptide (XP_002270149.1) SEQ ID NO:45 Soybean WRI1 peptide (XP_003533548.1) SEQ ID NO:46 Soybean WRI1 peptide (XP_003551723.1) SEQ ID NO:47 Tribulus terrestris ( Medicago truncatula WRI1 peptide (XP_003621117.1) SEQ ID NO:48 Populus tomentosa WRI1 polypeptide (XP_002323836.1) SEQ ID NO:49 Castor WRI1 polypeptide (XP_002517474.1) SEQ ID NO:50 Grape WRI1 polypeptide (CAN79925.1) SEQ ID NO:51 *Brachys bipinnatus* WRI1 polypeptide (XP_003572236.1) SEQ ID NO:52 Rice WRI1 polypeptide (BAD10030.1) SEQ ID NO:53 Two-color sorghum WRI1 polypeptide (XP_002444429.1) SEQ ID NO:54 Maize WRI1 polypeptide (NP_001170359.1) SEQ ID NO:55 WRI1 polypeptide of Arabidopsis thaliana subspecies *Fiddleleaf* (XP_002889265.1) SEQ ID NO:56 Arabidopsis thaliana WRI1 peptide (AAF68121.1) SEQ ID NO:57 Arabidopsis thaliana WRI1 peptide (NP_178088.2) SEQ ID NO:58 WRI1 polypeptide of Arabidopsis thaliana subspecies *Fiddleleaf* (XP_002890145.1) SEQ ID NO:59 Salt mustard ( Thellungiella halophila WRI1 polypeptide (BAJ33872.1) SEQ ID NO:60 Arabidopsis thaliana WRI1 polypeptide (NP_563990.1) SEQ ID NO:61 Soybean WRI1 peptide (XP_003530350.1) SEQ ID NO:62 *Brachys bipinnatus* WRI1 polypeptide (XP_003578142.1) SEQ ID NO:63 Rice WRI1 polypeptide (EAZ09147.1) SEQ ID NO:64 Two-color sorghum WRI1 polypeptide (XP_002460236.1) SEQ ID NO:65 Maize WRI1 peptide (NP_001146338.1) SEQ ID NO:66 Soybean WRI1 peptide (XP_003519167.1) SEQ ID NO:67 Soybean WRI1 peptide (XP_003550676.1) SEQ ID NO:68 Tribulus terrestris WRI1 peptide (XP_003610261.1) SEQ ID NO:69 Soybean WRI1 peptide (XP_003524030.1) SEQ ID NO:70 Soybean WRI1 peptide (XP_003525949.1) SEQ ID NO:71 Populus pilosa WRI1 polypeptide (XP_002325111.1) SEQ ID NO:72 Grape WRI1 peptide (CBI36586.3) SEQ ID NO:73 Grape WRI1 polypeptide (XP_002273046.2) SEQ ID NO:74 Populus tomentosa WRI1 polypeptide (XP_002303866.1) SEQ ID NO:75 Grape WRI1 peptide (CBI25261.3) SEQ ID NO:76 Sorbi-WRL1 SEQ ID NO: 77 Lupan-WRL1 SEQ ID NO:78 Ricco-WRL1 SEQ ID NO:79 Narrow-leaved lupin ( Lupin angustifolius WRI1 peptide SEQ ID NO:80 Aspergillus fumigatus ( Aspergillus fumigatus DGAT1 peptide (XP_755172.1) SEQ ID NO:81 Castor bean DGAT1 polypeptide (AAR11479.1) SEQ ID NO:82 Tung oil DGAT1 polypeptide (ABC94472.1) SEQ ID NO:83 *Chrysanthemum indicum* ( Vernonia galamensis DGAT1 peptide (ABV21945.1) SEQ ID NO:84 DGAT1 polypeptide of *Cymbidium goeringii* (ABV21946.1) SEQ ID NO:85 Euonymus japonicus ( Euonymus alatus DGAT1 peptide (AAV31083.1) SEQ ID NO:86 Caenorhabditis elegans (C. elegans) Caenorhabditis elegans DGAT1 peptide (AAF82410.1) SEQ ID NO:87 Brown rat ( Norway rat DGAT1 peptide (NP_445889.1) SEQ ID NO:88 Homo sapiens DGAT1 peptide (NP_036211.2) SEQ ID NO:89 WRI1 motif (RGVT / SRHRWTGR) SEQ ID NO:90 WRI1 motif (F / YEAHLWDK) SEQ ID NO:91 WRI1 motif (DLAALKYWG) SEQ ID NO:92 WRI1 motif (SXGFS / ARGX) SEQ ID NO:93 WRI1 motif (HHH / QNGR / KWEARIGR / KV) SEQ ID NO:94 WRI1 motif (QEEAAAXYD) SEQ ID NO:95 Rapeseed oil protein polypeptide (CAA57545.1) SEQ ID NO:96 Rapeseed oil protein S1-1 polypeptide (ACG69504.1) SEQ ID NO:97 Rapeseed oil protein S2-1 polypeptide (ACG69503.1) SEQ ID NO:98 Rapeseed oil protein S3-1 polypeptide (ACG69513.1) SEQ ID NO:99 Rapeseed oil protein S4-1 polypeptide (ACG69507.1) SEQ ID NO:100 Rapeseed oil protein S5-1 polypeptide (ACG69511.1) SEQ ID NO:101 Peanuts Peanut Olein protein 1 polypeptide (AAZ20276.1) SEQ ID NO:102 Peanut oil protein 2 polypeptide (AAU21500.1) SEQ ID NO:103 Peanut oil protein 3 polypeptide (AAU21501.1) SEQ ID NO:104 Peanut oil protein 5 polypeptide (ABC96763.1) SEQ ID NO:105 Castor oil protein 1 polypeptide (EEF40948.1) SEQ ID NO:106 Castor oil protein 2 polypeptide (EEF51616.1) SEQ ID NO:107 Soybean oil protein isotype α polypeptide (P29530.2) SEQ ID NO:108 Soybean oil protein isotype b polypeptide (P29531.1) SEQ ID NO:109 Linoleic acid protein low molecular weight isotype polypeptide (ABB01622.1) SEQ ID NO:110 Amino acid sequence of high molecular weight isotype polypeptide of linoleic acid protein (ABB01624.1) SEQ ID NO:111 Sunflower oil protein polypeptide (CAA44224.1) SEQ ID NO:112 Corn oil protein polypeptide (NP_001105338.1) SEQ ID NO:113 Rapeseed oil steroid protein polypeptide (ABM30178.1) SEQ ID NO:114 Rapeseed oil steroid protein SLO1-1 polypeptide (ACG69522.1) SEQ ID NO:115 Rapeseed oil steroid protein SLO2-1 polypeptide (ACG69525.1) SEQ ID NO:116 Sesame oil sterol protein polypeptide (AAL13315.1) SEQ ID NO:117 Corn oil body sterol protein polypeptide (NP_001152614.1) SEQ ID NO:118 CLO-1 polypeptide (ACG69529.1) of rapeseed oil body calciprotein SEQ ID NO:119 Rapeseed oil body calciprotein CLO-3 polypeptide (ACG69527.1) SEQ ID NO:120 Sesame oil calcitonin polypeptide (AAF13743.1) SEQ ID NO:121 Corn oil body calciprotein polypeptide (NP_001151906.1) SEQ ID NO:122 pJP3502 TDNA (inserted into the genome) sequence SEQ ID NO:123 pJP3507 vector sequence SEQ ID NO:124 Connector sequence SEQ ID NO:125 Selected partial Nicotiana benthamiana CGI-58 sequence (pTV46) for hpRNAi silencing. SEQ ID NO:126 Selected partial safflower AGPase sequence (pTV35) for hpRNAi silencing. SEQ ID NO:127 GXSXG lipase motif SEQ ID NO:128 HX(4)D Acyltransferase Motif SEQ ID NO:129 VX(3) HGF possible lipid binding motif SEQ ID NO:130 Arabidopsis thaliana CGi58 polynucleotide (NM_118548.1) SEQ ID NO:131 *Brachys bipinnatus* CGi58 polynucleotide (XM_003578402.1) SEQ ID NO:132 Soybean CGi58 polynucleotide (XM_003523590.1) SEQ ID NO:133 Maize CGi58 polynucleotide (NM_001155541.1) SEQ ID NO:134 Bicolor sorghum CGi58 polynucleotide (XM_002460493.1) SEQ ID NO:135 Castor bean CGi58 polynucleotide (XM_002510439.1) SEQ ID NO:136 Tribulus terrestris CGi58 polynucleotide (XM_003603685.1) SEQ ID NO:137 Arabidopsis thaliana LEC2 polynucleotide (NM_102595.2) SEQ ID NO:138 Alfalfa Tribulus terrestris LEC2 Polynucleotide ( X60387.1 ) SEQ ID NO:139 European rapeseed LEC2 polynucleotide ( HM370539.1 ) SEQ ID NO:140 Arabidopsis thaliana BBM polynucleotide (NM_121749.2) SEQ ID NO:141 Tribulus terrestris BBM polynucleotide ( AY899909.1 ) SEQ ID NO:142 Arabidopsis thaliana LEC2 polypeptide (NP_564304.1) SEQ ID NO:143 Alfalfa Tribulus terrestris LEC2 peptide (CAA42938.1) SEQ ID NO:144 Rapeseed LEC2 peptide (ADO16343.1) SEQ ID NO:145 Arabidopsis thaliana BBM peptide (NP_197245.2) SEQ ID NO:146 Tribulus terrestris alfalfa BBM peptide (AAW82334.1) SEQ ID NO:147 Inducible Aspergillus niger ( Black Aspergillus alcA promoter SEQ ID NO:148 AlcR inducer for activating the AlcA promoter in the presence of ethanol SEQ ID NO:149 Arabidopsis thaliana LEC1; (AAC39488) SEQ ID NO:150 Arabidopsis thaliana (Fiddleleaf Arabidopsis) Arabidopsis lyrata LEC1 (XP_002862657) SEQ ID NO:151 European rapeseed LEC1 (ADF81045) SEQ ID NO:152 Castor bean LEC1 (XP_002522740) SEQ ID NO:153 Soybean LEC1 (XP_006582823) SEQ ID NO:154 Alfalfa Tribulus terrestris LEC1 (AFK49653) SEQ ID NO:155 Maize LEC1 (AAK95562) SEQ ID NO:156 Peanut LEC1 (ADC33213) SEQ ID NO:157 Arabidopsis thaliana LEC1 sample (AAN15924) SEQ ID NO:158 European rapeseed LEC1 sample (AHI94922) SEQ ID NO:159 Pork Bean ( Phaseolus coccineus LEC1 sample (AAN01148) SEQ ID NO:160 Arabidopsis thaliana FUS3 (AAC35247) SEQ ID NO:161 European rapeseed FUS3 SEQ ID NO:162 Tribulus terrestris FUS3 SEQ ID NO:163 Arabidopsis thaliana SDP1 cDNA sequence, accession number NM_120486, 3275nt SEQ ID NO:164 Rapeseed SDP1 cDNA; Accession number GN078290 SEQ ID NO:165 *Bruguiera gymnorhiza* SDP1 cDNA, 2670 nt SEQ ID NO:166 Populus hairgrass SDP1 cDNA, 3884nt SEQ ID NO:167 Alfalfa SDP1 cDNA; XM_003591377; 2490nt SEQ ID NO:168 Soybean SDP1 cDNA XM_003521103; 2783nt SEQ ID NO:169 Dichromatic sorghum SDP1 cDNA XM_002458486; 2724nt SEQ ID NO:170 Maize SDP1 cDNA, NM_001175206; 2985nt SEQ ID NO:171 Small Bowl Moss ( Physcomitrella patens ) SDP1 cDNA, XM_001758117; 1998nt SEQ ID NO:172 Barley SDP1 cDNA, AK372092; 3439nt SEQ ID NO:173 Nicotiana benthamiana SDP1 cDNA, Nbv5tr6404201 SEQ ID NO:174 hpRNAi silencing targeting of the SDP1 cDNA region in Nicotiana benthamiana. SEQ ID NO:175 Arabidopsis thaliana SDP1 Gene promoter, 1.5kb The nucleotide sequence of the pSSU-Oleosin gene complement in the T-DNA of SEQ ID NO:176 pJP3502. The sequence (complementary sequence) is as follows: soybean lectin terminator 348 nt, 3' exon 255 nt, UBQ10 intron 304 nt, 5' exon 213 nt, SSU promoter 1751 nt. SEQ ID NO:177 Arabidopsis plastid GPAT cDNA, NM_179407 SEQ ID NO:178 Arabidopsis plastid GPAT polypeptide, NM_179407 SEQ ID NO:179 Populus hairy fruit plasmid GPAT cDNA, XP_006368351 SEQ ID NO:180 Jatropha curcas GPAT cDNA, ACR61638 SEQ ID NO:181 Ricinus plastid GPAT cDNA, XP_002518993 SEQ ID NO:182 Sunflower plastid GPAT cDNA, ADV16382 SEQ ID NO:183 GPAT cDNA of *Alfalfa tribulus* plasmid, XP_003612801 SEQ ID NO:184 soybean plastid GPAT cDNA, XP_003516958 SEQ ID NO:185 Safflower plasmid GPAT cDNA, CAHG3PACTR SEQ ID NO:186 Potato plastid GPAT cDNA, XP_006352898 SEQ ID NO:187 Rice, Japonica rice plasmid GPAT cDNA, NM_001072027 SEQ ID NO:188 Bicolor sorghum plastid GPAT cDNA, XM_002467381 SEQ ID NO:189 Maize plastid GPAT cDNA, NM_001158637 SEQ ID NO:190 Barley plastid GPAT cDNA, AK371419 SEQ ID NO:191 GPAT cDNA of *Bryophyta pulmonata*, XM_001771247 SEQ ID NO:192 Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii )Plastosome GPAT cDNA, XM_001694925 SEQ ID NO:193 Camphor tree ( Cinnamomum camphora 14:0-ACP thioesterase (Cinca-TE), chloroplast, 382aa, (accession number Q39473.1) SEQ ID NO:194 Coconut acyl-ACP thioesterase FatB1 (Cocnu-TE1; 417aa, accession number AEM72519.1) SEQ ID NO:195 Coconut acyl-ACP thioesterase FatB2 (Cocnu-TE2; 423aa, accession number AEM72520.1) SEQ ID NO:196 Coconut acyl-ACP thioesterase FatB3 (Cocnu-TE3; 414aa, accession number AEM72521.1) SEQ ID NO:197 Lanceolate-leaved calyx spur ( Cuphea lanceolata Acyl-(ACP)thioesterase type B (Cupla-TE, 419aa, accession number CAB60830.1) SEQ ID NO:198 Adhesive-hairy calyx spur ( Cuphea viscosissima FatB1 (Cupvi-TE; 419aa, Registry ID AEM72522.1) SEQ ID NO:199 California Laurel ( Umbellularia californica 12:0-ACP thioesterase (lauroyl-acyl carrier protein thioesterase) (Umbca-TE, 382aa; accession number Q41635.1) SEQ ID NO:200 Coconut LPAAT (Cocnu-LPAAT, 308aa, Registry No. Q42670.1) SEQ ID NO:201 Arabidopsis plastid LPAAT1 (Arath-PLPAAT; 356aa, accession number AEE85783.1) SEQ ID NO:202 Arabidopsis thaliana FATA1 SEQ ID NO:203 Arabidopsis thaliana FATA2 SEQ ID NO:204 Arabidopsis thaliana FATB SEQ ID NO:205 Arabidopsis thaliana WRI3 SEQ ID NO:206 Arabidopsis thaliana WRI4 SEQ ID NO:207 Oat WRI1 SEQ ID NO:208 Two-color sorghum WRI1 SEQ ID NO:209 Maize WRI1 SEQ ID NO:210 Ulva ( Triadica sebifera ) WRI1 SEQ ID NO:211 Potato ( S. tuberosum Patatin B33 promoter sequence Oligonucleotide primers of SEQ ID NOs 212-215 and 245-254 SEQ ID NO:216 Corn ( Z. mays SEE1 boot sub-region (from login number AJ494982, 1970nt) SEQ ID NO:217 *Hypericum perforatum* ( A. littoralis AlSAP startup subsequence, accession number DQ885219 SEQ ID NO:218 Agrobacterium rhizogenes ( A. rhizogenes ArRolC boot sequence, login number DQ160187 SEQ ID NO:219 An hpRNAi construct containing a 732bp fragment of the GPAT fragment of the tobacco plasmid *Nicotiana benthamiana*. SEQ ID NO:220 Oil palm ( Elaeis guineensis Oil palm (DGAT1) SEQ ID NO:221 Cultivated soybean MYB73, Registry No. ABH02868 SEQ ID NO:222 Arabidopsis thaliana bZIP53, accession number AAM14360 SEQ ID NO:223 Arabidopsis thaliana AGL15, accession number NP_196883 SEQ ID NO:224 Arabidopsis thaliana MYB118, accession number AAS58517 SEQ ID NO:225 Arabidopsis thaliana MYB115, accession number AAS10103 SEQ ID NO:226 Arabidopsis thaliana TANMEI, accession number BAE44475 SEQ ID NO:227 Arabidopsis thaliana WUS, accession number NP_565429 SEQ ID NO:228 European rapeseed ( B. napus GFR2a1, accession number AFB74090 SEQ ID NO:229 European rapeseed GFR2a2, accession number AFB74089 SEQ ID NO:230 Arabidopsis thaliana PHR1, accession number AAN72198 SEQ ID NO:231 Tobacco Benzovia TGD1 fragment SEQ ID NO:232 Potato SDP1 amino acids SEQ ID NO:233 Potato SDP1 nucleotide sequence SEQ ID NO:234 ​​Potato AGPase small subunit SEQ ID NO:235 Potato AGPase small subunit nucleotide sequence SEQ ID NO:236 Chinese tallow tree ( Sapium sebiferum LDAP-1 nucleotide sequence SEQ ID NO:237 LDAP-1 amino acid sequence of *Sapium seminaria* SEQ ID NO:238 DL-2 nucleotide sequence of *Sapium sebiferum* SEQ ID NO:239 LDAP-2 amino acid sequence of *Sapium seminaria* SEQ ID NO:240 LDAP-3 nucleotide sequence of *Sapium sebiferum* SEQ ID NO:241 DL-3 amino acid sequence of *Sapium seminaria* SEQ ID NO:242 Two-color sorghum ( S. bicolor SDP1 (Login ID XM_002463620) SEQ ID NO:243 Wheat ( T. aestivum SDP1 nucleotide sequence (accession number AK334547) SEQ ID NO:244 Two-color sorghum SDP1 hpRNAi fragment Invention Details General Technology Unless otherwise expressly defined, all technical terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, plant biology, cell biology, protein chemistry, lipid and fatty acid chemistry, biofuel production and biochemistry).

[0281] Unless otherwise specified, the recombinant proteins, cell culture and immunological techniques used in this invention are standard procedures well known to those skilled in the art. Such techniques are described and explained in the following literature resources: J. Perbal, Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); DM Glover and BD Hames (ed.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); FM Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory (1988); and JE Coligan et al. (ed.). Immunology, John Wiley & Sons (including all updates to date).

[0282] Selected definition The term "transgenic non-human organism" refers to, for example, a whole plant, algae, non-human animal, or organism suitable for fermentation such as yeast or fungi that contains one or more exogenous polynucleotides (transgenics) or polypeptides. In one embodiment, the transgenic non-human organism is not an animal or a part thereof. In one embodiment, the transgenic non-human organism is a phototrophic organism (e.g., a plant or algae) capable of obtaining energy from sunlight to synthesize organic compounds as nutrients.

[0283] In the context of polynucleotides or peptides, the term "exogenous" refers to a polynucleotide or peptide that is present in cells that do not naturally contain said polynucleotide or peptide. Such cells are referred to herein as "recombinant cells" or "transgenic cells." In one embodiment, the exogenous polynucleotide or peptide originates from a different genus than the cell containing said exogenous polynucleotide or peptide. In another embodiment, the exogenous polynucleotide or peptide originates from a different species. In one embodiment, the exogenous polynucleotide or peptide is expressed in a host plant or plant cell, and the exogenous polynucleotide or peptide originates from a different species or genus. The exogenous polynucleotide or peptide may be non-naturally occurring, for example, a synthetic DNA molecule produced by a recombinant DNA method. The DNA molecule may (generally preferably) include a protein-coding region that has been codon-optimized for expression in the cell, thereby producing a peptide having the same amino acid sequence as a naturally occurring peptide, although the nucleotide sequence of said protein-coding region is not naturally occurring. The exogenous polynucleotide may encode, or the exogenous polypeptide may be: diacylglycerol acyltransferase (DGAT) such as DGAT1 or DGAT2, Wrinkled 1 (WRI1) transcription factor, OBC such as olein or preferably LDAP, fatty acid thioesterase such as FATA or FATB polypeptide, or silencing repressor polypeptide.

[0284] As used herein, the term "extracted lipids" refers to a composition extracted from a genetically modified organism or a portion thereof containing at least 60% (w / w) lipids.

[0285] As used herein, the term "nonpolar lipid" refers to fatty acids and their derivatives that are soluble in organic solvents but insoluble in water. Fatty acids can be free fatty acids and / or in esterified form. Examples of esterified forms include, but are not limited to, triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG). Nonpolar lipids also include sterols, sterol esters, and wax esters. Nonpolar lipids are also referred to as "neutral lipids." Nonpolar lipids are typically liquid at room temperature. Preferably, nonpolar lipids primarily (>50%) comprise fatty acids with a length of at least 16 carbons. More preferably, at least 50% of the total fatty acids in nonpolar lipids are C18 fatty acids, such as oleic acid. Preferably, at least 5% of the total fatty acids in nonpolar lipids are C12 or C14 fatty acids, or both. In one embodiment, at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% of the fatty acids in the nonpolar lipids of the present invention may be present as TAGs. The nonpolar lipids may be further purified or processed, for example, by hydrolysis with a strong alkali to release free fatty acids or by fractionation, distillation, or other methods. The nonpolar lipids may be present in or derived from plant parts such as seeds, leaves, tubers, roots, or fruits, from recombinant cells, or from non-human organisms such as yeast. If the nonpolar lipids of the present invention are derived from seeds, they may form part of a "seed oil".

[0286] The concentrations of free and esterified sterols (e.g., sitosterol, campesterol, stigmasterol, brassinosteroids, D5-avenasterol, sitostanol, campestanol, and cholesterol) in the extracted lipids are as described in Phillips et al., 2002. Sterols in vegetable oils are present as: free alcohols, esters with fatty acids (esterified sterols), sterol glycosides, and acylated glycosides. The concentration of sterols in naturally occurring vegetable oils (seed oils) ranges from a maximum of approximately 1100 mg / 100g. Hydrogenated palm oil has one of the lowest concentrations of naturally occurring vegetable oils, approximately 60 mg / 100g. The recovered or extracted seed oils of this invention preferably have a total sterol content of about 100 to about 1000 mg / 100g of oil. For use as food or feed, it is preferred that the sterols are present primarily in free or esterified forms rather than glycosylated forms. In the seed oils of the present invention, preferably at least 50% of the sterols are present as esterified sterols, except for soybean seed oil (where about 25% of the sterols are esterified). The rapeseed oil and rapeseed oil of the present invention preferably have about 500 to about 800 mg of total sterols / 100g, wherein sitosterol is the major sterol and rapeseed sterol is the minor sterol. The corn seed oil of the present invention preferably has about 600 to about 800 mg of total sterols / 100g, wherein sitosterol is the major sterol. The soybean seed oil of the present invention preferably has about 150 to about 350 mg of total sterols / 100g, wherein sitosterol is the major sterol and stigmasterol is the minor sterol, and has more free sterols than esterified sterols. The cottonseed oil of the present invention preferably has about 200 to about 350 mg of total sterols / 100g, wherein sitosterol is the major sterol. The coconut oil and palm oil of the present invention preferably have about 50 to about 100 mg of total sterols per 100g, wherein phytosterol is the major sterol. The safflower seed oil of the present invention preferably has about 150 to about 250 mg of total sterols per 100g, wherein phytosterol is the major sterol. The peanut seed oil of the present invention preferably has about 100 to about 200 mg of total sterols per 100g, wherein phytosterol is the major sterol. The sesame seed oil of the present invention preferably has about 400 to about 600 mg of total sterols per 100g, wherein phytosterol is the major sterol. The sunflower seed oil of the present invention preferably has about 200 to 400 mg of total sterols per 100g, wherein phytosterol is the major sterol. Oils derived from the nutrient-rich plant parts of the present invention preferably have less than 200 mg of total sterols per 100g, more preferably less than 100 mg of total sterols per 100g, and most preferably less than 50 mg of total sterols per 100g, wherein the majority of the sterols are free sterols.

[0287] As used herein, the term "seed oil" refers to a composition obtained from the seeds / grains of a plant containing at least 60% (w / w) lipids, or obtainable from the seeds / grains while the seed oil is still present in the seeds / grains. That is, the seed oils of the present invention include seed oils present in seeds / grains or portions thereof, as well as seed oils extracted from seeds / grains. The seed oil is preferably extracted seed oil. The seed oil is generally liquid at room temperature. Preferably, the total fatty acid (TFA) content in the seed oil is primarily (>50%) fatty acids with a length of at least 16 carbons. More preferably, at least 50% of the total fatty acids in the seed oil are C18 fatty acids, such as oleic acid. The fatty acids are generally in esterified form, such as TAG, DAG, acyl-CoA, or phospholipids. The fatty acids can be free fatty acids and / or in esterified form. In one embodiment, at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% of the fatty acids in the seed oil of the present invention may be present as TAGs. In one embodiment, the seed oil of the present invention is a "substantially purified" or "purified" oil that has been isolated from one or more other lipids, nucleic acids, polypeptides, or other contaminating molecules associated with it in the seeds or crude extracts. Preferably, the substantially purified seed oil is free of at least 60%, more preferably at least 75%, and more preferably at least 90% of other components associated with it in the seeds or extracts. The seed oil of the present invention may further contain non-fatty acid molecules, such as, but not limited to, sterols. In one embodiment, the seed oil is rapeseed oil (Brassica oleracea var. spp., such as...). Brassica carinata , Brassica juncea , Brassica napobrassica European rapeseed), mustard oil (mustard) Brassica juncea Other rapeseed oils (e.g., Brassica napobrassica, Brassica camellia), sunflower oil (from the genus *Helianthus* such as sunflower), linseed oil (flax), soybean oil (soybean), safflower oil (safflower), corn oil (corn), tobacco oil (from the genus *Nicotiana* such as safflower tobacco or *Nicotiana benthamiana*), peanut oil (peanut), palm oil (oil palm), cottonseed oil (upland cotton), coconut oil (coconut), avocado oil (avocado... Persea americana Olive oil (olive) Olea europaea Cashew oil (cashew ( Anacardium occidentale Macadamia nut oil (Macadamia nuts ( Macadamia intergrifolia Almond oil (almonds) Prunus amygdalusOat seed oil (oats), rice oil (rice spp. such as paddy rice and glutinous rice), Arabidopsis seed oil (Arabidopsis thaliana), or oil from the following seeds: spiky coconut (coconut) Acrocomia aculeate ),peanut( Aracinis hypogaea ), Murumuru, Star Palm ( Astrocaryum vulgare ,tucumã), Attalea geraensis (Indaiá-rateiro), Attalea humilis (American oil palm) Attalea oleifera (andaiá), Attalea phalerata (uricuri), Attalea speciosa (babassu), beets, flaxseed, Caryocar brasiliense (pequi), sea cabbage (Abyssiniankale), melon, barley, jatropha (physic nut), Joannesia princeps (arara nut-tree), Oiticica, Lupinus, Curved-leaved Palm, Moti Palm, Miscanthus species such as allopolyploid Miscanthus 'Qigang' and Miscanthus sinensis, Pakapa palmatum, Pakapa palmatum, Oenocarpus distichus (bacaba-de-leque), switchgrass ( Panicum virgatum Switchgrass Paraqueiba paraensis (Mari), avocado, water safflower (Indian beech), hairy poplar, castor bean, sugarcane, sesame, potato, sorghum such as bicolor sorghum, sorghum, large-flowered cocoa, clover, Brazilian needle palm, and wheat such as wheat. Seed oil can be extracted from seeds / grains by any method known in the art. This typically involves extraction using nonpolar solvents such as diethyl ether, petroleum ether, chloroform / methanol, or butanol mixtures, usually associated with the first crushing of seeds. Starch-related lipids in grains can be extracted using water-saturated butanol. Seed oil can be “degummed” by methods known in the art to remove polysaccharides, or otherwise treated to remove contaminants or improve purity, stability, or color. TAGs and other esters in seed oil can be hydrolyzed to release free fatty acids, or the seed oil can be hydrogenated, chemically treated, or enzymatically treated as known in the art.

[0288] As used herein, the term "fatty acid" refers to a saturated or unsaturated carboxylic acid having a long aliphatic tail with a length of at least 8 carbon atoms. Preferred fatty acids have carbon-carbon bonds with a length of at least 12 carbon atoms. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetates with two carbon atoms. Fatty acids can be in a free state (non-esterified) or in esterified form, such as TAG, DAG, MAG, acyl-CoA (thioester) bonds, acyl-ACP bonds, or other covalently bonded forms. When covalently bonded in esterified form, fatty acids are referred to herein as "acyl" groups. Fatty acids can be esterified into phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or diphosphatidylglycerol. Saturated fatty acids do not contain any double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen, because all carbons (except for the carboxylic acid [-COOH] group) contain as many hydrogen atoms as possible. In other words, the omega (ω) end contains 3 hydrogen atoms (CH3-), and each carbon atom in the chain contains 2 hydrogen atoms (-CH2-). Unsaturated fatty acids have a similar form to saturated fatty acids, except that one or more olefin functional groups are present along the chain, and each olefin replaces the single bond "-CH2-CH2-" portion of the chain with the "-CH=CH-" portion of the double bond (i.e., the carbon atom that is double-bonded to another carbon). The two adjacent carbon atoms in the chain that are bonded to either side of the double bond can exist in either cis or trans configuration.

[0289] As used herein, the term "monounsaturated fatty acid" or "MUFA" refers to a fatty acid containing at least 12 carbon atoms in its carbon chain and only one olefinic group (carbon-carbon double bond), which may be in esterified or non-esterified (free) form. As used herein, the term "polyunsaturated fatty acid" or "PUFA" refers to a fatty acid containing at least 12 carbon atoms in its carbon chain and at least two olefinic groups (carbon-carbon double bonds), which may be in esterified or non-esterified (free) form.

[0290] As used herein, fatty acids of medium chain length, also referred to as "MCFA," comprise an acyl chain of 8-14 carbons. The acyl chain may be modified (e.g., it may contain one or more double bonds, hydroxyl groups, epoxy groups, etc.) or unmodified (saturated). This term includes at least one or more of caprylic acid (C10:0), lauric acid (C12:0), and myristic acid (C14:0).

[0291] "Monoacylglycerol" or "MAG" is a glycerol ester in which glycerol is esterified by a fatty acid. As used herein, MAG contains... sn -1 / 3 (also referred to in this article) sn- 1 MAG or 1-MAG or 1 / 3-MAG) or sn-The hydroxyl group at position 2 (also referred to as 2-MAG in this text) means that MAG does not contain phosphorylated molecules such as PA or PC. MAG is therefore a component of neutral lipids in cells.

[0292] "Diaacylglycerol ester" or "DAG" is a glycerol ester in which glycerol is esterified by two fatty acids, which may be the same or preferably different. As used herein, DAG comprises... sn- 1, 3 or sn- The hydroxyl group at position 2 means that DAG does not contain phosphorylated molecules such as PA or PC. Therefore, DAG is a component of neutral lipids in cells. In the Kennedy pathway of DAG synthesis (… Figure 1 ), precursor sn 3-glycerol-3-phosphate (G3P) in sn- In the first reaction catalyzed by glycerol-3-phosphoacyltransferase (GPAT), esterification to two acyl groups (each from fatty acid coenzyme A ester) forms LysoPA, followed by a reaction catalyzed by lysophosphatidylacyltransferase (LPAAT). sn- The second acylation at position 2 forms phosphatidic acid (PA). This intermediate is then dephosphorylated via PAP to form DAG. DAG can also be formed from TAG by removing the acyl group with a lipase, or essentially from PC by removing the choline head group with any of the enzymes PDCT, PLC, or PLD. Figure 1 ).

[0293] "Triacylglycerol ester" or "TAG" is a glycerol ester in which glycerol is esterified by three fatty acids, which can be the same (as in triolein) or, more commonly, different. In the Kennedy pathway of TAG synthesis, DAG is formed as described above, and then the third acyl group is esterified to the glycerol backbone via the activity of DGAT. Alternative pathways for TAG formation include the PDAT-catalyzed pathway (…). Figure 1 (and the MGAT approach described in this article).

[0294] As used herein, the term "wild-type" or variations thereof refers to a nutrient plant part, cell, or non-human object or part thereof such as a tuber or root, which, according to the invention, has not been genetically modified, such as by containing exogenous polynucleotides.

[0295] The term "corresponding" refers to a nutrient plant part, cell, seed, or non-human organism or part thereof (such as a tuber or root) that has the same or similar genetic background as the nutrient plant part, cell, seed, or non-human organism or part thereof of the present invention, but has not been modified as described herein (e.g., lacking exogenous polynucleotides and / or lacking genetic modifications). In a preferred embodiment, the corresponding nutrient plant part, eukaryotic cell, seed, or non-human organism or part thereof is at the same developmental stage as the nutrient plant part, eukaryotic cell, seed, or non-human organism or part thereof of the present invention. For example, if the non-human organism is a flowering plant, the corresponding plant is preferably also flowering. The corresponding nutrient plant part, eukaryotic cell, seed, or non-human organism or part thereof can be used as a control to compare nucleic acid or protein expression levels, or the degree and nature of trait modifications, such as nonpolar lipid production and / or content, with the nutrient plant part, eukaryotic cell, seed, or non-human organism or part thereof modified as described herein. Those skilled in the art can readily determine the appropriate “corresponding” nutrient plant part, eukaryotic cell, seed or non-human object or part, tissue, organ or organism for such comparison.

[0296] As used herein, “compared to” or “relative to” refers to the comparison level of nonpolar lipids, total nonpolar lipid content, fatty acid content, or other parameters of a nutrient plant part, eukaryotic cell, seed, non-human organism or part thereof (such as tuber or root) expressing one or more exogenous polynucleotides or exogenous polypeptides with that of a nutrient plant part, eukaryotic cell, seed, non-human organism or part thereof lacking one or more exogenous polynucleotides or exogenous polypeptides.

[0297] As used herein, "enhanced ability to produce nonpolar lipids" is a relative term, referring to an increase in the total amount of nonpolar lipids produced by the nutrient plant parts, eukaryotic cells, seeds, or non-human organisms or their parts (such as tubers or roots) of the present invention relative to the corresponding nutrient plant parts, eukaryotic cells, seeds, or non-human organisms or their parts. In one embodiment, the content of TAGs and / or polyunsaturated fatty acids or the oleic acid content in the total fatty acid content of nonpolar lipids is increased, or the linolenic acid content in the total fatty acid content of nonpolar lipids is decreased, for example, by at least 2% in absolute terms.

[0298] As used herein, “synergistic,” “synergistic,” “synergistically acting,” and related terms are all comparative terms that indicate a combination of elements present in the cells, plants, or parts thereof of the present invention, for example, the effect of the combination of elements A and B is greater than the sum of the effects of the elements individually in the corresponding cells, plants, or parts thereof, for example, the sum of the effects of A and B. When two or more elements are present in the cells, plants, or parts thereof, for example, elements A, B, and C, it indicates that the effect of the combination of all elements is greater than the sum of the effects of the elements individually. In a preferred embodiment, it indicates that the effect of the combination of elements A, B, and C is greater than the sum of the combined effects of elements A and B and the effect of element C. In such a case, it can be said that element C synergizes with elements A and B. As will be understood, the effect is measured in the corresponding cells, plants, or parts thereof, growing under the same conditions and at the same stage of biological development, for example.

[0299] As used herein, "germinating at substantially the same rate as the corresponding wild-type plant" means that the seeds of the plants of the present invention are relatively capable of germination compared to the seeds of wild-type plants with undefined exogenous polynucleotides. Germination can be measured in vitro on tissue culture media or in soil when present in the field. In one embodiment, for example, when grown under optimal greenhouse conditions for the plant species, the number of germinated seeds is at least 75%, more preferably at least 90%, compared to the corresponding wild-type seeds. In another embodiment, for example, when grown under optimal greenhouse conditions for the plant species, the number of germinated seeds that produce seedlings growing at a certain rate is at least 75%, more preferably at least 90%, compared to the corresponding wild-type seeds. This is referred to as "seedling vigor." In one embodiment, the initial root growth and shoot growth rates of the seedlings of the present invention are substantially the same as those of the corresponding wild-type seedlings grown under the same conditions. In one embodiment, the leaf matter (dry weight) of the plants of the present invention is at least 80%, preferably at least 90%, relative to the corresponding wild-type plants grown under the same conditions, preferably in the field. In one embodiment, the height of the plant of the present invention is at least 70%, preferably at least 80%, and more preferably at least 90% of the height of the corresponding wild-type plant, which is preferably grown and matured in the field under the same conditions.

[0300] As used herein, the term "exogenous polynucleotide that downregulates the production and / or activity of endogenous polypeptides" or variations thereof refers to a polynucleotide encoding an RNA molecule that downregulates production and / or activity (e.g., encoding amiRNA or hpRNAi), or that itself downregulates the production and / or activity of endogenous polypeptides (e.g., is an amiRNA or hpRNA that can be directly delivered to, for example, cells), said endogenous polypeptide being, for example, SDP1 TAG lipase, plassomal GPAT, plassomal LPAAT, TGD polypeptide, AGPase, or δ-12 fatty acid desaturase (FAD2), or a combination of two or more thereof. Typically, said RNA molecule reduces the expression of the endogenous gene encoding the polypeptide.

[0301] As used herein, the term "on a weight basis" refers to the weight of a substance (e.g., TAG, DAG, fatty acids) as a weight percentage of a composition containing the substance (e.g., seeds, leaves). For example, if a genetically modified seed has 25 μg of total fatty acids per 120 μg of seed weight, then the percentage of total fatty acids on a weight basis is 20.8%.

[0302] As used herein, the term "on a relative basis" refers to a percentage comparison of parameters such as the amount of a substance in a composition containing that substance with parameters of the corresponding composition. For example, a reduction from 3 units to 2 units represents a 33% reduction on a relative basis.

[0303] As used in this article, a "plast" is an organelle in plants (including algae) that is the site from which carbon-based compounds, including sugars, starch, and fatty acids, are produced during photosynthesis. Plasts include chloroplasts, which contain chlorophyll and perform photosynthesis; xanthophyll, the precursor to chloroplasts; and specialized plasts such as chromoplasts, which are used for pigment synthesis and storage; gerntoplasts, which control the dismantling of photosynthetic apparatus during senescence; starch-producing bodies, which are used for starch synthesis and storage; oleoplasts, which are used for lipid storage; and protein bodies, which are used for protein storage and modification.

[0304] As used herein, the term "biofuel" refers to any type of fuel commonly used in power machinery such as automobiles, airplanes, ships, trucks, or petroleum-powered engines, whose energy is delivered from biomass carbon fixation. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels, and biogas. Examples of biofuels include bioethanol, biodiesel, synthetic diesel, vegetable oils, bioethers, biogas, syngas, solid biofuels, algae-derived fuels, biohydrogen, biomethanol, 2,5-dimethylfuran (DMF), biodimethyl ether (bioDME), Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols, and wood diesel.

[0305] As used herein, the term "bioethanol" refers to biologically produced alcohols, such as ethanol, propanol, and butanol. Bioethanols are produced by microorganisms and / or enzymes through the fermentation of sugars, hemicellulose, or cellulose.

[0306] As used herein, the term "biodiesel" refers to a composition containing methyl or ethyl esters of fatty acids (derived from lipids via transesterification).

[0307] As used in this article, the term "synthetic diesel" refers to a form of diesel fuel derived from renewable feedstocks rather than the fossil feedstocks used in most diesel fuels.

[0308] As used herein, the term "vegetable oil" includes pure vegetable oil (or straight vegetable oil) or waste vegetable oil (byproduct of other industries), including oil produced from nutrient-rich plant parts or seeds.

[0309] As used herein, the term "biogas" refers to methane or a combustible mixture of methane and other gases produced by the anaerobic digestion of organic materials by anaerobic organisms.

[0310] As used herein, the term "syngas" refers to a gaseous mixture containing varying amounts of carbon monoxide and hydrogen, and possibly other hydrocarbons, produced by the partial combustion of biomass. Syngas can be converted to methanol in the presence of a catalyst (typically copper-based), followed by methanol dehydration in the presence of a different catalyst (e.g., silicon-aluminum).

[0311] As used herein, the term "Fischer-Tropsch" refers to a series of chemical reactions that convert a mixture of carbon monoxide and hydrogen into liquid hydrocarbons. The syngas can first be modulated to achieve the desired H₂ / CO ratio using, for example, water gas shift. The conversion takes place in the presence of a catalyst, typically iron or cobalt. Temperature, pressure, and catalyst determine whether light or heavy synthetic oils are produced. For example, at 330°C, gasoline and paraffin are primarily produced, while at 180°–250°C, diesel and wax are primarily produced. The liquid produced from the syngas (which contains various hydrocarbon fractions) is very clean (sulfur-free) straight-chain hydrocarbons.

[0312] As used herein, the term “biochar” refers to char produced from biomass, for example, through the pyrolysis of biomass.

[0313] As used herein, the term "raw material" refers to the material used to produce a product (e.g., biofuels such as biodiesel or synthetic diesel), such as biomass or its conversion products (e.g., syngas).

[0314] As used herein, the term "industrial product" refers to hydrocarbon products consisting primarily of carbon and hydrogen, such as methyl and / or ethyl esters of fatty acids or alkanes such as methane, mixtures of longer-chain alkanes (typically liquid at room temperature), biofuels, carbon monoxide and / or hydrogen, or bio-alcohols such as ethanol, propanol, or butanol, or biochar. The term "industrial product" is intended to include intermediate products that can be converted into other industrial products; for example, syngas itself is considered an industrial product, and it can be used to synthesize hydrocarbon products that are also considered industrial products. As used herein, the term industrial product includes the pure form of the aforementioned compounds, or more commonly, mixtures of various compounds and components, such as hydrocarbon products which can contain a range of carbon chain lengths, as is well known in the art.

[0315] As used in this article, “offspring” refers to the middle and all subsequent generations of offspring that are descended from the parents, such as the second, third or later generations of offspring.

[0316] Throughout this specification, the word “(comprise)” or variations such as “comprises” or “comprising” shall be understood to simply include the said element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0317] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be used to provide clear support for both meanings or either meaning.

[0318] As used herein, unless otherwise stated otherwise, terms refer to approximately + / - 10%, more preferably + / - 5%, more preferably + / - 2%, more preferably + / - 1%, and more preferably + / - 0.5% of the specified value.

[0319] Production of nonpolar lipids and triglycerides This invention is based on the discovery that the content of nonpolar lipids in recombinant eukaryotic cells can be increased by a combination of modifications selected from those named herein as follows: (A) push, (B) pull, (C) protect, (D) package, (E) plastid export, (F) plastid import, and (G) prokaryotic pathway. As described herein, cells without plastids can contain various combinations of AD, while cells with plastids, such as plant and algal cells, can contain various combinations of AG.

[0320] Therefore, the recombinant cells, transgenic non-human animals or parts thereof, and transgenic plants or parts thereof of the present invention have many combinations of exogenous polynucleotides and / or genetic modifications, each providing one type of modification. These exogenous polynucleotides and / or genetic modifications include: (A) Exogenous polynucleotides encoding transcription factor polypeptides that increase the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in cells, transgenic nonhuman animals or parts thereof, or transgenic plants or parts thereof, providing a "push" modification. (B) Exogenous polynucleotides encoding polypeptides involved in the biosynthesis of one or more nonpolar lipids in cells, transgenic nonhuman animals or parts thereof, or transgenic plants or parts thereof, providing "pull" modifications. (C) Genetic modification that, when compared with corresponding cells, transgenic nonhuman animals or portions thereof lacking the genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in cells, transgenic nonhuman animals or portions thereof, or transgenic plants or portions thereof, providing a “protective” modification. (D) Exogenous polynucleotides encoding oil-body-coated (OBC) peptides provide "packaging" modifications. (E) An exogenous polynucleotide encoding a polypeptide that, when compared with a corresponding cell, transgenic nonhuman animal or part thereof, or transgenic plant or part thereof lacking the exogenous polynucleotide, increases fatty acid exporting cells, transgenic nonhuman animals or parts thereof, or transgenic plants or parts thereof, providing a "plastosome exporting" modification. (F) Genetic modification that, compared with the corresponding cells, transgenic nonhuman animals or portions thereof, or transgenic plants or portions thereof lacking the genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the importation of fatty acids into plastids of cells, transgenic nonhuman animals or portions thereof, or transgenic plants or portions thereof, providing a "plastosome importation" modification, and G) Genetic modification that, when compared with the corresponding cells, transgenic nonhuman animals or parts thereof lacking the genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in plastids of cells, transgenic nonhuman animals or parts thereof, or transgenic plants or parts thereof, providing a “prokaryotic pathway” modification.

[0321] The preferred combination of exogenous polynucleotides and / or genetic modifications of the present invention (also referred to herein as the group) is; 1) A, B, and one of the following: C, D, E, F, or G; 2) A, C, and one of the optional D, E, F, or G; 3) A, D, and one of the optional E, F, or G; 4) A, E, and either F or G, which may be present at any time; 5) A, F, and the arbitrarily existing G; 6) A and G; 7) A, B, C, and one of the randomly existing D, E, F, or G; 8) A, B, D, and one of the optional E, F, or G; 9) A, B, E, and either F or G, which may be present at any time; 10) A, B, F, and the arbitrarily existing G; 11) A, B, C, D, and one of the optional E, F, or G; 12) A, B, C, E, and either F or G (whichever exists). 13) A, B, C, F, and any arbitrarily existing G; 14) A, B, D, E, and either F or G (whichever exists). 15) A, B, D, F, and the arbitrarily existing G; 16) A, B, E, F, and the arbitrarily existing G; 17) A, C, D, and one of the optional E, F, or G; 18) A, C, E, and either F or G, which may be present at any time; 19) A, C, F, and the arbitrarily existing G; 20) A, C, D, E, and either F or G (whichever exists). 21) A, C, D, F, and the arbitrarily existing G; 22) A, C, E, F, and the optional fifth modifier G; 23) A, D, E, and either F or G, whichever exists; 24) A, D, F, and the arbitrarily existing G; 25) A, D, E, F, and the arbitrarily existing G; 26) A, E, F, and the arbitrarily existing G; 27) Six of the following: A, B, C, D, E, F, and G, omitting one of A, B, C, D, E, F, or G, and... 28) Any of 1-26 above, wherein two or more exogenous polynucleotides encode two or more different transcription factor polypeptides that increase the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in cells, such as an exogenous polynucleotide encoding WRI1 and another exogenous polynucleotide encoding LEC2.

[0322] In each of the above preferred combinations, there may be at least two different exogenous polynucleotides encoding at least two different transcription factor polypeptides that increase the expression of one or more glycolysis and / or fatty acid biosynthesis genes in cells, transgenic animals or parts thereof, or transgenic plants or parts thereof.

[0323] These modifications are described as follows: A. The "push" modification is characterized by an increase in total fatty acid synthesis in the plastids of eukaryotic cells. In one embodiment, this occurs by increasing the expression and / or activity of transcription factors that regulate fatty acid synthesis in the plastids. In one embodiment, this can be achieved by expressing a foreign polynucleotide in transgenic cells, the foreign polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cell. In one embodiment, the increased fatty acid synthesis is not caused by the supply of altered ACCase to the cells, the activity of which is less inhibited by fatty acids compared to endogenous ACCase in the cells. In one embodiment, the cells contain a foreign polynucleotide encoding a transcription factor, which is preferably under the control of a promoter other than a constitutive promoter. The transcription factors can be selected from the group consisting of WRI1, LEC1, LEC1-like, LEC2, BBM, FUS3, ABI3, ABI4, ABI5, Dof4, and Dof11, or the group consisting of MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2, and PHR1, with WRI1, LEC1, or LEC2 being preferred. In another embodiment, the increased total fatty acid synthesis is relative to the corresponding wild-type cells. In one embodiment, there are two or more exogenous polynucleotides encoding two or more different transcription factor polypeptides.

[0324] B. The “pull” modification is characterized by increased expression and / or activity of fatty acylacyltransferases that catalyze the synthesis of TAG, DAG, or MAG in cells, such as DGAT, PDAT, LPAAT, GPAT, or MGAT, preferably DGAT or PDAT. In one embodiment, this can be achieved by expressing a foreign polynucleotide in transgenic cells, said foreign polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. In one embodiment, the acyltransferase is a membrane-bound acyltransferase that uses an acyl-CoA substrate as an acyl donor in the case of DGAT, LPAAT, GPAT, or MGAT, or uses an acyl group from a PC as an acyl donor in the case of PDAT. The pull modification can be relative to the corresponding wild-type cells, or preferably, relative to the corresponding cells with the push modification. In one embodiment, said cells contain a foreign polynucleotide encoding a fatty acylacyltransferase.

[0325] C. The “protective” modification is characterized by reduced catabolism of triacylglycerols (TAGs) in the cell. In one embodiment, this can be achieved through a genetic modification in the cell that downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the cell compared to corresponding cells lacking the genetic modification. In an embodiment, the cells have reduced expression and / or activity of endogenous TAG lipases, preferably SDP1 lipase, Cgi58 peptide, acyl-CoA oxidases such as ACX1 or ACX2, or peptides involved in the β-oxidation of fatty acids in the cell such as PXA1 peroxisome ATP-binding cassette transporter. This can occur by expressing exogenous polynucleotides encoding RNA molecules in the cell that reduce the expression of endogenous genes encoding, for example, TAG lipases such as SDP1 lipase, acyl-CoA oxidases, or peptides involved in the β-oxidation of fatty acids in the cell, or by mutations in endogenous genes encoding, for example, TAG lipases, acyl-CoA oxidases, or peptides involved in the β-oxidation of fatty acids. In one embodiment, the reduced expression and / or activity is relative to the corresponding wild-type cells or relative to the corresponding cells with push modification.

[0326] D. The “packaging” modification is characterized by increased expression and / or accumulation of oil body coating (OBC) peptides. In one embodiment, this can be achieved by expressing exogenous polynucleotides encoding oil body coating (OBC) peptides in transgenic cells. The OBC peptide can be an olein, such as polyolein, olein calcitonin, or olein sterol, or preferably LDAP. In one embodiment, the level of olein accumulated in the eukaryotic cells is at least 2-fold higher than that of the corresponding cells containing the olein gene from pJP3502's T-DNA. In one embodiment, the increased expression or accumulation of the OBC peptide is not solely caused by the push modification. In one embodiment, the expression and / or accumulation is relative to the corresponding wild-type cells, or preferably, relative to the corresponding cells with the push modification.

[0327] E. The “plastosome export” modification is characterized by an increased rate of total fatty acid export from the plasmids of eukaryotic cells. In one embodiment, this can be achieved by expressing a foreign polynucleotide in transgenic cells that encodes a polypeptide that, when compared to a corresponding cell lacking the foreign polynucleotide, increases fatty acid export from the plasmid. In one embodiment, this occurs through increased expression and / or activity of fatty acid thioesterase (TE), fatty acid transporter polypeptides such as ABCA9 polypeptide, or long-chain acyl-CoA synthase (LACS). In one embodiment, the cells contain a foreign polynucleotide encoding TE, fatty acid transporter polypeptide, or LACS. TE may be a FATB polypeptide or preferably a FATA polypeptide. In one embodiment, the TE is preferably a TE specific for MCFA. In one embodiment, the plasmid export modification is relative to the corresponding wild-type cells, or preferably, relative to the corresponding cells with the export modification.

[0328] F. The “plastosome importation” modification is characterized by a reduced rate of fatty acid importation from the plastid into the cytoplasm. In one embodiment, this can be achieved through a genetic modification in the cell that downregulates the endogenous production and / or activity of peptides involved in fatty acid importation into the cytoplasm, compared to corresponding cells lacking the modification. For example, this can occur by expressing exogenous polynucleotides encoding RNA molecules in the cell that reduce the expression of endogenous genes encoding transporter peptides such as TGD peptides (e.g., TGD1, TGD2, TGD3, or TGD4 peptides), or by mutations in endogenous genes encoding TGD peptides. In one embodiment, the reduced importation rate is relative to the corresponding wild-type cells or relative to corresponding cells with the importation modification.

[0329] G. The “prokaryotic pathway” modification is characterized by a reduction in the amount or rate of DAG production in the cytoplasm. In one embodiment, this can be achieved through a genetic modification in the cell that downregulates the endogenous production and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in the plasmid, compared to corresponding cells lacking the genetic modification. In one embodiment, the reduced amount or rate of DAG production occurs through a decrease in the production of LPA from acyl-ACP and G3P in the plasmid. The reduced amount or rate of DAG production can occur through the expression of an exogenous polynucleotide encoding an RNA molecule in the cell that reduces the expression of an endogenous gene encoding plasmid GPAT, plasmid LPAAT, or plasmid PAP (preferably plasmid GPAT), or through a mutation in an endogenous gene encoding a plasmid polypeptide. In one embodiment, the reduced amount or rate of DAG production is relative to the corresponding wild-type cells, or preferably, relative to the corresponding cells with the DAG modification.

[0330] Push modifications are necessary for this invention, while pull modifications are preferred. Protective and packaging modifications can be complementary, i.e., one of the two can be sufficient. The cell may contain one, two, or all three of the following modifications: plasmid export, plasmid import, and prokaryotic pathway. In one embodiment, at least one exogenous polynucleotide in the cell, preferably at least encoding a transcription factor regulating fatty acid synthesis in plasmids, is expressed under the control of a promoter other than a constitutive promoter, such as a development-related promoter, a promoter preferably activated in photosynthetic cells, a tissue-specific promoter, a promoter modified by reducing its expression level relative to the corresponding natural promoter, or preferably a senescence-specific promoter. More preferably, the exogenous polynucleotide encoding at least a transcription factor regulating fatty acid synthesis in plasmids is expressed under the control of a promoter other than a constitutive promoter, and the exogenous polynucleotide encoding an RNA molecule encoding an endogenous production and / or activity of a polypeptide involved in triglyceride catabolism is also expressed under the control of a promoter other than a constitutive promoter, which may be the same or different.

[0331] Plants produce some, but not all, of their membrane lipids, such as MGDG, in plastids via the so-called prokaryotic pathway. Figure 1 In plants, there is also a eukaryotic pathway for the synthesis of galactopyritins and glycerides, which first synthesizes FA in plastids and then assembles FA into glycerides in the ER. MGDG synthesized via the eukaryotic pathway is contained in MGDG. sn C18:3 (ALA) fatty acids esterified at the -2 position. The DAG backbone, including ALA, used in MGDG synthesis via this pathway is assembled in the ER and then fed into the plastid. In contrast, MGDG synthesized via the prokaryotic pathway contains MGDG in... sn C16:3 fatty acids esterified at position -2. The proportion of prokaryotic to eukaryotic pathways in the production of MGDG (16:3) versus MGDG (18:3) is a characteristic and distinguishing feature of different plant species (Mongrand et al. 1998). This distinguishing fatty acid composition of MGDG allows all higher plants (angiosperms) to be classified as so-called 16:3 or 18:3 plants. Through Arabidopsis thaliana (… Arabidopsis16:3 species, exemplified by rapeseed and others, generally possess both prokaryotic and eukaryotic pathways for MGDG synthesis, while 18:3 species, exemplified by red tobacco, peas, and soybeans, generally possess only (or almost entirely) the eukaryotic pathway for MGDG synthesis, providing little or no accumulation of C16:3 fatty acids in vegetative tissues. As used herein, a “16:3 plant” or “16:3 species” is a plant with more than 2% C16:3 fatty acids in the total fatty acid content of its photosynthetic tissues. As used herein, an “18:3 plant” or “18:3 species” is a plant with less than 2% C16:3 fatty acids in the total fatty acid content of its photosynthetic tissues. As described herein, plants can be converted from 16:3 plants to 18:3 plants through appropriate genetic modifications. The flow ratio between prokaryotic and eukaryotic pathways is not conserved across different plant species or tissues. In 16:3 species, up to 40% of leaf flux occurs via the eukaryotic pathway (Browse et al., 1986), while in 18:3 species, such as peas and soybeans, approximately 90% of the FA synthesized in the plastids is exported to the ER to supply the source of FA for the eukaryotic pathway (Ohlrogge and Browse, 1995; Somerville et al., 2000).

[0332] Therefore, varying amounts of 18:3 and 16:3 fatty acids were found in the glycolipids of different plant species. This is used to distinguish 18:3 plants, whose fatty acids with three double bonds are almost entirely C18 fatty acids, from those containing C18 fatty acids with three double bonds. 16 -and C 18 - Fatty acids in 16:3 plants. In the chloroplasts of 18:3 plants, the enzymatic activities catalyzing the conversion of phosphatidic acid to diacylglycerol and diacylglycerol to monogalactosyldiacylglycerol (MGD) are significantly less active than in 16:3 chloroplasts. In the leaves of 18:3 plants, chloroplasts synthesize stearoyl-ACP2 in the stroma, introducing the first double bond into the saturated hydrocarbon chain, and then hydrolyzing the thioester by thioesterases (… Figure 1 The released oleic acid is exported through the chloroplast membrane into the membrane of the eukaryotic portion of the cell, possibly the endoplasmic reticulum, where it is incorporated into the protoplast (PC). The PC-linked oleyl group is desaturated in these membranes and subsequently returns to the chloroplast. The MGD-linked acyl group is the substrate used to introduce a third double bond in order to obtain an MGD with two linolenic acid residues. This galactolipid is characteristic of 18:3 plants such as those in the Asteraceae and Leguminosae families. In photosynthetically active cells of 16:3 plants, such as members of the Apiaceae and Brassicaceae families, both pathways operate in parallel to provide thylakoids with MGDs.

[0333] In one embodiment, the nutrient plant parts, eukaryotic cells, seeds, or transgenic non-human organisms or parts thereof (such as tubers or roots) of the present invention produce higher levels of nonpolar lipids such as TAG, or total fatty acid (TFA) content, preferably both, than corresponding nutrient plant parts, eukaryotic cells, seeds, or non-human organisms or parts thereof lacking genetic modification or exogenous polynucleotides. In one example, when compared with the corresponding seeds, leaves, or surfaces with a surface area of ​​at least 1 cm²... 2 Compared to the leaves, stems, or tubers, the plants of the present invention produce seeds, leaves, or tubers with increased nonpolar lipid content such as TAG or TFA (preferably both), or with a surface area of ​​at least 1 cm². 2 The leafy parts, stems and / or tubers.

[0334] In another embodiment, a nutrient plant part, a genetically modified non-human organism or a part thereof (such as a tuber or root), preferably a plant, tuber, root, or seed, produces a TAG enriched with one or more specific fatty acids. A broad spectrum of fatty acids can be incorporated into the TAG, including saturated and unsaturated fatty acids as well as short-chain and long-chain fatty acids. Some non-limiting examples of fatty acids that can be incorporated into the TAG and whose levels can be elevated include: caprylic acid (10:0), lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), isoleic acid (18:1), linoleic acid (18:2), tung oil acid (18:3), gamma-linolenic acid (18:3), alpha-linolenic acid (18:3ω3), octadecanoic acid (18:4ω3), peanut oil, etc. The fatty acids include oleic acid (20:0), eicosapentaenoic acid (20:2), dihomo-γ-linolenic acid (20:3), eicosatrienoic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:4), eicosapentaenoic acid (20:5ω3), docosanoic acid (22:0), docosapentaenoic acid (22:5ω), docosahexaenoic acid (22:6ω3), tetracosanoic acid (24:0), nervonic acid (24:1), ceric acid (26:0), and linolenic acid (28:0). In one embodiment of the invention, the nutrient plant parts, eukaryotic cells, seeds, or transgenic organisms or parts thereof (such as tubers or roots) are enriched with TAGs containing oleic acid, and / or linolenic acid (ALA) is reduced, preferably by at least 2% or at least 5% on an absolute basis.

[0335] Preferably, the vegetative plant parts, eukaryotic cells, seeds, or transgenic non-human organisms or parts thereof of the present invention are transformed with one or more chimeric DNAs (exogenous polynucleotides). In the case of multiple chimeric DNAs, these are preferably covalently linked to a single DNA molecule, such as a single T-DNA molecule, and preferably integrated into a single locus in the host cell genome. Alternatively, the chimeric DNA may be on two or more DNA molecules that are not linked in the host genome, or the DNA molecule may not be integrated into the host genome, as seen in transient expression experiments. The plant, vegetative plant parts, eukaryotic cells, seeds, or transgenic non-human organisms or parts thereof are preferably homozygous for the DNA molecule inserted into their genome.

[0336] transcription factors In eukaryotic cells, various transcription factors are involved in fatty acid synthesis and lipid incorporation into fatty acids such as TAGs, and therefore can be manipulated for push modification. A preferred transcription factor is WRI1. As used herein, the terms "Wrinkled 1," "WRI1," or "WRL1" refer to AP2 / ERWEBP-type transcription factors that regulate the expression of several enzymes involved in glycolysis and de novo fatty acid biosynthesis. WRI1 possesses two plant-specific (AP2 / EREB) DNA-binding domains. At least in Arabidopsis thaliana, WRI1 also regulates the cleavage of sucrose via glycolysis, thereby regulating the supply of precursors for fatty acid biosynthesis. In other words, it controls the carbon flow from photosynthesis to lipid storage. At least in Arabidopsis thaliana... wri1 The mutant exhibits a wrinkled seed phenotype due to a defect in incorporating sucrose and glucose into the TAG.

[0337] Examples of genes transcribed from WRI1 include, but are not limited to, those encoding one or more, preferably all of, the following genes: pyruvate kinase (At5g52920, At3g22960), pyruvate dehydrogenase (PDH) E1α subunit (At1g01090), acetyl-CoA carboxylase (ACCase), BCCP2 subunit (At5g15530), enoyl-ACP reductase (At2g05990; EAR), phosphoglycerate mutase (At1g22170), cytofructose kinase, and cytophosphoglycerate mutase, sucrose synthase (SuSy) (see, for example, Liu et al., 2010b; Baud et al., 2007; Ruuska et al., 2002).

[0338] WRI1 contains the conserved domain AP2 (cd00018). AP2 is a DNA-binding domain found in transcriptional regulators in plants, such as APETALA2 and EREBP (ethylene response element binding protein). In EREBP, this domain specifically binds to the 11 bp GCC box of the ethylene response element (ERE, an essential promoter element in the ethylene response). EREBP and the C-repetition sequence binding factor CBF1, which are involved in stress responses, contain a single copy of the AP2 domain. APETALA2-like proteins, which play a role in plant development, contain two copies.

[0339] Other sequence motifs that can be found in WRI1 and its functional homologs include: 1. RGVT / SRHRWTGR (SEQ ID NO:89). 2. F / YEAHLWDK (SEQ ID NO:90). 3. DLAALKYWG (SEQ ID NO:91). 4. SXGFS / ARGX (SEQ ID NO:92). 5. HHH / QNGR / KWEARIGR / KV (SEQ ID NO:93). 6. QEEAAAXYD (SEQ ID NO:94). As used herein, the term “Wrinkled 1” or “WRI1” also includes “Wrinkled 1-like” or “WRI1-like” proteins. Examples of the WRI1 protein include accession number Q6X5Y6 (Arabidopsis thaliana; SEQ ID NO:22), XP_002876251.1 (Arabidopsis thaliana subsp. fiddleleaf; SEQ ID NO:23), ABD16282.1 (Rabocium barbarum; SEQ ID NO:24), ADO16346.1 (Rabocium barbarum; SEQ ID NO:25), XP_003530370.1 (Soybean; SEQ ID NO:26), AEO22131.1 (Jatropha curcas; SEQ ID NO:27), XP_002525305.1 (Castor bean; SEQ ID NO:28), XP_002316459.1 (Populus pubescens; SEQ ID NO:29), CBI29147.3 (Grape; SEQ ID NO:30), and XP_003578997.1. (Bruguiera gymnorhiza; SEQ ID NO:31), BAJ86627.1 (Barley; SEQ ID NO:32), EAY79792.1 (Rice; SEQ ID NO:33), XP_002450194.1 (Brocinaria bicolor; SEQ ID NO:34), ACG32367.1 (Maize; SEQ ID NO:35), XP_003561189.1 (Bruguiera gymnorhiza; SEQ ID NO:36), ABL85061.1 (Brucinaria spp.; SEQ ID NO:37), BAD68417.1 (Rice; SEQ ID NO:38), XP_002437819.1 (Brocinaria bicolor; SEQ ID NO:39), XP_002441444.1 (Brocinaria bicolor; SEQ ID NO:39) SEQ ID NO:40), XP_003530686.1 (soybean; SEQ ID NO:41), XP_003553203.1 (soybean; SEQ ID NO:42), XP_002315794.1 (poplar; SEQ ID NO:43), XP_002270149.1 (grape; SEQ ID NO:44), XP_003533548.1 (soybean; SEQ ID NO:45), XP_003551723.1 (soybean; SEQ ID NO:46), XP_003621117.1 (alfalfa; SEQ ID NO:47), XP_002323836.1 (poplar; SEQ ID NO:48), XP_002517474.1 (castor bean; SEQ ID NO:40). NO:49), CAN79925.1 (Grape; SEQ ID NO:50), XP_003572236.1 (Broccaria divaricata; SEQ ID NO:51), BAD10030.1 (Rice; SEQ ID NO:52), XP_002444429.1 (Broccaria divaricata; SEQ ID NO:53), NP_001170359.1 (Maize; SEQ ID NO:54), XP_002889265.1 (Arabidopsis thaliana, subspecies lyreinae; SEQ ID NO:55), AAF68121.1 (Arabidopsis thaliana; SEQ ID NO:56), NP_178088.2 (Arabidopsis thaliana; SEQ ID NO:57), XP_002890145.1 (Arabidopsis thaliana, subspecies lyreinae; SEQ ID NO:58), BAJ33872.1 (Salix babylonica; SEQ ID NO:50). NO:59), NP_563990.1 (Arabidopsis thaliana; SEQ ID NO:60), XP_003530350.1 (Soybean; SEQ ID NO:61), XP_003578142.1 (Broccaria divaricata; SEQ ID NO:62), EAZ09147.1 (Rice; SEQ ID NO:63), XP_002460236.1 (Sorghum bicolor; SEQ ID NO:64), NP_001146338.1 (Maize; SEQ ID NO:65), XP_003519167.1 (Soybean; SEQ ID NO:66), XP_003550676.1 (Soybean; SEQ ID NO:67), XP_003610261.1 (Alfalfa tribulus; SEQ ID NO:69). Examples include: XP_003524030.1 (soybean; SEQ ID NO:69), XP_003525949.1 (soybean; SEQ ID NO:70), XP_002325111.1 (hairy poplar; SEQ ID NO:71), CBI36586.3 (grape; SEQ ID NO:72), XP_002273046.2 (grape; SEQ ID NO:73), XP_002303866.1 (hairy poplar; SEQ ID NO:74), and CBI25261.3 (grape; SEQ ID NO:75). Other examples include Sorbi-WRL1 (SEQ ID NO:76), Lupan-WRL1 (SEQ ID NO:77), Ricco-WRL1 (SEQ ID NO:78), and Narrow-leaved lupin WRI1 (SEQ ID NO:79). The preferred WRI1 is either maize WRI1 or sorghum WRI1.

[0340] Recently, a subset of WRI1-like transcription factors has been reclassified as WRI2, WRI3, or WRI4 transcription factors, characterized by preferential expression in the stems and / or roots of plants rather than in developing seeds (To et al., 2012). Despite their reclassification, they are included in the definition of “WRI1” herein. Preferred WRI1-like transcription factors are those that can complement plant expression. wri1 Mutations in the function of WRI1-like transcription factors, particularly in seeds of plants like Arabidopsis thaliana, can disrupt the development of these transcription factors. wri1 Functions in mutants. The functions of WRI1-like peptides can also be determined in the transient assay of *Benjamin* as described in this article.

[0341] As used herein, the polypeptides “LEAFY COTYLEDON” or “LEC” refer to transcription factors, which are LEC1, LEC1-like, LEC2, ABI3, or FUS3 transcription factors, exhibiting broad control over seed maturation and fatty acid synthesis. LEC2, FUS3, and ABI3 are related polypeptides, each containing a 120-amino acid B3 DNA-binding domain found only in plant proteins (Yamasaki et al., 2004). They can be distinguished by phylogenetic analysis to determine their amino acid sequence relevance to Arabidopsis polypeptide members with the following accession numbers: LEC2, accession number AAL12004.1; FUS3 (also known as FUSCA3), accession number AAC35247. LEC1 belongs to a different class of polypeptides and is homologous to the HAP3 polypeptide of the CBF-binding factor class (Lee et al., 2003). The LEC1, LEC2, and FUS3 genes are required during early embryogenesis to maintain embryonic cell fate and designate cotyledonary identity, and are also required later in the initiation and maintenance of embryonic maturation (Santos-Mendoza et al., 2008). They also induce the expression of genes encoding seed storage proteins and oleanolic protein genes by binding to the RY motif present in the promoter. They can also be distinguished by their expression patterns during seed development or by their ability to complement corresponding mutations in Arabidopsis.

[0342] As used herein, the term "Leafy Cotyledon 1" or "LEC1" refers to the NF-YB transcription factor involved in zygote development and somatic embryogenesis. The endogenous gene is specifically expressed in the embryo and endosperm of the seed. LEC1 activates genes encoding WRI1 as well as a large class of fatty acid synthesis genes. Ectopic expression of LEC1 also induces rapid activation of auxin-responsive genes and can lead to the formation of somatic ligands. Examples of LEC1 peptides include those from Arabidopsis thaliana (AAC39488, SEQ ID NO:149), Alfalfa truncatum (AFK49653, SEQ ID NO:154), and rapeseed (ADF81045, SEQ ID NO:151), and Arabidopsis thaliana (Fiddleleaf Arabidopsis thaliana). A. lyrata (XP_002862657, SEQ ID NO:150), castor bean ( R. communis (XP_002522740, SEQ ID NO:152), cultivated soybean (XP_006582823, SEQ ID NO:153), peanut ( A. hypogaea Proteins from (ADC33213, SEQ ID NO:156) and corn (AAK95562, SEQ ID NO:155).

[0343] LEC1-like proteins (L1L) are closely related to LEC1 but exhibit different gene expression patterns and are expressed earlier during embryogenesis (Kwong et al., 2003). Examples of LEC1-like peptides include proteins from Arabidopsis thaliana (AAN15924, SEQ ID NO:157), rapeseed (AHI94922, SEQ ID NO:158), and LEC1-like proteins from broad bean (AAN01148, SEQ ID NO:159).

[0344] As used herein, the term "Leafy Cotyledon 2" or "LEC2" refers to a B3 domain transcription factor involved in zygote development and somatic embryogenesis, and which activates the expression of a gene encoding WRI1. Its ectopic expression contributes to embryogenesis from vegetative plant tissues (Alemanno et al., 2008). Examples of LEC2 peptides include proteins from Arabidopsis thaliana (accession number NP_564304.1, SEQ ID NO:142), alfalfa (accession number CAA42938.1, SEQ ID NO:143), and rapeseed (accession number ADO16343.1, SEQ ID NO:144).

[0345] In one embodiment, the exogenous polynucleotide of the present invention encoding LEC2 comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 142-144, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 142-144. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0346] As used herein, the term "FUS3" refers to a B3 domain transcription factor involved in zygote development and somatic embryogenesis, and is primarily detected in the protoepidermal tissue of the embryo (Gazzarrini et al., 2004). Examples of FUS3 peptides include proteins from Arabidopsis thaliana (AAC35247, SEQ ID NO:160), rapeseed (XP_006293066.1, SEQ ID NO:161), and alfalfa (XP_003624470, SEQ ID NO:162). Overexpression of any LEC1, L1L, LEC2, FUS3, and ABI derived from exogenous polynucleotides is preferably controlled by developmental regulatory promoters, such as senescence-specific promoters, inducible promoters, or promoters engineered to provide reduced levels of expression relative to natural promoters, particularly in plants other than Arabidopsis thaliana and the European rapeseed variety Westar, in order to avoid developmental abnormalities in plant development that are typically associated with overexpression of these transcription factors (Mu et al., 2008).

[0347] As used herein, the terms “BABY BOOM” or “BBM” refer to the AP2 / ERF transcription factor, which induces regeneration under culture conditions that typically do not support regeneration in wild-type plants. This applies to both *Raphanus pensilis* and *Arabidopsis thaliana*. BBM ( BnBBM Ectopic expression of the gene induces spontaneous somatic embryogenesis and organogenesis in seedlings grown on hormone-free basal medium (Boutilier et al., 2002). In tobacco, ectopic expression... BBMExpression is sufficient to induce adventitious shoot and root regeneration on basal medium, but somatic embryo (SE) formation requires exogenous cytokinin (Srinivasan et al., 2007). Examples of BBM peptides include proteins from Arabidopsis thaliana (accession number NP_197245.2, SEQ ID NO:145), maize (US7579529), bicolor sorghum (accession number XP_002458927), and alfalfa tribulus (accession number AAW82334.1, SEQ ID NO:146).

[0348] In one embodiment, unless otherwise stated, the exogenous polynucleotide of the present invention encoding BBM comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in one of SEQ ID NO: 145 or 146, or a polypeptide whose amino acid sequence is at least 30% identical to one or both of SEQ ID NO: 145 or 146. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0349] The ABI3 polypeptide (Arabidopsis thaliana accession number NP_189108) is associated with the maize VP1 protein, expressed at low levels in vegetative tissues, and affects plastid development. The ABI4 polypeptide (Arabidopsis thaliana accession number NP_181551) belongs to a family of transcription factors containing plant-specific AP2 domains (Finkelstein et al., 1998) and functions downstream of ABI3. ABI5 (Arabidopsis thaliana accession number NP_565840) is a bZIP family transcription factor that affects ABA sensitivity and controls the expression of some LEA genes in seeds. It binds to ABA-responsive elements.

[0350] Each of the following transcription factors was selected based on its function in embryogenesis in plants. Accession numbers are provided in Table 10. Homologues of each can be readily identified in many other plant species and tested as described in Example 10.

[0351] MYB73 is a transcription factor identified in soybean that is involved in stress response.

[0352] bZIP53 is a transcription factor in the bZIP protein family identified in Arabidopsis thaliana.

[0353] AGL15 (Agamous-like 15) is a MADS-box transcription factor naturally expressed during embryogenesis. AGL15 is also expressed in leaf primordia, shoot apical meristems, and young flower buds, indicating that it may also play a role in post-budding development. AGL15 plays a role in embryogenesis and gibberellic acid catabolism. Its target is a B3 domain transcription factor, a key regulator of embryogenesis.

[0354] MYB115 and MYB118 are transcription factors from the MYB family of Arabidopsis thaliana that are involved in embryogenesis.

[0355] Also known as EMB2757, TANMEI encodes a repeating protein called WD, which is required for embryonic development in Arabidopsis thaliana.

[0356] Also known as Wuschel, WUS is a homeobox gene that controls the pool of stem cells in the embryo. It is expressed in the stem cell organization center of meristems and is required to maintain stem cells in an undifferentiated state. This transcription factor binds to the core motif of the TAAT element.

[0357] GFR2a1 and GFR2a2 are transcription factors derived from at least soybean.

[0358] Fatty acyl acyl transferase As used herein, the term "fatty acyl acyltransferase" refers to a protein capable of transferring an acyl group from acyl-CoA, PC, or -ACP (preferably acyl-CoA or PC) to a substrate to form a TAG, DAG, or MAG. These acyltransferases include DGAT, PDAT, MGAT, GPAT, and LPAAT.

[0359] As used herein, the term "diacylglycerol acyltransferase" (DGAT) refers to a protein that transfers a fatty acyl group from an acyl-CoA to a DAG substrate to produce a TAG. Therefore, the term "diacylglycerol acyltransferase activity" refers to the transfer of an acyl group from an acyl-CoA to a DAG to produce a TAG. DGAT can also have MGAT function, but it is primarily used as DGAT; that is, when enzyme activity is expressed in nmol product / min / mg protein, its catalytic activity as DGAT is higher than its catalytic activity as MGAT (see, for example, Yen et al., 2005). DGAT activity can be rate-limiting in TAG synthesis in seeds (Ichihara et al., 1988). DGAT uses an acyl-CoA substrate as an acyl donor and transfers it to a DAG... sn- The enzyme forms a TAG at position 3. It functions in its native state in the endoplasmic reticulum (ER) of the cell.

[0360] There are three known classes of DGAT, referred to as DGAT1, DGAT2, and DGAT3. DGAT1 peptides are membrane proteins that typically have 10 transmembrane domains, DGAT2 peptides are also membrane proteins but typically have 2 transmembrane domains, while DGAT3 peptides typically lack transmembrane domains and are considered soluble in the cytoplasm and do not integrate into the membrane. Plant DGAT1 peptides typically have approximately 510–550 amino acid residues, while DGAT2 peptides typically have approximately 310–330 residues. DGAT1 is the major enzyme responsible for the production of TAG from DAG in the seeds of most developing plants, while DGAT2 from plant species that produce large amounts of rare fatty acids, such as the tung tree (Vernicia fordii) and castor bean (Castor bean), appears to play an important role in the accumulation of rare fatty acids in TAG. Overexpression of AtDGAT1 in tobacco leaves leads to a 6–7 fold increase in TAG content (Bouvier-Nave et al., 2000).

[0361] Examples of DGAT1 peptides include DGAT1 proteins from Aspergillus fumigatus (XP_755172.1; SEQ ID NO:80), Arabidopsis thaliana (CAB44774.1; SEQ ID NO:1), castor bean (AAR11479.1; SEQ ID NO:81), tung oil tree (ABC94472.1; SEQ ID NO:82), daisy (ABV21945.1 and ABV21946.1; SEQ ID NO:83 and SEQ ID NO:84, respectively), Euonymus japonicus (AAV31083.1; SEQ ID NO:85), Caenorhabditis elegans (AAF82410.1; SEQ ID NO:86), brown rat (NP_445889.1; SEQ ID NO:87), and Homo sapiens (NP_036211.2; SEQ ID NO:88), as well as their variants and / or mutants. Examples of DGAT2 polypeptides include proteins encoded by the DGAT2 gene from Arabidopsis thaliana (NP_566952.1; SEQ ID NO:2), castor bean (AAY16324.1; SEQ ID NO:3), tung oil tree (ABC94474.1; SEQ ID NO:4), *Morchella ramaniforme* (AAK84179.1; SEQ ID NO:5), *Homo sapiens* (Q96PD7.2; SEQ ID NO:6) (Q58HT5.1; SEQ ID NO:7), cattle (Q70VZ8.1; SEQ ID NO:8), and house mouse (AAK84175.1; SEQ ID NO:9), as well as their variants and / or mutants. The amino acid sequences of DGAT1 and DGAT2 show little homology. In increasing oil content (TAG), the efficiency of expressing exogenous DGAT2 in leaves is twice that of DGAT1. Furthermore, compared to DGAT1, Arabidopsis DGAT2 shows a greater preference for linoleyl-CoA and linolenic acid-CoA as acyl donors, relative to oleoyl-CoA. This substrate preference, in addition to their amino acid sequences, can be used to distinguish between these two DGAT classes.

[0362] Examples of DGAT3 peptides include those derived from peanuts ( Arachis hypogaea The DGAT3 gene (Saha, et al., 2006) encodes proteins, as well as their variants and / or mutants. DGAT has virtually no detectable MGAT activity, for example, below 300 pmol / min / mg protein, preferably below 200 pmol / min / mg protein, more preferably below 100 pmol / min / mg protein.

[0363] In one embodiment, the exogenous polynucleotide of the present invention encoding DGAT1 comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in SEQ ID NO:1 or 80-88, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO:1 or 80-88. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0364] In one embodiment, the exogenous polynucleotide of the present invention encoding DGAT2 comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 2-9, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 2-9. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0365] As used herein, the term "phospholipid: diacylglycerol acyltransferase" (PDAT; EC 2.3.1.158) or its synonym "phospholipid: 1,2-diacyl- sn "-Glycerol O-acyltransferase" refers to an acyltransferase that transfers an acyl group from a phospholipid (usually PC) to the sn-3 position of a DAG to form a TAG. This reaction is independent of DGAT and uses a phospholipid as an acyl donor. Several forms of PDAT exist in plant cells, including PDAT1, PDAT2, or PDAT3 (Ghosal et al., 2007).

[0366] As used herein, the term "monoacylglycerol acyltransferase" or "MGAT" refers to the enzyme that transfers a fatty acyl group from acyl-CoA to a MAG substrate, such as... sn -2 MAG to produce DAG. Therefore, the term "monoacylglycerol acyltransferase activity" refers at least to the transfer of an acyl group from acyl-CoA to MAG to produce DAG. As used herein, the term "MGAT" includes proteins that act on... sn- 1 / 3 MAG and / or sn- 2 MAG substrates were used to form sn- 1,3 DAG and / or sn- Enzymes of 1,2 / 2,3-DAG. In a preferred embodiment, relative to... sn -1 MAG, MGAT sn- 2. MAG substrates have preferences, or are basically only used. sn- 2. MAG as a substrate. As used herein, MGAT does not include enzymes that preferentially transfer acyl groups to LysoPA relative to MAG; such enzymes are called LPAATs. That is, MGAT preferentially uses non-phosphorylated monoacyl substrates, even if they may have lower catalytic activity to LysoPA. Preferred MGATs do not have detectable activity to acylate LysoPA. MGAT can also have DGAT function, but it is primarily used as MGAT, i.e., its catalytic activity as MGAT is higher than its catalytic activity as DGAT when enzyme activity is expressed in nmol product / min / mg protein (see also Yen et al., 2002). There are three known classes of MGAT, referred to as MGAT1, MGAT2, and MGAT3. Examples of MGAT1, MGAT2, and MGAT3 peptides are described in WO2013 / 096993.

[0367] As used in this article, the "MGAT pathway" refers to a biosynthetic pathway that forms TAG, distinct from the Kennedy pathway, in which DAG is catalyzed by MGAT. sn- 1 MAG or preferred sn- 2. MAG is formed through acylation. DAG can then be used to form TAG or other lipids. WO2012 / 000026 first confirmed that plant leaf tissues can synthesize MAG from G-3-P, thus MAG is usable by exogenous MGAT expressed in leaf tissues. Second, MGAT from various sources can function in plant tissues, requiring successful interaction with other plant factors involved in lipid synthesis. Third, the DAG produced by exogenous MGAT activity is usable by plant DGAT or exogenous DGAT to produce TAG. MGAT and DGAT activities can be achieved by introducing constructs encoding enzymes (or candidate enzymes) into Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae The strain H1246 was used to determine TAG accumulation.

[0368] Certain motifs that have been shown to be important for catalytic activity in some DGAT2 are also conserved in MGAT acyltransferases. Of particular interest is a putative neutral lipid-binding domain with the conserved sequence FLXLXXXN (SEQ ID NO:14) (where each X is independently any nonproline amino acid and N is any nonpolar amino acid, and this sequence is located in the N-terminal transmembrane region), followed by a putative glycerol / phosphatidyltransferase domain. The FLXLXXXN motif (SEQ ID NO:14) is present in mouse DGAT2 (amino acids 81-88) and MGAT1 / 2 but not in yeast or plant DGAT2. It is important for the activity of mouse DGAT2. Other DGAT2 and / or MGAT1 / 2 sequence motifs include: 1. The highly conserved YFP tripeptide (SEQ ID NO:10), present in most DGAT2 peptides and also in MGAT1 and MGAT2, exists, for example, as amino acids 139-141 in mouse DGAT2. Mutating this motif in yeast DGAT2 through non-conservative substitution will cause the enzyme to lose function.

[0369] 2. The highly conserved HPHG tetrapeptide (SEQ ID NO: 11) in MGAT and in the DGAT2 sequence from animals and fungi, for example as amino acids 161-164 in mouse DGAT2, is of significant importance for catalytic activity, at least in yeast and mouse DGAT2. Plant DGAT2 acyltransferases, on the other hand, possess a conserved EPHS (SEQ ID NO: 12) sequence and therefore can accept conserved changes to the first and fourth amino acids.

[0370] 3. A relatively long conserved motif as part of a putative glycerophospholipid domain. An example of this motif is RXGFX(K / R)XAXXXGXXX(L / V)VPXXXFG(E / Q) (SEQ ID NO:13), which is present as amino acids 304-327 in mouse DGAT2. This motif is less conserved in the amino acid sequence than others, as can be expected from its length, but homologues can be identified through motif searches. The spacing between more conserved amino acids can vary; that is, there may be additional or fewer X amino acids within the motif compared to the sequence above.

[0371] An important component in the synthesis of glycerides from fatty acid esterification to ACP or CoA is the enzyme. snGlyceryl-3-phosphoacyltransferase (GPAT) is another polypeptide involved in the biosynthesis of nonpolar lipids. This enzyme participates in various metabolic pathways and physiological functions. It catalyzes the following reaction: G3P + fatty acyl-ACP or -CoA  LPA + free -ACP or -CoA. GPAT-catalyzed reactions occur in three different plant subcellular compartments: plastids, endoplasmic reticulum (ER), and mitochondria. These reactions are catalyzed by three different types of GPAT enzymes, using acyl-ACP as its natural acyl matrix in a soluble form located in the plastid matrix. Figure 1 PGPAT in the ER and two membrane-bound forms in the mitochondria using acyl-CoA and acyl-ACP as natural acyl donors, respectively (Chen et al., 2011).

[0372] As used herein, the term "glycerol-3-phosphoacyltransferase" (GPAT; EC 2.3.1.15) and its synonym "glycerol-3-phosphate" are used interchangeably. O "Acyltransferase" refers to a protein that acylates glycerol-3-phosphate (G-3-P) to form LysoPA and / or MAG. If GPAT also possesses phosphatase activity against LysoPA, the latter product is formed. If GPAT is ER-type GPAT (also known as "microsomal GPAT"), then the acyl-CoA group is also considered. sn If GPAT is a glycerol-3-phosphate 1-O-acyltransferase, the transferred acyl group comes from acyl-CoA, or if GPAT is plastid-type GPAT (PGPAT), the transferred acyl group comes from acyl-ACP. Therefore, the term "glycerol-3-phosphate acyltransferase activity" refers to G-3-P acylation forming LysoPA and / or MAG. The term "GPAT" encompasses the formation of G-3-P acylation to form LysoPA and / or MAG. sn- 1 LPA and / or sn- 2 LPA preferred sn- 2. An enzyme of LPA. Preferably, the GPAT that can be overexpressed in push modification is a membrane-bound GPAT that functions in the cellular ER, more preferably GPAT9, while the plastid GPAT downregulated in the prokaryotic pathway is a soluble GPAT (“plastid GPAT”). In a preferred embodiment, GPAT has phosphatase activity. In a most preferred embodiment, GPAT is an enzyme that produces sn- 2 MAG phosphatase activity sn- 2 GPAT.

[0373] As used in this article, the term " sn- 1-Glycerol-3-phosphoacyltransferase sn- 1. GPAT) refers to the preferential formation of a 1-acyl group by acylation of sn-glycerol-3-phosphate (G-3-P). sn -glycerol-3-phosphate ( sn- 1. LPA) protein. Therefore, the term " sn- "1-glycerol-3-phosphoacyltransferase activity" refers to the activity of glycerol-3-phosphoacyltransferase. sn -Glyceryl-3-phosphorylation forms 1-acyl- sn- Glyceryl-3-phosphate (GSP) sn- 1 LPA).

[0374] As used in this article, the term " sn -2-glycerol-3-phosphoacyltransferase" sn -2 GPAT) refers to the preferential formation of a 2-acyl group by acylation of sn-glycerol-3-phosphate (G-3-P). sn -glycerol-3-phosphate ( sn -2 LPA) protein. Therefore, the term " sn "-2-glycerol-3-phosphoacyltransferase activity" refers to the activity of glycerol-3-phosphoacyltransferase. sn -Glyceryl-3-phosphoacylation forms 2-acyl- sn- Glyceryl-3-phosphate (GSP) sn -2 LPA).

[0375] The GPAT family is a large family, and all known members contain two conserved domains: the plsC acyltransferase domain (PF01553; SEQ ID NO:15) and the HAD-like hydrolase (PF12710; SEQ ID NO:16) superfamily domain and its variants. Furthermore, at least in Arabidopsis, GPATs in subclasses GPAT4–GPAT8 contain an N-terminal region homologous to the phosphoserine phosphatase domain (PF00702; SEQ ID NO:17), and GPATs that produce MAG as a product can be identified by the presence of such homologous regions. Some GPATs endogenously expressed in leaf tissues contain the conserved amino acid sequence GDLVICPEGTTCREP (SEQ ID NO:18). Both GPAT4 and GPAT6 contain conserved residues known to be crucial for phosphatase activity, specifically conserved amino acids in motif I (DXDX[T / V][L / V]; SEQ ID NO:19) and motif III (K-[G / S][D / S]XXX[D / N]; SEQ ID NO:20) at the N-terminus (Yang et al., 2010).

[0376] Homologs of Arabidopsis GPAT4 (accession number NP_171667.1) and GPAT6 (NP_181346.1) include AAF02784.1 (Arabidopsis thaliana), AAL32544.1 (Arabidopsis thaliana), AAP03413.1 (rice), and ABK25381.1 (North American spruce). Picea sitchensis ), ACN34546.1 (maize), BAF00762.1 (Arabidopsis thaliana), BAH00933.1 (rice), EAY84189.1 (rice), EAY98245.1 (rice), EAZ21484.1 (rice), EEC71826.1 (rice), EEC76137.1 (rice), EEE59882.1 (rice), EFJ08963.1 (Selaginella tamariscina) Selaginella moellendorffii )), EFJ11200.1 (Selaginella tamariscina), NP_001044839.1 (Rice), NP_001045668.1 (Rice), NP_001147442.1 (Maize), NP_001149307.1 (Maize), NP_001168351.1 (Maize), AFH02724.1 (European rapeseed), NP_191950.2 (Arabidopsis thaliana), XP_001765001.1 (Bryophytum comosum), XP_001769671.1 (Bryophytum comosum), (Grape), XP_002275348.1 (Grape), XP_002276032.1 (Grape), XP_002279091.1 (Grape), XP_002309124.1 (Populus tomentosa), XP_002309276.1 (Populus tomentosa), XP_002322752.1 (Populus tomentosa), XP_002323563.1 (Populus tomentosa), XP_002439887.1 (Bicolor sorghum), XP_002458786.1 (Bicolor sorghum), XP_002463916.1 (Bicolor sorghum), XP_002464630.1 (Bicolor sorghum), XP_002511873.1 (Castor bean), XP_002517438.1 (Castor bean), XP_002520171.1 (Castor bean), ACT32032.1 (Tung oil tung), NP_001051189.1 (Rice), AFH02725.1 (European rapeseed), XP_002320138.1 (hairy poplar), XP_002451377.1 (bicolor sorghum), XP_002531350.1 (castor bean) and XP_002889361.1 (fiddle-leaved Arabidopsis).

[0377] The soluble polymorphs GPAT (PGPAT, also known as ATS1 in Arabidopsis thaliana) have been purified, and the genes encoding them have been cloned from several plant species such as peas. Pisumsativum Login ID: P30706.1), Spinach ( Spinacia oleracea Login ID: Q43869.1), squash ( Cucurbita moschate Login ID: P10349.1), cucumber ( Cucumis sativus (Accession number: Q39639.1) and Arabidopsis thaliana (accession number: Q43307.2). The soluble plastid GPAT is the first guaranteed step in the prokaryotic pathway for glycerol synthesis and is operable only in plastids (accession number: Q39639.1). Figure 1 The so-called prokaryotic pathway is located only in plant plastids, and the assembly of the DAG is used to contain glycerol in the backbone. sn Synthesis of galactosides (MGDG and DGMG) of C16:3 fatty acids esterified at the -2 position.

[0378] Conserved motifs and / or residues can be used as sequence-based diagnostics for GPAT enzymes. Alternatively, more rigorous function-based assays can be employed. Such assays involve, for example, supplying labeled glycerol-3-phosphate to cells or microsomes, followed by quantifying the level of the labeled product using thin-layer chromatography or similar techniques. GPAT activity results in the production of labeled LPA, while GPAT / phosphatase activity results in the production of labeled MAG.

[0379] As used herein, the term "lysophosphatidyl acyltransferase" (LPAAT; EC 2.3.1.51) and its synonyms "1-acyl-glycerol-3-phosphoacyltransferase", "acyl-CoA:1-acyl- sn "-glycerol-3-phosphate 2-O-acyltransferase" and "1-acylglycerol-3-phosphate" O "Acyltransferase" refers to a protein that acylates lysophosphatidic acid (LPA) to form phosphatidic acid (PA). If LPAAT is ER-type LPAAT, the transferred acyl group comes from acyl-CoA; or if LPAAT is plastid-type LPAAT (PLPAAT), the transferred acyl group comes from acyl-ACP. Therefore, the term "lysophosphatidic acid acyltransferase activity" refers to the acylation of LPA to form PA.

[0380] Oil-coated peptides Plant seeds and pollen accumulate TAGs in subcellular structures called oil bodies, typically 0.5–2.5 μm in diameter. Lipid droplets, also referred to as “oil bodies” as used herein, are lipid-rich organelles used for storing or exchanging natural lipids, primarily TAGs. Lipid droplet sizes can vary considerably, from about 20 nm to 100 μm. These organelles have a TAG core surrounded by a phospholipid monolayer containing several embedded proteins involved in lipid metabolism and storage, as well as lipid transport to other membranes; if the oil bodies originate from plant seeds or flower tissues, they include olein proteins (Jolivet et al., 2004). They typically consist of 0.5–3.5% protein, with the remainder being lipids. They are among the least dense organelles in most cells and are therefore easily separated by flotation centrifugation. Olein proteins represent the most abundant (at least 80%) proteins in the oil body membranes from seeds.

[0381] As used herein, the term "oil protein" refers to amphiphilic proteins present in the oil body membrane of the storage tissue of seeds (see, e.g., Huang, 1996; Lin et al., 2005; Capuano et al., 2007; Lui et al., 2009; Shimada and Hara-Nishimura, 2010) and artificially produced variants (see, e.g., WO2011 / 053169 and WO2011 / 127118).

[0382] Oily proteins have low... M r (15-26,000), corresponding to approximately 140-230 amino acids, which allows them to tightly adhere to the surface of the oil body. Within each seed species, there are usually two or more different... M r Oily proteins. Each oily protein molecule contains a relatively hydrophilic variable N-terminal domain (e.g., about 48 amino acid residues), a completely hydrophobic central domain (e.g., about 70-80 amino acid residues), which is particularly rich in aliphatic amino acids such as alanine, glycine, leucine, isoleucine, and valine, and an amphiphilic α-helical domain of about 30-40 amino acid residues located at or near the C-terminus. The central hydrophobic domain is typically contained within a proline motif of about 12 residues at its center. Generally, the central segment of hydrophobic residues is inserted into the lipid core, and the amphiphilic N-terminus and / or amphiphilic C-terminus are located on the surface of the oil body, with positively charged residues embedded in a phospholipid monolayer and negatively charged residues exposed to the outside.

[0383] As used herein, the term "olein" encompasses polyolein, which comprises multiple olein polypeptides fused together in a head-to-tail manner to form a single polypeptide (WO2007 / 045019), such as 2x, 4x, or 6x olein peptides; and encompasses oleobody caloproteins, which bind calcium and are the smallest protein component of the protein that coats the oil body in a seed (Froissard et al., 2009); and encompasses oleobody sterol proteins, which bind sterols (WO2011 / 053169). However, the majority (at least 80%) of olein proteins in general oil bodies are not oleobody caloproteins and / or oleobody sterol proteins. The term "olein" also encompasses artificially modified olein polypeptides, such olein proteins in which one or more amino acids of a natural olein protein are artificially replaced by cysteine ​​residues, as described in WO2011 / 053169. Typically, 4-8 residues are artificially replaced, preferably 6 residues, but up to 2-14 residues can be substituted. Preferably, the amphiphilic N-terminal and C-terminal domains contain cysteine ​​substitutions. This modification increases the cross-linking ability of the oleoprotein and enhances its thermal stability and / or resistance to protease degradation.

[0384] Numerous oleoprotein sequences and the nucleotide sequences encoding them are known from many different plant species. Examples include, but are not limited to, oleoproteins from Arabidopsis thaliana, rapeseed, maize, rice, peanut, castor bean, soybean, flax, grape, cabbage, cotton, sunflower, sorghum, and barley. Examples of oleoproteins (with their accession numbers) include rapeseed oleoprotein (CAA57545.1; SEQ ID NO:95), rapeseed oleoprotein S1-1 (ACG69504.1; SEQ ID NO:96), rapeseed oleoprotein S2-1 (ACG69503.1; SEQ ID NO:97), rapeseed oleoprotein S3-1 (ACG69513.1; SEQ ID NO:98), and rapeseed oleoprotein S4-1 (ACG69507.1; SEQ ID NO:99). Europe rapeseedPeanut olein S5-1 (ACG69511.1; SEQ ID NO:100), peanut olein 1 (AAZ20276.1; SEQ ID NO:101), peanut olein 2 (AAU21500.1; SEQ ID NO:102), peanut olein 3 (AAU21501.1; SEQ ID NO:103), peanut olein 5 (ABC96763.1; SEQ ID NO:104), castor oil protein 1 (EEF40948.1; SEQ ID NO:105), castor oil protein 2 (EEF51616.1; SEQ ID NO:106), soybean olein isotype a (P29530.2; SEQ ID NO:107), soybean olein isotype b (P29531.1; SEQ ID NO:107). SEQ ID NO:108), low molecular weight isotype of linseed oil protein (ABB01622.1; SEQ ID NO:109), high molecular weight isotype of linseed oil protein (ABB01624.1; SEQ ID NO:110), sunflower oil protein (CAA44224.1; SEQ ID NO:111), corn oil protein (NP_001105338.1; SEQ ID NO:112), rapeseed oil steroid protein (ABM30178.1; SEQ ID NO:113), rapeseed oil steroid protein SLO1-1 (ACG69522.1; SEQ ID NO:114), rapeseed oil steroid protein SLO2-1 (ACG69525.1; SEQ ID NO:115), sesame oil steroid protein (AAL13315.1; SEQ ID NO:109), sesame oil steroid protein (AAL13315.1; SEQ ID NO:115), sesame oil steroid protein (AAL13315.1; SEQ ID NO:115). (SEQ ID NO:116), corn oil body sterol protein (NP_001152614.1; SEQ ID NO:117), rapeseed oil body calcinin CLO-1 (ACG69529.1; SEQ ID NO:118), rapeseed oil body calcinin CLO-3 (ACG69527.1; SEQ ID NO:119), sesame oil body calcinin (AAF13743.1; SEQ ID NO:120), corn oil body calcinin (NP_001151906.1; SEQ ID NO:121), and soybean oil body calcinin (AAB71227). Other lipid-encapsulated peptides with equivalent functions are plastoglobulin and MLDP peptide (WO2011 / 127118).

[0385] In one embodiment, unless otherwise stated, the exogenous polynucleotide of the present invention encoding oleic protein comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 95-112, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 95-112. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0386] In one embodiment, unless otherwise stated, the exogenous polynucleotide of the present invention encoding an oleobody sterol protein comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 113-117, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 113-117. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0387] As used herein, “lipid droplet-associated protein” or “LDAP” refers to a polypeptide associated with lipid droplets in a plant, found in tissues or organs other than seeds, anthers, and pollen (such as fruit tissues including pericarps and mesocarps). LDAP can be associated with oil bodies in seeds, anthers, or pollen, as well as in tissues or organs other than seeds, anthers, or pollen. They are distinct from olein proteins, which are polypeptides associated with the surface of lipid droplets in seed tissues, anthers, and pollen. LDAP as used herein includes naturally occurring LDAP polypeptides in plant tissues as well as artificially generated amino acid sequence variants. The function of such variants can be tested as exemplified in Example 15.

[0388] Horn et al. (2013) identified two LDAP genes expressed in avocado pericarps. The encoded avocado LDAP1 and LDAP2 peptides were 62% identical in amino acids and showed homology with peptides encoded by Arabidopsis thaliana At3g05500 and SRPP-like proteins from rubber trees. Gidda et al. (2013) identified [a specific gene expression] in oil palm ([a specific gene expression]). Elaeis guineensisThree LDAP genes expressed in the pericarp but not in the kernel were identified, and it was concluded that LDAP genes are plant-specific and conserved across all plant species. LDAP peptides may contain additional structural domains (Gidda et al., (2013)). Genes encoding LDAP are generally upregulated in non-seed tissues rich in lipids and are thus identifiable but not considered to be expressed in all non-seed cells that produce oil (including those used for short-term storage). Horn et al. (2013) presented a phylogenetic tree of SRPP-like proteins in plants. Exemplary LDAP peptides are described in Example 15 herein. Homologues of LDAP in other plant species can be readily identified by those skilled in the art.

[0389] In one embodiment, unless otherwise stated, the exogenous polynucleotide of the present invention encoding LDAP comprises one or more of the following: i) A nucleotide encoding a polypeptide or a biologically active fragment thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 237, 239 or 241, or a polypeptide whose amino acid sequence is at least 30% identical to any one or more of SEQ ID NO: 237, 239 or 241. ii) Nucleotides whose sequence is at least 30% identical to that in i), and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0390] As used herein, the term "peptide involved in starch biosynthesis" refers to any polypeptide that, when downregulated to below normal (wild-type) levels in cells, results in reduced starch synthesis and consequently, reduced starch levels. An example of such a polypeptide is AGPase.

[0391] As used herein, the term "ADP-glucose phosphorylase" or "AGPase" refers to an enzyme that regulates starch biosynthesis, catalyzing the conversion of glucose-1-phosphate and ATP to ADP-glucose (which serves as a building block of starch polymers). The active form of AGPase consists of two large subunits and two small subunits.

[0392] In plants, ADPase exists primarily as a tetramer, composed of two large subunits and two small subunits. While these subunits differ in their catalytic and regulatory functions depending on the species (Kuhn et al., 2009), the small subunit generally exhibits catalytic activity in plants. The small subunit has a molecular weight of approximately 50-55 kDa, while the large subunit has a molecular weight of approximately 55-60 kDa. This plant enzyme is strongly activated by 3-phosphoglycerate (PGA), a product of carbon dioxide fixation; in the absence of PGA, the enzyme exhibits only about 3% of its activity. Plant ADPase is also strongly inhibited by inorganic phosphate (Pi). In contrast, bacterial and algal ADPase exists as a 50 kDa homotetramer. Algal enzymes (similar to their plant counterparts) are activated by PGA and inhibited by Pi, while bacterial enzymes are activated by fructose-1,6-bisphosphate (FBP) and inhibited by AMP and Pi.

[0393] TAG lipase and β-oxidase As used herein, the term "peptide involved in lipid degradation and / or reduction of lipid content" refers to any peptide that causes lipid catabolism, and whose downregulation in cells to below normal (wild-type) levels results in increased levels of oils (such as fatty acids and / or TAGs) in those cells (preferably cells of the vegetative parts of plants, tubers, roots, or seeds). Examples of such peptides include, but are not limited to, lipases, or lipases such as the CTi58 (comparative genetic identifier-58-like) peptide, the SUGAR-DEPENDENT 1 (SDP1) triacylglycerol lipase (see, for example, Kelly et al., 2011), and the lipases described in WO2009 / 027335.

[0394] As used herein, the term "TAG lipase" (EC.3.1.1.3) refers to a protein that hydrolyzes TAG into one or more fatty acids and any one of DAG, MAG, or glycerol. Therefore, the term "TAG lipase activity" refers to the hydrolysis of TAG to glycerol and fatty acids.

[0395] As used herein, the term "CGi58" refers to the soluble acyl-CoA-dependent lysophosphatidyl acyltransferase encoded by the At4g24160 gene in Arabidopsis thaliana and its homologues in other plants, as well as "Ict1p" and its homologues in yeast. Plant genes, such as those from the Arabidopsis locus At4g24160, are expressed as two alternative transcripts: a longer full-length isoform (At4g24160.1) and a shorter isoform lacking the 3' end (At4g24160.2) (see James et al., 2010; Ghosh et al., 2009; US 201000221400). Both mRNAs encode proteins that are homologous to the human CGI58 protein and other orthologous members of this α / β hydrolase family (ABHD). In one embodiment, the CGI58 (At4g24160) protein contains three conserved motifs across plant species: the GXSXG lipase motif (SEQ ID NO:127), the HX(4)D acyltransferase motif (SEQ ID NO:128), and a possible lipid-binding motif, VX(3)HGF (SEQ ID NO:129). The human CGI-58 protein possesses lysophosphatidyl acyltransferase (LPAAT) activity but not lipase activity. Conversely, plant and yeast proteins possess the standard lipase sequence motif GXSXG (SEQ ID NO:127), which is absent in vertebrate (human, shrub, and zebrafish) proteins, and exhibit both lipase and phospholipase activity (Ghoshet al., 2009). While plant and yeast CGI58 proteins appear to possess detectable amounts of TAG lipase and phospholipase A activity in addition to LPAAT activity, this is not the case with the human protein.

[0396] Homologous in Arabidopsis CGI-58 Genetic disruption leads to the accumulation of natural lipid droplets in mature leaves. (Source: [Original Source Name]) cgi-58 Mass spectrometry of isolated lipid droplets from loss-of-function mutants revealed that they contained triacylglycerols with conventional leaf-specific fatty acids. Maturation cgi-58 The plant's leaves showed a significantly elevated absolute triglyceride level, more than 10 times higher than that in the wild plant. cgi-58 The lipid levels in the oil-storing seeds of loss-of-function plants were unchanged, and unlike the mutations in β-oxidation, this... cgi-58 Seeds germinate and grow normally without the need for sucrose (James et al., 2010).

[0397] Examples of nucleotides encoding the CGi58 polypeptide include those from: Arabidopsis thaliana (NM_118548.1 encoding NP_194147.2; SEQ ID NO:130), Brachypodium distichum (XP_003578450.1; SEQ ID NO:131), soybean (XM_003523638.1 encoding XP_003523590.1; SEQ ID NO:132), maize (NM_001155541.1 encoding NP_001149013.1; SEQ ID NO:133), bicolor sorghum (XM_002460538.1 encoding XP_002460493.1; SEQ ID NO:134), and castor bean (XM_002510485.1 encoding XP_002510439.1; SEQ ID NO:130). NO:135), alfalfa (coded XP_003603733.1 and XM_003603685.1; SEQ ID NO:136), and rice (coded EAZ09782.1).

[0398] In one embodiment, the genetic modification of the present invention downregulates the endogenous generation of CTi58, wherein CTi58 is encoded by one or more of the following: i) Nucleotides containing a sequence as shown in any one of SEQ ID NO:130-136, ii) Nucleotides comprising at least 30% identical sequences to any one or more of those in SEQ ID NO:130-136, and iii) Hybridize to one or both of the polynucleotides in i) or ii) under strict conditions.

[0399] Other lipases with lipase activity against TAG include SUGAR-DEPENDENT1 triglyceride lipase (SDP1, see, for example, Eastmond et al., 2006; Kelly et al., 2011) and SDP1-like peptides found in plant species, yeast (TGL4 peptide), and animal cells, which are involved in the breakdown of stored TAG. SDP1 and SDP1-like peptides appear to be responsible for initiating TAG breakdown in seeds after germination (Eastmond et al., 2006). In the absence of exogenous WRI1 and DGAT1, SDP1Mutant plants exhibit elevated levels of PUFA in their TAGs. As used herein, “SDP1 polypeptide” includes SDP1 polypeptide, SDP1-like polypeptides, and their homologues in plant species. SDP1 and SDP1-like polypeptides in plants are 800–910 amino acid residues in length, possess a patatin-like acyl hydrolase domain that can be associated with the oil body surface, and preferentially hydrolyze TAGs to DAGs or MAGs. SDP1 is believed to play a crucial role in the hydrolysis of TAGs. sn The acyl group at the -2 position has a preference. Arabidopsis contains at least three genes encoding SDP1 lipase, namely... SDP1 (Accession number NP_196024, nucleotide sequence SEQ ID NO:163 and homologues in other species) SDP1L (Accession number NM_202720 and its congeners in other species, Kelly et al., 2011) and ATGLL (At1g33270) (Eastmond et al, 2006). Particular attention is paid to reducing gene activity expressed in vegetative tissues of plants, such as leaves, stems, and roots. SDP1 Genes. Therefore, the level of nonpolar lipids in nutrient plant parts can be increased by reducing the activity of the SDP1 peptide, for example, through mutations in the endogenous gene encoding the SDP1 peptide or by introducing a gene encoding a gene that reduces endogenous activity. SDP1 Exogenous genes that silence gene expression RNA molecules. Such reduction is particularly beneficial in tuber crops such as sugar beets and potatoes, and in "high-sucrose" plants such as sugarcane and sugar beets.

[0400] The genes encoding SDP1 homologs (including SDP1-like homologs) in selected plant species can be compared with known homologs. SDP1 Gene sequence homology is readily identified. Known SDP1 nucleotide or amino acid sequences include accession numbers: GN078290 (SEQ ID NO:164), GN078281, and GN078283 in *Brassica napus*; and *Capsella bursa-pastoris*. Capsella rubella ), XP_006287072; Cocoa ( Theobroma cacao ), XP_007028574.1; Populus hairy-fruited, XP_002308909 (SEQ ID NO:166); Peach tree ( Prunus persica ), XP_007203312; Plum Blossom ( Prunus mume ), XP_008240737; Apple ( Malus domestica ), XP_008373034; castor bean, XP_002530081; tribulus terrestris, XP_003591425 (SEQ ID NO:167); tomato ( Solanum lycopersicum), XP_004249208; common beans, XP_007162133; soybeans, XP_003554141 (SEQ ID NO:168); potatoes, XP_006351284; soybeans, XP_003521151; chickpeas ( Cicer arietinum ), XP_004493431; cucumber, XP_004142709; melon, XP_008457586; jatropha, KDP26217; grape, CBI3074; rice, Japonica rice group BAB61223; rice, Indica rice group EAY75912; rice, Japonica rice group NP_001044325; bicolor sorghum, XP_002458531 (SEQ ID NO:169); two-spike short-stalked grass, XP_003567139 (SEQ ID NO:165); maize, AFW85009; barley, BAK03290 (SEQ ID NO:172); jointed goatgrass ( Aegilops tauschii ), EMT32802; bicolor sorghum, XP_002463665; corn, NP_001168677 (SEQ ID NO:170); barley, BAK01155; jointed barley, EMT02623; Urartu wheat ( Triticum urartu EMS67257; *Moss sclerotium*, XP_001758169 (SEQ ID NO:171). The preferred SDP1 sequence used in the genetic construct for suppressing endogenous gene expression is derived from the cDNA corresponding to the gene most highly expressed in cells, vegetative plant parts, or seeds (whichever is to be modified). If it is desired to reduce the activity of all members of the gene family in the species, it is preferred to use cDNA corresponding to all members of the plant species. SDP1 Highly conserved nucleotide sequences between the cDNA of genes.

[0401] In one embodiment, the genetic modification of the present invention downregulates the endogenous generation of SDP1, wherein SDP1 is encoded by one or more of the following: i) Nucleotides with sequences as shown in any one of SEQ ID NO:163-174, ii) Nucleotides whose sequence is at least 30% identical to any one or more sequences shown in SEQ ID NO:163-174, and iii) Hybridize to one or both of the nucleotide sequences in i) or ii) under strict conditions.

[0402] As illustrated in the examples, a reduction in the expression and / or activity of SDP1 TAG lipase in plant leaves significantly increases TAG content, both in terms of the amount of TAG accumulated during plant development and at an earlier time, against the backdrop of co-expression of the transcription factor WRI1 and fatty acyltransferase. Specifically, an increase was observed in plants prior to flowering and reached up to approximately 70% on a weight basis (%dry weight) at the onset of senescence. This increase is relative to TAG levels observed in the leaves of corresponding plants converted with exogenous polynucleotides encoding WRI1 and fatty acyltransferases but lacking modifications that reduce SDP1 expression and / or activity.

[0403] Reducing the expression of other TAG catabolism genes in plant parts can also increase TAG content, such as those encoding acyl-CoA oxidase. ACX Genes such as Acx1 (Homologous to At4g16760 and other plant species) or Acx2 (At5g65110 and its homologues in other plant species) gene. Another polypeptide involved in lipid catabolism is PXA1, which is a peroxisome ATP-binding cassette transporter required for the input of β-oxidized fatty acids (Zolman et al. 2001).

[0404] Fatty acids are exported from plastids. As used herein, the term "polypeptide that increases fatty acid export from cytoplasm" refers to any polypeptide that assists in the transfer of fatty acids from within the plastid (a cell containing plastids, such as the cells of vegetative parts, tubers, roots, or plant seeds) to outside the plastid (which can be any other part of the cell, such as the endoplasmic reticulum (ER)). Examples of such polypeptides include, but are not limited to, C16 or C18 fatty acid thioesterases such as the FATA or FATB polypeptides, C8-C14 fatty acid thioesterases (which are also FATB polypeptides), fatty acid transporters such as the ABCA9 polypeptide, or long-chain acyl-CoA synthases (LACS).

[0405] As used herein, the term "fatty acid thioesterase" or "FAT" refers to an enzyme that catalyzes the hydrolysis of the thioester bond between the acyl moiety of an acyl-ACP and the acyl carrier protein (ACP), resulting in the release of free fatty acids. These enzymes typically function in the plastid of organisms that synthesize fatty acids de novo. As used herein, the term "C16 or C18 fatty acid thioesterase" refers to an enzyme that catalyzes the hydrolysis of the thioester bond between the C16 and / or C18 acyl moiety of an acyl-ACP and the ACP, resulting in the release of free C16 or C18 fatty acids. Upon exiting the plastid, the free fatty acids are then re-esterified to CoA within the plastid membrane. The substrate specificity of fatty acid thioesterases (FATs) in plastids is involved in determining the chain length and saturation profile of fatty acids exported from the plastid. Based on their substrate specificity and nucleotide sequence, FATs can be classified into two classes, FATA and FATB (EC 3.1.2.14) (Jones et al., 1995). FATA peptides preferably use oleoyl-ACP as a substrate, while FATB peptides exhibit higher activity towards saturated acyl-ACP of different chain lengths, such as acting on palmitoyl-ACP to produce free palmitic acid. Examples of FATA peptides that can be used in this invention include, but are not limited to, those from Arabidopsis thaliana (NP_189147), peanut (GU324446), sunflower (AAL79361), and safflower (AAA33020). Morus notabilis FATA polypeptides from (XP_010104178.1), rapeseed (CDX77369.1), castor bean (XP_002532744.1), and flaxseed (AFQ60946.1). Examples of FATB polypeptides that can be used in this invention include, but are not limited to, those from maize (AIL28766), rapeseed (ABH11710), sunflower (AAX19387), Arabidopsis thaliana (AEE28300), California laurel (AAC49001), peanut (AFR54500), castor bean (EEF47013), and short-stalked sedge (ABL85052.1).

[0406] One subclass of FATB peptides are fatty acid thioesterases that are hydrolyzed by the C8-C14 saturated acyl moieties of ACP linked by thioester bonds. These enzymes are also known as medium-chain fatty acid (MCFA) thioesterases or MC-FAT enzymes. These enzymes can also exhibit thioesterase activity towards C16-ACP; in fact, they can have greater thioesterase activity towards C16 acyl-ACP substrates than towards MCFA-ACP substrates, although they are considered MCFA thioesterases herein if they produce at least 0.5% MCFA in the total fatty acid content when exogenously expressed in plant cells. Examples of MCFA thioesterases are given in Example 9 of this document.

[0407] As used herein, the term "fatty acid transporter" refers to polypeptides present in the plastid membrane that participate in the active transfer of fatty acids from the plastid to the outside of the plastid. Examples of ABCA9 (ABC transporter A family member 9) polypeptides that can be used in this invention include, but are not limited to, those from Arabidopsis thaliana (Q9FLT5), Capsella bursa-pastoris (XP_006279962.1), Arabis alpine (KFK27923.1), shepherd's purse (XP_010457652.1), rapeseed (CDY23040.1), and turnip ( Brassica rapa Those ABCA9 polypeptides in (XP_009136512.1).

[0408] As used herein, the term "acyl-CoA synthase" or "ACS" (EC 6.2.1.3) refers to a polypeptide belonging to the ligase family that catalyzes a two-step process via an adenylate intermediate to form fatty acyl-CoA, using non-esterified fatty acids, CoA, and ATP as substrates to produce acyl-CoA esters, AMP, and pyrophosphate as products. As used herein, the term "long-chain acyl-CoA synthase" (LACS) refers to an ACS that is active against at least C18 free fatty acid substrates, although it can have broader activity against any C14-C20 free fatty acids. Endogenous plastid LACS enzymes are located in the outer membrane of the plastid and function together with fatty acid thioesterases for the export of fatty acids from the plastid (Schnurr et al., 2002). In Arabidopsis, at least nine LACS genes have been identified (Shockey et al., 2002). Preferred LACS polypeptides are of the LACS9 subclass, which is the major plastid LACS in Arabidopsis. Examples of LACS peptides that can be used in this invention include, but are not limited to, those from Arabidopsis thaliana (Q9CAP8), Capsella bursa-pastoris (XP_010416710.1), Capsella bursa-pastoris (XP_006301059.1), rapeseed (CDX79212.1), turnip (XP_009104618.1), and Gossypium raymondii (…). Gossypium raimondii The LACS peptides of (XP_012450538.1) and grape (XP_002285853.1). Homologues of the peptides mentioned above in other species can be readily identified by those skilled in the art.

[0409] Peptides involved in the formation of diacylglycerols (DAG) in plastids The level of nonpolar lipids in nutrient plant parts can also be increased, for example, by reducing the activity of polypeptides involved in the production of diacylglycerols (DAG) in plastids in plant parts, for example, by mutating endogenous genes encoding such polypeptides or by introducing exogenous genes encoding silent RNA molecules that reduce the expression of target genes involved in the production of diacylglycerols (DAG) in plastids.

[0410] As used herein, the term "peptide involved in diacylglycerol (DAG) generation in plastids" refers to any polypeptide in plastids (in cells containing plastids, such as those of vegetative parts, tubers, roots, or plant seeds) that is directly involved in diacylglycerol synthesis. Examples of such polypeptides include, but are not limited to, plastid GPAT, plastid LPAAT, or plastid PAP.

[0411] GPAT is described elsewhere in this document. Examples of plasmid GPAT peptides that can be targeted for downregulation in this invention include, but are not limited to, those from Arabidopsis thaliana (BAA00575), shepherd's purse (XP_006306544.1), flaxseed (010499766.1), rapeseed (CDY43010.1), turnip (XP_009145198.1), sunflower (ADV16382.1), and mandarin orange (…). Citrus unshiu Those plastid GPAT polypeptides of (BAB79529.1). Homologues in other species can be readily identified by those skilled in the art.

[0412] LPAAT is described elsewhere in this document. Those skilled in the art will understand that plastid LPAAT targeted for downregulation to reduce DAG synthesis in plastids is not endogenous LPAAT that functions outside the plastid, such as those in the ER, which, for example, can be used to generate TAGs containing medium-chain fatty acids as described herein. Examples of plastid LPAAT peptides that can be targeted for downregulation in this invention include, but are not limited to, those from rapeseed (ABQ42862), turnip (XP_009137939.1), Arabidopsis (NP_194787.2), capernaum (XP_010432969.1), soybean (XP_006592638.1), and potato (XP_006343651.1). Homologues of the peptides mentioned above in other species can be readily identified by those skilled in the art.

[0413] As used herein, the term "phosphatidylphosphatase" (PAP) (EC 3.1.3.4) refers to the hydrolysis of 3- sn-The phosphate group on the phosphatidylcholine produces a protein containing 1,2-diacyl-sn-glycerol (DAG) and phosphate. Examples of plastid PAP peptides that can be targeted for downregulation in this invention include, but are not limited to, those from Arabidopsis thaliana (Q6NLA5), shepherd's purse (XP_006288605.1), flaxseed (XP_010452170.1), rapeseed (CDY10405.1), turnip (XP_009122733.1), soybean (XP_003542504.1), and potato (XP_006361792.1). Homologues of the peptides mentioned above in other species can be readily identified by those skilled in the art.

[0414] Fatty acid input plasmid The level of nonpolar lipids in nutrient-rich plant parts can also be increased by reducing the activity of TGD peptides in plant parts, for example, through mutations in endogenous genes encoding TGD peptides or by introducing genes that reduce the activity of endogenous TGD peptides. TGD Exogenous genes of silenced RNA molecules in gene expression. As used in this paper, “trigalactosyldiglycerol (TGD) polypeptide” is a polypeptide involved in lipid transport from the ER to the chloroplast (Xu et al., 2010) and in the formation of protein complexes with lipid permease function. Four such polypeptides are known to form or be associated with TGD permease: TGD-1 (accession number At1g19800 and homologues in other species), TGD-2 (accession number At2g20320 and homologues in other species), TGD-3 (accession number NM-105215 and homologues in other species), and TGD-4 (accession number At3g06960 and homologues in other species) (US20120237949). TGD-1, -2, and -3 polypeptides are considered to be components of ATP-binding cassette (ABC) transporters associated with the inner membrane of the chloroplast. TGD-2 and TGD-4 peptides bind to phosphatidic acid, while the TGD-3 peptide functions as an ATPase in the chloroplast stroma. As used in this article, the "endogenous TGD gene" is the gene encoding the TGD peptide in plants. In Arabidopsis thaliana... TGD-1 Mutations in the gene lead to the accumulation of triglycerides, oligogalactosides and phosphatidic acid (PA) (Xu et al., 2005). TGD Gene or SDP1 Mutations in genes, or in fact, in any desired gene in a plant, can be introduced in a site-specific manner using techniques such as artificial zinc finger nucleases (ZFNs), TAL effectors (TALENs), or CRISPR technologies (using Cas9-type nucleases). Preferred exogenous genes encoding silent RNA are those encoding double-stranded RNA molecules such as hairpin RNA or precursors of artificial microRNAs.

[0415] Fatty acid modifying enzymes As used herein, the term "FAD2" refers to a membrane-bound Δ-12 fatty acid desaturase, which desaturates oleic acid (C18:1...). Δ9 Desaturation to produce linoleic acid (C18: 2) Δ9,12 ).

[0416] As used herein, the term "cyclooxygenase" or "fatty acid cyclooxygenase" refers to an enzyme that introduces an epoxy group into a fatty acid, resulting in the production of an epoxy-containing fatty acid. In a preferred embodiment, an epoxy group is introduced onto the 12th carbon of the fatty acid chain, in which case the cyclooxygenase is a Δ12-cyclooxygenase, particularly for C16 or C18 fatty acid chains. The cyclooxygenase may be a Δ9-cyclooxygenase, a Δ15-cyclooxygenase, or, as known in the art, act at different positions in the acyl chain. The cyclooxygenase may be of the P450 class. Preferred cyclooxygenases are monooxygenases as described in WO98 / 46762. Many cyclooxygenases or putative cyclooxygenases have been cloned and are known in the art. Other examples of cyclooxygenases include proteins containing the amino acid sequence provided in SEQ ID NO:21 of WO 2009 / 129582, derived from *Codonopsis pilosula* (a type of cyclooxygenase). ...

Claims

1. A method for producing extracted lipids, the method comprising the following steps: i) Obtaining a genetically modified plant or a part thereof, which contains a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following: d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts, and wherein, prior to flowering, the cells of the plant's leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids. (ii) Extracting lipids from the plant or its parts, and (iii) Recover the extracted lipids, This produces the extracted lipids.

2. The method of claim 1, wherein the transgenic plant or a portion thereof further comprises one or more of the following: a) The fifth exogenous polynucleotide, which encodes an oil-body-coated (OBC) polypeptide. b) A second genetic modification that, when compared to cells of the corresponding leaf or stem lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the transport of fatty acids into the plastids of the leaf or stem cells, and c) A third genetic modification that, when compared to cells of the corresponding leaf or stem lacking the third genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in plastids.

3. The method of claim 1, wherein one or more or all of the promoters are expressed at a higher level in the leaves or stems of the transgenic plant than in the seeds of the plant.

4. The method of claim 1, wherein one or more or all of the following characteristics are applicable: i) The cells of the leaf or stem have increased total fatty acid synthesis relative to the corresponding leaf or stem cells lacking the first exogenous polynucleotide, or decreased total fatty acid catabolism relative to the corresponding leaf or stem cells lacking the first exogenous polynucleotide, or both, thereby having elevated levels of total fatty acids relative to the corresponding leaf or stem cells lacking the first exogenous polynucleotide. ii) Relative to cells of a corresponding leaf or stem having a first exogenous polynucleotide and lacking an exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids, the cells of said leaf or stem have increased expression and / or activity of fatty acyltransferases that catalyze the synthesis of TAG, DAG, or MAG, preferably TAG. iii) Relative leaf or stem cells having a first exogenous polynucleotide and lacking genetic modifications that downregulate the endogenous generation and / or activity of polypeptides involved in the production of diacylglycerol (DAG) in plastids of plants or parts thereof, said leaf or stem cells having reduced production of lysophosphatidic acid (LPA) from acyl-ACP and G3P in their plastids. iv) Compared to the corresponding leaf or stem cells lacking the exogenous polynucleotide and / or genetic modification, the leaf or stem cells have an altered C16:3-C18:3 fatty acid ratio in their total fatty acid content and / or their galactolipid content, preferably a reduced ratio. v) The cells of the leaf or stem contain at least 8%, at least 10%, at least 11%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, 8%-75%, 10%-75%, 11%-75%, 15%-75%, 20%-75%, 30%-75%, 40%-75%, 50%-75%, 60%-75%, or 25%-50% (w / w dry weight) of total nonpolar lipids. vi) The cells of the leaves or stems contain at least 8%, at least 10%, at least 11%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, 8%-75%, 10%-75%, 11%-75%, 15%-75%, 20%-75%, 30%-75%, 40%-75%, 50%-75%, 60%-75%, or 25%-50% (w / w dry weight) of TAG. vii) The transcription factor polypeptides are selected from Wrinkled 1 (WRI1), Leafy Cotyledon 1 (LEC1), LEC1-like, Leafy Cotyledon 2 (LEC2), BABY BOOM (BBM), FUS3, ABI3, ABI4, ABI5, Dof4, and Dof11. viii) Oleic acid comprises at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%), or 20%-65%, of the total fatty acid content in the cells of the said leaf or stem. (ix) The nonpolar lipids in the cells of the leaf or stem comprise fatty acids, said fatty acids comprising hydroxyl groups, epoxy groups, cyclopropane groups, two-carbon bonds, three-carbon bonds, conjugated double bonds, branched chains such as methylated or hydroxylated branches, or combinations of two or more of these, or any two, three, four, five or six of the aforementioned groups, bonds or branches. x) The nonpolar lipids in the cells of the leaf or stem comprise one or more polyunsaturated fatty acids selected from eicosapentaenoic acid (EDA), arachidonic acid (ARA), octadecanoic acid (SDA), eicosatrienoic acid (ETE), eicosapentaenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or combinations of two or more of these. xi) One or more promoters are expressed at higher levels in the leaves or stems of the plant relative to the seeds of the plant. xii) One or more promoters are selected from tissue-specific promoters such as leaf and / or stem-specific promoters, developmental regulatory promoters such as senescence-specific promoters such as the SAG12 promoter, inducible promoters, or circadian rhythm regulatory promoters. xiii) The cells of the leaf or stem contain a total fatty acid content and optionally present exogenous polynucleotides encoding LPAAT, wherein the total fatty acid content comprises medium-chain fatty acids, preferably C12:0, C14:0, or both, at a level of at least 5% of the total fatty acid content, and wherein the LPAAT has preferential activity toward fatty acids with a medium chain length (C8-C14), preferably C12:0 or C14:

0. xiv) The cells of the leaf or stem contain a total fatty acid content in which the oleic acid level is increased by at least 2% relative to the corresponding leaf or stem cells lacking the exogenous polynucleotides and / or genetic modifications, and / or the α-linolenic acid (ALA) level is decreased by at least 2% relative to the corresponding leaf or stem cells lacking the exogenous polynucleotides and / or genetic modifications. (xv) Nonpolar lipids in the cells of the corresponding leaves or stems that lack the exogenous polynucleotides and / or genetic modifications, comprising modified levels of total sterols, preferably free (non-esterified) sterols, stearoyl esters, stearoyl glycosides, relative to the nonpolar lipids in the corresponding leaves or stems lacking the exogenous polynucleotides and / or genetic modifications. (xvi) The nonpolar lipids in the cells of the leaves or stems comprise waxes and / or wax esters. (xvii) On a weight basis, the levels of one or more nonpolar lipids and / or total nonpolar lipids in the cells of the leaf or stem are at least 2% higher than those in the corresponding leaf or stem cells containing exogenous polynucleotides encoding Arabidopsis thaliana WRI1 (SEQ ID NO:21) and Arabidopsis thaliana DGAT1 (SEQ ID NO:1), and xviii) The total polyunsaturated fatty acid (PUFA) content reduced relative to the total PUFA content of the corresponding leaf or stem cells lacking the aforementioned exogenous polynucleotides and / or genetic modifications.

5. The method of claim 1, wherein one or more or all of the following characteristics apply when relevant: i) The polypeptide involved in the biosynthesis of one or more nonpolar lipids is a fatty acyl acyl transferase, such as DGAT, PDAT, LPAAT, GPAT, or MGAT, preferably DGAT or PDAT, involved in the biosynthesis of TAG, DAG, or monoacylglycerol (MAG) in the cells of the leaf or stem. ii) The polypeptide involved in the catabolism of triacylglycerol (TAG) in the cells of the leaf or stem is SDP1 lipase, Cgi58 polypeptide, acyl-CoA oxidase such as ACX1 or ACX2, or a polypeptide involved in the β-oxidation of fatty acids in the cells of the leaf or stem such as PXA1 peroxisome ATP-binding cassette transporter, preferably SDP1 lipase. iii) The oil-body-coated (OBC) polypeptide is an olein protein, such as polyolein or olein-body calcitonin, or lipid droplet-associated protein (LDAP). iv) The polypeptides in the cytoplasm of the leaves or stems that increase fatty acid export are C16 or C18 fatty acid thioesterases such as FATA or FATB polypeptides, fatty acid transporters such as ABCA9 polypeptides, or long-chain acyl-CoA synthases (LACS). v) The polypeptide involved in transporting fatty acids into the cytoplasm of the leaf or stem is a fatty acid transporter or its subunit, preferably a TGD polypeptide, and vi) The polypeptides involved in the formation of diacylglycerol (DAG) in plasmids are plasmid GPAT, plasmid LPAAT, or plasmid PAP.

6. The method of claim 1, wherein the cells of the leaf or stem are: i) Derived from or in 16:3 plants, and contains one or more of the following; a) An exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking the exogenous polynucleotide, increases fatty acid efflux from the plant's plastids. b) A first genetic modification that, compared to a corresponding plant lacking the first genetic modification, downregulates the endogenous generation and / or activity of polypeptides involved in the input of fatty acids into the plastids of the plant. c) A second genetic modification that, compared to a corresponding plant lacking the second genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the formation of diacylglycerol (DAG) in the plastids of said plant. The exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the vegetative plant part, or ii) From or in 18:3 plants.

7. The method of claim 1, wherein prior to flowering of the transgenic plant containing the cells of the leaf or stem, the cells of the leaf or stem contain at least 10%, at least 11%, 8%-15%, or 9%-12% (w / w dry weight) of total nonpolar lipids.

8. The method of claim 1, wherein the genetic modification is an endogenous gene mutation, such as a point mutation, insertion, or deletion, that partially or completely inactivates the gene, or the genetic modification is an exogenous polynucleotide encoding an RNA molecule that represses the expression of the endogenous gene, wherein the exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant.

9. The method of claim 1, comprising recovering extracted lipids by collecting them in a container and / or degumming, deodorizing, decolorizing, drying, grading, separating one or more of the extracted lipids, flowering and / or wax esters from the extracted lipids, or analyzing the fatty acid composition of the extracted lipids.

10. The method of claim 1, wherein the method further comprises converting the extracted lipids into an industrial product.

11. The method of claim 10, wherein the industrial product is a hydrocarbon product such as a fatty acid ester, preferably a methyl fatty acid ester and / or an ethyl fatty acid ester, an alkane such as methane, ethane or a longer chain alkane, a mixture of longer chain alkanes, an alkene, a biofuel, carbon monoxide and / or hydrogen, a bio-alcohol such as ethanol, propanol or butanol, biochar, or a combination of carbon monoxide, hydrogen and biochar.

12. A method for obtaining recombinant plant cells, the method comprising the following steps: i) Introducing at least one exogenous polynucleotide and / or at least one genetic modification from a set of exogenous polynucleotides and / or genetic modifications into plant cells to produce plant cells containing said set of exogenous polynucleotides and / or genetic modifications. ii) Express the exogenous polynucleotide in the cell or its progeny cells. iii) Analyze the lipid content of the cells or daughter cells, and iv) Select cells having the aforementioned set of exogenous polynucleotides and / or genetic modifications, and in plants where said cells are present, the cells of their leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids before flowering, and The set of exogenous polynucleotides and / or genetic modifications includes a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, compared with corresponding cells lacking the genetic modification, downregulates the endogenous production and / or activity of peptides involved in the catabolism of triacylglycerols (TAGs) in the cells, and optionally includes one or both of the following. d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding cell lacking a fourth exogenous polynucleotide, increases fatty acid efflux from the cell's plastids, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the cells. Each exogenous polynucleotide is operatively linked to a promoter that can direct the expression of the polynucleotide in the cell.

13. The method of claim 12, further comprising producing a plant from said cells.

14. A method for selecting plants having a set of exogenous polynucleotides and / or genetic modifications integrated into their genome, the method comprising the following steps i) Screening for the presence or absence of the aforementioned set of exogenous polynucleotides and / or genetic modifications in one or more plants, and ii) Select plants containing the aforementioned set of exogenous polynucleotides and / or genetic modifications. The set of exogenous polynucleotides and / or genetic modifications includes a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, compared with a corresponding plant lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant, and optionally includes one or both of the following. d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant lacking a fourth exogenous polynucleotide, increases fatty acid efflux from the plant's plastids, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant, and wherein, prior to flowering, the cells of the plant's leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids. This produces the plant.

15. A method for producing an industrial product, the method comprising the following steps: i) Obtaining a genetically modified plant or a part thereof, which contains a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts, and wherein, prior to flowering, the cells of the plant's leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids. as well as ii) a) Converting at least some of the lipids in the plant or its parts from step i) into an industrial product by in situ application of heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the plant or its parts. b) Physically processing the plant or its parts in step i), and subsequently or simultaneously converting at least some of the lipids in the processed plant or its parts into an industrial product by applying heat, chemical or enzymatic methods, or any combination thereof, to the lipids in the processed plant or its parts, and iii) Recycle the industrial products. Thus, the industrial products are produced.

16. The method of claim 15, wherein the plant part is a nutrient plant part.

17. The method of claim 15, further comprising the following steps: (a) Extracting at least some nonpolar lipids from the plant or its parts, wherein the content of nonpolar lipids is nonpolar lipids, and (b) Recovery of extracted nonpolar lipids, Steps (a) and (b) are performed prior to the step of converting at least some lipids in the plant or its parts into an industrial product.

18. Lipids obtained from a genetically modified plant or a portion thereof, or recoverable or extractable by the method of any one of claims 1-9, wherein the genetically modified plant or a portion thereof comprises: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or a part thereof, and wherein, prior to flowering, the cells of the plant’s leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids.

19. Use of a genetically modified plant or a portion thereof for the production of an industrial product, said genetically modified plant or a portion thereof comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts, and wherein, prior to flowering, the cells of the plant's leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids, or Contains the recovered or extracted lipids of claim 18.

20. A method for producing fuel, the method comprising: i) Reacting the lipid of claim 18 optionally with an alcohol in the presence of a catalyst to produce an alkyl ester, and ii) Optionally, the alkyl ester is mixed with a petroleum-based fuel.

21. A method for producing synthetic diesel fuel, the method comprising: i) Converting lipids in a genetically modified plant or a portion thereof into bio-oil by means including pyrolysis or hydrothermal treatment, or into syngas by gasification, wherein the genetically modified plant or a portion thereof comprises: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or its parts, and wherein, prior to flowering, the cells of the plant's leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids. as well as ii) Converting the bio-oil into synthetic diesel fuel by a method including staged separation, preferably at 150 o C-200 o C or 200 o C-300 o Hydrocarbons condensed under C2C, or syngas converted into biofuels using metal catalysts or microbial catalysts.

22. A method for producing biofuel, the method comprising converting lipids in a genetically modified plant or a portion thereof into biooil via pyrolysis, into bioethanol via fermentation, or into biogas via gasification or anaerobic digestion, wherein the genetically modified plant or a portion thereof comprises: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or a part thereof, and wherein, prior to flowering, the cells of the plant’s leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids.

23. The method of claim 22, wherein the portion is a nutrient plant part.

24. A method of producing feed, the method comprising mixing a genetically modified plant or a portion thereof, or a recovered or extracted lipid of claim 18, or an extract or portion thereof, with at least one other food ingredient, said genetically modified plant or a portion thereof comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or a part thereof, and wherein, prior to flowering, the cells of the plant’s leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids.

25. A method of feeding an animal, the method comprising providing the animal with a transgenic plant or a portion thereof, or a recovered or extracted lipid of claim 18, said transgenic plant or a portion thereof comprising: a) A first exogenous polynucleotide encoding a transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. b) A second exogenous polynucleotide encoding a polypeptide involved in the biosynthesis of one or more nonpolar lipids. c) Genetic modification that, when compared with a corresponding plant or part thereof lacking the genetic modification, downregulates the endogenous production and / or activity of polypeptides involved in the catabolism of triacylglycerols (TAGs) in the plant or part thereof, and optionally includes one or both of the following d) A third exogenous polynucleotide encoding a polypeptide that, when compared to a corresponding plant or part thereof lacking a fourth exogenous polynucleotide, increases the export of fatty acids from the plastids of the plant or part thereof, and e) A fourth exogenous polynucleotide encoding a second transcription factor polypeptide that increases the expression of one or more genes involved in glycolysis and / or fatty acid biosynthesis in the plant or its parts. Each exogenous polynucleotide is operatively linked to a promoter capable of directing the expression of the polynucleotide in the plant or a part thereof, and wherein, prior to flowering, the cells of the plant’s leaves or stems contain at least 8% (w / w dry weight) of total nonpolar lipids.

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