Application of a gene that promotes the accumulation of very long-chain fatty acids in plant seeds

Abstract

The present invention belongs to the field of plant genetic engineering and discloses the use of a gene for promoting the accumulation of very-long-chain fatty acids in plant seeds. The gene fragment comprises a nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5, or a complementary sequence thereof, or a derivative nucleotide sequence with at least 95% homology to these nucleotide sequences and the same function. The carbon chain of the very-long-chain fatty acid has greater than 18 carbon atoms, and the gene fragment is capable of increasing the proportion of very-long-chain fatty acids in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) in plant seeds. The present invention utilizes the LPAT2 gene (nucleotide sequence, for example, as shown in SEQ ID No. 1 to SEQ ID No. 5) from a cruciferous plant to increase the percentage of very-long-chain fatty acids in phosphatidylcholine and phosphatidylethanolamine in plant seeds, overcoming technical bottlenecks encountered in the current field.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and more specifically, relates to the application of a gene for promoting the accumulation of very long-chain fatty acids in plant seeds. Background Art

[0002] Organic chemical raw materials, such as petroleum and natural gas, play a crucial role in the chemical industry. However, as humans continue to exploit the Earth's mineral resources, non-renewable resources like petroleum and natural gas are decreasing and will eventually become depleted. Furthermore, their toxicity and environmental pollution are a major concern.

[0003] Plant fatty acid synthesis primarily occurs in plastids, using malonyl-CoA as a precursor and acetyl-CoA as a feedstock. The fatty acid synthase complex catalyzes the synthesis of fatty acids of varying carbon chain lengths (primarily C18). These C18 fatty acids are transported to the endoplasmic reticulum, where they undergo chain extension by the fatty acid elongase complex to produce C20 and C22 very long-chain fatty acids (VLCFAs). Transgenic overexpression of the fatty acid elongase complex in plants can typically increase VLCFA content in seeds, but this still falls short of meeting industrial requirements.

[0004] The esterification of fatty acids to form lipids is carried out in the endoplasmic reticulum through the Kennedy pathway: in this process, acyl-CoA is catalyzed by 3-phosphoglycerate acyltransferase to remove the acyl group at the sn-1 carbon position using glycerol-3-phosphate as the backbone to obtain lysophosphatic acid (LPA) (Jain, RK, Coffey, M and Lai, K, et al., 2000), which is then catalyzed by lysophosphatidyl acyltransferase (LPAT) to esterify at the sn-2 position to form phosphatidic acid (PA), which is then deacylated by phosphatidic acid phosphorylase and diacylglycerol acyltransferase to synthesize triglyceride (TAG). Lysophosphatidyl acyltransferase (LPAT) is a key enzyme that controls the production of phosphatidic acid from lysophosphatidic acid (Liang, M and Jiang, J, 2013).

[0005] The seeds of some varieties (lines) or germplasm resources of cruciferous plants, such as crambe and rapeseed, are rich in the VLCFA erucic acid, with concentrations exceeding 50%. Therefore, scientists have attempted to further increase the VLCFA content in cruciferous plants such as crambe. A higher percentage of VLCFAs in plant oils and fats reduces the cost of industrial purification, making the continuous improvement of VLCFA content in plant oils and fats crucial. Studies have found that despite high erucic acid content in triglycerides in cruciferous plant seeds, VLCFAs are primarily distributed at the sn-1 and sn-3 carbon positions of the triglycerides, with virtually no VLCFAs at the sn-2 position. Because LPATs specifically catalyze the accumulation of fatty acids at the sn-2 position of TAGs, scientists believed that cruciferous plant LPATs lacked a preference for VLCFAs. Consequently, scientists have been searching for LPATs with a VLCFA preference outside of the cruciferous plant family. For example, the introduction of meadowfoam-derived LPAT genes into Brassica napus (Lassner, MW, Levering, CK and Davies, HM, et al., 1995) has achieved some success in increasing VLCFA content. However, this has recently encountered new technical bottlenecks. For example, the low VLCFA content on phosphatidylcholine (PC) hinders further improvement of VLCFA accumulation in triacylglycerols (TAGs) (Guan, Rui, Lager, Ida, Li, Xueyuan, et al., 2014). Therefore, the search for LPATs that enhance VLCFA accumulation on PC has important applications in genetic engineering and synthetic biology.

[0006] For a long time, scientists have always believed that the ideal LPAT gene resources do not exist in cruciferous plants themselves, and have ignored them. The previous research results of the research group where the inventor of the present invention is located, "An application of a gene to promote the accumulation of linolenic acid in plant seeds" (Chinese patent CN 201810614691.2), can effectively promote the accumulation of linolenic acid in plant seeds by overexpressing the BnLPAAT2 gene in Brassica napus. However, the focus of this previous research result is linolenic acid (C18:3). Although the patent also compares the content ratios of eicosapentaenoic acid (C20:0), eicosapentaenoic acid (C20:1), eicosadienoic acid (C20:2), and erucic acid (C22:1) in the total fatty acids of seeds, the above results cannot be used to predict the effect of increasing the content of VLCFAs in phosphatidylcholine (or phosphatidylethanolamine). Summary of the Invention

[0007] In response to the above-mentioned deficiencies and improvements in the prior art, the present invention provides an application of a gene that promotes the accumulation of very-long-chain fatty acids in plant seeds. By using the LPAT2 gene from a cruciferous plant (nucleotide sequence, for example, as shown in any one of SEQ ID No. 1 to SEQ ID No. 5), the percentage of very-long-chain fatty acids in phosphatidylcholine and phosphatidylethanolamine in transgenic seeds can be significantly increased. The present invention reveals that LPAT2 can promote the accumulation of VLCFAs in phosphatidylcholine (and / or phosphatidylethanolamine), which is of great value in overcoming the technical bottleneck of low VLCFA content in phosphatidylcholine in the field.

[0008] To achieve the above objectives, according to one aspect of the present invention, there is provided a use of a gene fragment for increasing the proportion of very long-chain fatty acids in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) in plant seeds, characterized in that the gene fragment has a nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or its complementary sequence, or a derived nucleotide sequence having a homology of not less than 95% with the nucleotide sequence shown in any one of SEQ ID No. 1 to SEQ ID No. 5 and the same function as the nucleotide sequence as a result of adding, deleting or replacing one or more nucleotides;

[0009] The carbon number of the carbon chain of the very long chain fatty acid is greater than 18, and the gene fragment can increase the content ratio of the very long chain fatty acid in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) of plant seeds.

[0010] According to another aspect of the present invention, the present invention provides a use of a protein for increasing the proportion of very long-chain fatty acids in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) in plant seeds, characterized in that the protein has an amino acid sequence as shown in any one of SEQ ID No. 6 to SEQ ID No. 10, or a derivative protein having equivalent activity to the amino acid sequence as shown in any one of SEQ ID No. 6 to SEQ ID No. 10 resulting from the addition, deletion or substitution of one or more amino acids;

[0011] The carbon number of the carbon chain of the very long chain fatty acid is greater than 18, and the protein can increase the content ratio of the very long chain fatty acid in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) of plant seeds.

[0012] According to another aspect of the present invention, the present invention provides a use of a recombinant vector in breeding improvement to increase the content of very long-chain fatty acids in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) in plant seeds, characterized in that the recombinant vector comprises a gene fragment having a nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or a complementary sequence thereof, or a derived nucleotide sequence having a homology of not less than 95% with the nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5 and having the same function as the nucleotide sequence as a result of adding, deleting or replacing one or more nucleotides;

[0013] The carbon number of the carbon chain of the very long chain fatty acid is greater than 18, and the gene fragment can increase the content ratio of the very long chain fatty acid in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) of plant seeds.

[0014] According to another aspect of the present invention, the present invention provides a use of a recombinant strain in breeding and improvement to increase the content of very long-chain fatty acids in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) in plant seeds, characterized in that the recombinant strain comprises a gene fragment, wherein the gene fragment has a nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or a complementary sequence thereof, or a derived nucleotide sequence resulting from the addition, deletion or substitution of one or more nucleotides and having a homology of not less than 95% with the nucleotide sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5 and having the same function;

[0015] The carbon number of the carbon chain of the very long chain fatty acid is greater than 18, and the gene fragment can increase the content ratio of the very long chain fatty acid in phosphatidylcholine (PC) and / or phosphatidylethanolamine (PE) of plant seeds.

[0016] As a further preferred embodiment of the present invention, the very long chain fatty acid is preferably a very long chain fatty acid with a carbon chain carbon number of 20 or 22.

[0017] As a further preferred embodiment of the present invention, the plant includes monocotyledonous plants or dicotyledonous plants, preferably any one of Arabidopsis thaliana, rapeseed, peanut, soybean, tea oil, jatropha, palm, corn, rice, wheat, sesame, sunflower, and olive.

[0018] The above technical solutions conceived by the present invention, compared with the prior art, have discovered new uses for known gene fragments. Based on molecular biological methods, the present invention isolated the gene LPAT2 (Lysophosphatidic AcidAcyltransferase 2) from developing seeds of cruciferous plants, which specifically catalyzes the esterification of very-long-chain fatty acids to form phosphatidic acid. LPAT2 encodes an acyltransferase that can significantly increase the percentage of very-long-chain fatty acids in phosphatidylcholine and phosphatidylethanolamine in transgenic seeds, and also improve the accumulation of very-long-chain fatty acids in the total fatty acids of plant seeds.

[0019] Very long-chain fatty acids (VLCFAs) refer to fatty acids with a carbon chain length greater than 18 carbons, for example, C20:0, C20:1, C20:2, C20:3, C22:0, C22:1 (the Arabic numerals immediately after C indicate the carbon chain length, and the Arabic numerals immediately after the colon indicate the number of unsaturated bonds in the fatty acid, for example, C22:1 represents erucic acid). They all belong to very long-chain fatty acids. Very-long-chain fatty acids offer numerous advantages. For example, erucic acid, a common member of the very-long-chain fatty acids, has a long carbon chain, strong hydrophobicity and water resistance, excellent lubricity, and a slower oxidation-polymerization rate than short-chain fatty acids. It is an important oleochemical product with a wide range of applications in metallurgy, machinery, rubber, chemicals, paints, textiles, manufacturing, and medicine. Using erucic acid as a raw material, derivatives such as erucamide, behenic acid, tridecane dicarboxylic acid, and nonanoic acid can be produced. These products are used as anti-sticking agents, slip agents, and antistatic agents for polyethylene and polypropylene resins; lubricants and release agents for plastics processing; adhesion and anti-fouling agents for printing inks; softeners and water-proofing agents for various fibers; dispersants for various high-grade lubricants, dyes, and coatings; and as cleaning agents and rust inhibitors for metal machinery. It can also be used in food packaging materials. Currently, industrial erucic acid is primarily extracted from natural plant seeds, which falls far short of meeting industrial demand. The present invention uses LPAT2 from the Cruciferae family, such as Arabidopsis thaliana (abbreviated as At in the accompanying drawings below), Brassica napus (abbreviated as Bna in the accompanying drawings below), Camelina sativa (abbreviated as Csa in the accompanying drawings below), Brassica rapa (abbreviated as Bra in the accompanying drawings below) and Brassica oleracea (abbreviated as Bol in the accompanying drawings below), and the nucleotide sequences of their LPAT2 genes are as shown in SEQ ID No. 1 to SEQ ID No. 5, respectively. The LPAT2 genes of the Cruciferae family have high homology with each other. Taking the nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 5 as an example, they have high homology with each other (as shown in SEQ ID No. 1 to SEQ ID No. 5).Figure 1 shown).

[0020] The present invention reveals that LPAT2 from the Cruciferae family can significantly increase the percentage of very-long-chain fatty acids in phosphatidylcholine and phosphatidylethanolamine in transgenic seeds, while also promoting the accumulation of very-long-chain fatty acids (particularly significantly increasing the C20 and C22 very-long-chain fatty acids). By expressing the LPAT2 genes of rapeseed, Arabidopsis thaliana, camellia sinensis, Chinese cabbage, and cabbage separately, and using the following examples as an example, overexpression analysis in Arabidopsis thaliana demonstrates that LPAT2 from the Cruciferae family can significantly increase the percentage of very-long-chain fatty acids in phosphatidylcholine and phosphatidylethanolamine, and can also promote the accumulation of very-long-chain fatty acids in the total fatty acids of plant seeds and increase the content of very-long-chain fatty acids.

[0021] As previously mentioned, because VLCFAs rarely accumulate at the sn-2 position of TAG in cruciferous plants, most researchers believe that LPATs from cruciferous plants lack a preference for VLCFAs. Furthermore, scientists have long screened LPATs with a preference for VLCFAs by extracting enzymes from immature seeds and testing their preference in vitro. While this method can quickly and effectively detect LPATs with a preference for VLCFAs at the sn-2 position of TAG, it overlooks a class of LPATs that can increase the content of VLCFAs in phospholipids. For these reasons, these technical bottlenecks have limited researchers' ability to explore the effects of LPATs from cruciferous plants on the content of VLCFAs in phospholipids. The present invention, however, employs a research method that overexpresses LPAT in Arabidopsis seeds. Despite the relatively long experimental period (requiring more than six months), it allows for a more direct and comprehensive examination of the role of LPAT in promoting the accumulation of VLCFAs in phospholipids. Taking the following examples as an example, the inventors cloned LPAT genes from cruciferous plants represented by Brassica napus, Arabidopsis thaliana, Camelina oleracea, Cabbage, and Brassica rapa, and overexpressed them in seeds, discovering that the relevant LPATs can promote the accumulation of VLCFAs in seed phosphatidylcholine (PC). In addition to phosphatidylcholine, the content of VLCFAs in total seed oil and phosphatidylethanolamine was also significantly increased. Therefore, the LPAT discovered by the present invention will be of great significance in breaking through bottleneck problems in the field, providing a new understanding for the academic community on the role of LPAT from the cruciferous family in promoting the accumulation of VLCFAs, and has important application value. In other words, the present invention discovered and reported for the first time that the LPAT2 gene can significantly increase the content of VLCFAs in phospholipids, providing sufficient substrate for promoting the accumulation of VLCFAs in TAG.

[0022] In summary, the novel use of the LPAT2 gene from cruciferous plants disclosed in the present invention has important practical significance for genetically engineering oil crops to produce VLCFAs. It can actually increase the yield of VLCFAs, and is particularly significant for breaking the technical bottleneck of insufficient accumulation of very long-chain fatty acids in phospholipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is an alignment of the amino acid sequences of LPAT2 from Brassica napus, Arabidopsis thaliana, Capsella oleracea, Brassica rapa, and Cabbage oleracea. In the figure, the black background represents the fully identical portions of sequence. As can be seen from the figure, the amino acid sequences of LPAT2 from five cruciferous oilseed crops—Brassica napus, Arabidopsis thaliana, Capsella oleracea, Brassica rapa, and Cabbage oleracea—are highly conserved.

[0024] Figure 2 The results of overexpression of LPAT2 from Arabidopsis thaliana, Brassica napus, Brassica rapa and Brassica oleracea in Arabidopsis seeds were used to increase the production of very long chain fatty acids. Figure 2 A in the figure indicates the percentage of very long chain fatty acid C20:0. Figure 2 B in the figure shows the percentage of very long chain fatty acid C20:2. Figure 2 The C in the chart indicates the percentage of very long chain fatty acid C20:3. Figure 2 D in the figure shows the percentage of very-long-chain fatty acid C22:1. In the figure, Control#At represents the wild-type control in the Arabidopsis thaliana LPAT2 overexpression group; Control#Bna represents the wild-type control in the Brassica napus LPAT2 overexpression group; Control#Bra represents the wild-type control in the Brassica rapa LPAT2 overexpression group; and Control#Bol represents the wild-type control in the Brassica oleracea LPAT2 overexpression group.

[0025] Figure 3 The results of overexpression of LPAT2 from camelina indica in Arabidopsis seeds were used to increase the production of very long chain fatty acids. Figure 3 A in the figure indicates the percentage of very long chain fatty acid C20:0. Figure 3 B in the figure shows the percentage of very long chain fatty acid C20:2. Figure 3 The C in the chart indicates the percentage of very long chain fatty acid C20:3. Figure 3 The percentage of very-long-chain fatty acid C22:1 is shown in D. In the figure, Vector control represents the empty vector control; WT control represents the wild-type control. Three stars indicate extremely significant differences (P < 0.001, n > 6).

[0026] Figure 4 The changes in fatty acid components in phosphatidylcholine and phosphatidylethanolamine. Figure 4Figure A shows the changes in fatty acid composition in phosphatidylcholine (PC). It can be seen from the figure that the composition of very long chain fatty acids is greatly increased; Figure 4 Figure B shows the changes in the fatty acid composition of phosphatidylethanolamine (PE). The figure shows a significant increase in the composition of very long-chain fatty acids. Three stars (***) indicate a highly significant difference, p < 0.001, n = 3. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0028] (I) Cloning of LPAT2 genes from Arabidopsis thaliana, Brassica napus, Capsella oleracea, Brassica rapa, and Brassica oleracea

[0029] (I) RNA extraction from developing seeds of Arabidopsis thaliana, Brassica napus, Camelina oleracea, Brassica rapa, and Cabbage

[0030] The pipette tips, centrifuge tubes, and solutions involved in the following operations are all free of RNase contamination, and gloves must be worn during all operations.

[0031] (1) Preparation of extraction solution: Take 500 μl of RL lysis solution, then add 5 μl of β-mercaptoethanol and mix well.

[0032] (2) Homogenization: Remove the pods from the developing seeds in liquid nitrogen, quickly grind into powder, add 500 μl of RL lysis buffer, and vortex vigorously to mix. Centrifuge at 12,000 rpm for 5 minutes, and aspirate the supernatant.

[0033] (3) Transfer all the solutions to the CS filter column, centrifuge at 12000 rpm for 2 min, and carefully pipette the supernatant from the collection tube into a new RNase-free centrifuge tube, trying to avoid contact between the pipette tip and the cell debris precipitate in the collection tube.

[0034] (4) Add 0.5 times the volume of the supernatant to the centrifuge tube and mix thoroughly. Then transfer the supernatant to the adsorption column CR3 and centrifuge at 12000 rpm for 1 min. Pour away the waste liquid in the collection tube and put the adsorption column CR3 back into the collection tube.

[0035] (5) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube.

[0036] (6) Add 80 μl of DNase I digestion solution to the center of adsorption column CR3 and leave at room temperature for 15 minutes to digest the DNA.

[0037] (7) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube.

[0038] (8) Add 500 μl of rinse solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube. Repeat step 8 once.

[0039] (9) Centrifuge at 12000 rpm for 2 min and discard the waste liquid. Place the adsorption column CR3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0040] (10) Place the adsorption column CR3 in a new RNase-free centrifuge tube. Add 50 μl of RNase-free water to the middle of the adsorption membrane. Let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to obtain the RNA solution. Measure the RNA concentration and the ratio of OD values ​​260 to 280. If the ratio is between 1.8 and 2.0, the RNA is considered of acceptable quality and stored in a -70°C freezer until use. However, it should not be stored for more than one week.

[0041] (2) Synthesis of cDNA chain from reverse transcription of total RNA

[0042] The ReverTra Ace-α reverse transcription kit from Toyobo was used.

[0043] (1) Heat denaturation of RNA: Add 10 μl of RNA (1 μg / μl), 2 μl of Oligo(dT) (10 pmol / μl), and 12 μl of DEPC water to an ice box, mix gently, centrifuge in a palm centrifuge to remove residual nightshade on the tube wall, react in a metal bath at 65°C for 5 min, and immediately place on ice. This step is optional and is used to disrupt the tertiary or secondary structure of RNA and unwind the strands.

[0044] (2) Preparation of reaction solution: On ice, add 8 μl of 5× Reaction Buffer, 2 μl of RNase Inhibitor (10 U / μl), 4 μl of dNTPs Mixture (10 mM), and 2 μl of ReverTra Ace-α-to a total of 40 μl. Mix gently and set the reaction time in a metal bath at 42°C for 1 h. Heat denaturation at 85°C for 10 min to terminate the reaction. After the reaction is complete, store the reaction product in a -20°C refrigerator until use.

[0045] (III) PCR amplification and sequencing of the full-length gene and coding region

[0046] Reference sequences for primer design were mainly from the genome databases of Brassica napus, Camelina oleracea, Arabidopsis thaliana, Brassica rapa, and Cabbage. Specific primers were designed for amplification based on the sequence information predicted in the genome databases.

[0047] On ice, add 10×PCR Buffer 2μl, 25mM MgSO4 2μl, dNTP (2.5mM) 2μl, LPAT2 upstream primer (25μM) 0.3μl, LPAT2 downstream primer (25μM) 0.3μl, cDNA (200ng / μl) 1μl, KOD plus (1U / μl) 1μl, and ddH2O 11.8μl in sequence to a total volume of 20μl.

[0048] The reaction procedure is as follows:

[0049] Pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 68°C for 40 s-1 min, and extension at 68°C for another 10 min, for a total of 30 cycles.

[0050] After amplification, the amplified product was subjected to agarose gel electrophoresis. After electrophoresis, the target band was examined under UV light. The target band was excised with a razor blade and transferred to a centrifuge tube. The DNA fragment was recovered using an agarose gel extraction kit and ligated to the T vector. On ice, 5 μl of Solution I, 4 μl of the PCR product, and 1 μl of the pMD18-T vector (50 ng / μl) were added to a PCR tube and mixed thoroughly. The tube was placed in a metal bath and ligated overnight at 16°C. The ligation product was added to competent E. coli, mixed thoroughly, and incubated on ice for 30 minutes. Heat-shocked in a 42°C water bath for 90 seconds, then immediately returned to ice for 3 minutes. 700 μl of LB medium was added and incubated at 37°C in a shaker at 200 rpm for 30 minutes. 200 μl of the culture was transferred to solid LB medium supplemented with ampicillin (100 μg / ml) and spread evenly with a spreader. The plate was inverted and placed in a 37°C incubator for overnight incubation. The obtained positive plaques were sent to the company for sequencing. The five sequence-differentiated fragments obtained by sequencing were named: AtLPAT2, BnaLPAT2, CsaLPAT2, BraLPAT2, and BolLPAT2 (these nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.5; these sequences are identical to the corresponding sequences published in the genome database). Comparison of the amino acid sequences of the above genes revealed that the LPAT2 amino acid sequences of Brassica napus, Arabidopsis thaliana, Capsella chinensis, Brassica rapa, and Brassica oleracea from the Cruciferae family are extremely conserved, indicating that their functions are similar (e.g. Figure 1shown).

[0051] (II) Construction of overexpression vector

[0052] (1) Construction of plant expression vector and Agrobacterium transformation

[0053] The cloned gene sequence was used as a template for amplification using TOYOBO's high-fidelity enzyme KOD plus. The resulting gene fragment was recovered from an agarose gel for future use. 40 μl of the plant expression vector pBinGlyRed3 plasmid was added to 4 μl of EcoRI and 5 μl of enzyme digestion buffer, mixed evenly, and digested at 37°C for 30 minutes. The vector was then recovered from an agarose gel for future use. 2 μl of each gene and plasmid fragment was added to 1 μl of infusion kit mix, mixed evenly, and ligated at 50°C for 20 minutes. The ligation product was transformed into Escherichia coli, resulting in positive plaques. The plasmid was then extracted for future use. The recombinant plasmid carries the soybean globulin promoter, driving seed-specific expression of the LPAT2 gene. The recombinant plasmid was then transformed into Agrobacterium tumefaciens GV3101, and positive Agrobacterium was screened.

[0054] (2) Agrobacterium infection and transformation of Arabidopsis thaliana

[0055] The resulting positive strain was cultured overnight at 28°C and 200 rpm in LB medium supplemented with gentamicin (50 μg / ml), kanamycin (50 μg / ml), and rifampicin (25-50 μg / ml). The cells were collected by centrifugation at 5000 rpm for 3 minutes, and the Agrobacterium was diluted to an OD value of approximately 0.8 using the transformation infiltration solution. Arabidopsis plants in the early flowering stage were selected for floral transformation. Before transformation, the siliques of the Arabidopsis were trimmed off. The inflorescences of the Arabidopsis were immersed in the transformation solution for 0.5-1 minute. The Arabidopsis seedlings were placed in the dark and humid conditions overnight. The Arabidopsis were then returned to the culture room and the transformation was repeated after 3-5 days. The seeds were harvested after the Arabidopsis matured. Because the plant expression vector carries the DsRed3 fluorescent protein, the seeds are excited under 520 nm green light to release fluorescence. Red seeds that pass through the filter are transgenic T1 generations.

[0056] (III) Preliminary screening of transgenic T1 generation single-copy lines

[0057] After the T1 generation plants mature, the seeds are collected, which are the T2 generation, and the seeds are screened for copy number. The transgenic T2 seeds are excited under 520nm green light, and after passing through a red filter, bright field photos and excitation light source photos are collected respectively. This step is completed under a fluorescent stereo microscope. The collected photos are used to count the number of seeds using statistical software such as Wanshen CG seed testing software or Image J, and the ratio of red seeds to non-red seeds is finally obtained. The red seeds of the strains with a calculated ratio close to 3:1 are selected for sowing, and the seeds are collected after the seeds mature as T3 generation seeds.

[0058] (IV) Fatty acid analysis and expression analysis of overexpression lines

[0059] (1) Extraction and methylation of oils and fats

[0060] 1. Weigh 5-10 mg of Arabidopsis T3 seeds and place them in a 10 ml glass test tube.

[0061] 2. Add 1.5 ml of 2.5% H2SO4 methanol solution (containing 0.01% BHT), 200 μl of C17:0 toluene solution (concentration of 2 mg / ml), and 0.4 ml of toluene solution in sequence. After filling with nitrogen, be sure to tighten the lid to prevent high temperature volatilization.

[0062] Place in a 3.99℃ water bath for 1 hour, take out and let cool, then add 1.8ml of double-distilled water and 1ml of n-hexane in sequence.

[0063] 4. After mixing, let it stand for 12 hours, aspirate the supernatant with a 1 ml syringe, and then filter it through a 0.45 μm pore size into a sample bottle for testing.

[0064] (2) Gas chromatography analysis of fatty acid methyl esters

[0065] The gas chromatograph used was an Agilent 7890A, and the columns were Agilent J&W GC Columns. The injection volume was 1 μl. The oven heating program was: initial temperature 180°C, heating at a rate of 10°C / min to 225°C, and holding for 7 minutes. The column constant flow rate was 1 ml / min, and the carrier gas was nitrogen.

[0066] The peak area was calculated using the software provided by the Agilent ChemStation, and the oil content was calculated using the internal standard method (C17:0 was used as the internal standard for determining the total amount of fatty acids). The calculation formula was: m (C17:0 mass) / s (C17:0 peak area) = m (oil mass) / s (peak area of ​​other fatty acids except C17:0).

[0067] The results of fatty acid composition of AtLPAT2, BnaLPAT2, CsaLPAT2, BraLPAT2 and BolLPAT2 transgenic lines showed that the percentage of very long chain fatty acids including C20:0, C20:2, C20:3 and C22:1 in AtLPAT2, BnaLPAT2, CsaLPAT2, BraLPAT2 and BolLPAT2 overexpression lines was significantly increased compared with the wild-type control. Figure 2 、 Figure 3 As shown. Specifically:

[0068] (1) For C20:0:

[0069] like Figure 2 As shown, the C20:0 content in dry seeds of Arabidopsis thaliana AtLPAT2-OE strains ranged from 2.62±0.19% to 2.9±0.11%, which was increased by 24% to 37% compared with the wild type (2.11±0.05%).

[0070] The percentage of C20:0 in dry seeds of rapeseed BnaLPAT2-OE strain was 2.74±0.07% to 3±0.4%, which was increased by 22% to 33% compared with the wild type (2.24±0.06%).

[0071] The percentage of C20:0 in dry seeds of BraLPAT2-OE strain of Chinese cabbage ranged from 2.84±0.01% to 3.13±0.22%, which was increased by 29% to 42% compared with the wild type (2.2±0.21%).

[0072] The percentage of C20:0 in dry seeds of the Brassica oleracea BolLPAT2-OE strain ranged from 2.82±0.05% to 2.93±0.18%, which was 23% to 28% higher than that of the wild type (2.28±0.2).

[0073] Another example Figure 3 As shown, the percentage of C20:0 in dry seeds of CsaLPAT2-OE strain was 2.56±0.15% to 2.78±0.06%, which was increased by 90% to 106% compared with the wild type (1.35±0.1%).

[0074] (2) For C20:2:

[0075] like Figure 2 As shown, the C20:2 content in dry seeds of Arabidopsis thaliana AtLPAT2-OE strains ranged from 2.46±0.26% to 2.68±0.09%, which was increased by 78% to 94% compared with the wild type (1.38±0.18%).

[0076] The percentage of C20:2 in dry seeds of rapeseed BnaLPAT2-OE strain was 2.85±0.14% to 3.04±0.2%, which was 53% to 63% higher than that of the wild type (1.86±0.01%).

[0077] The percentage of C20:2 in dry seeds of BraLPAT2-OE strain of Chinese cabbage was 2.73±0.08% to 2.82±0.24%, which was increased by 57% to 62% compared with the wild type (1.74±0.08%).

[0078] The percentage of C20:2 in dry seeds of the Brassica oleracea BolLPAT2-OE strain ranged from 2.66±0.07% to 2.92±0.09%, which was 43% to 57% higher than that of the wild type (1.86±0.23%).

[0079] Another example Figure 3 As shown, the percentage of C20:2 in dry seeds of CsaLPAT2-OE strain was 2.64±0.13% to 2.74±0.12%, which was increased by 23% to 27% compared with the wild type (2.15±0.08%).

[0080] (3) For C20:3:

[0081] like Figure 2 As shown, the C20:3 content in dry seeds of Arabidopsis thaliana AtLPAT2-OE strains ranged from 1.19±0.09% to 1.55±0.18%, which was increased by 114% to 179% compared with the wild type (0.55±0.05%).

[0082] The percentage of C20:3 in dry seeds of rapeseed BnaLPAT2-OE strain was 1.02±0.07% to 1.18±0.17%, which was increased by 228% to 279% compared with the wild type (0.31±0.08%).

[0083] The C20:3 content in dry seeds of BraLPAT2-OE strain of Chinese cabbage ranged from 0.88±0.10% to 1.20±0.21%, which was increased by 45% to 98% compared with the wild type (0.60±0.01%).

[0084] The percentage of C20:3 in dry seeds of the Brassica oleracea BolLPAT2-OE strain ranged from 0.84±0.11% to 1.11±0.02%, which was increased by 23% to 62% compared with the wild type (0.68±0.09%).

[0085] Another example Figure 3As shown, the percentage of C20:3 in dry seeds of CsaLPAT2-OE strain was 1.24±0.06% to 1.78±0.09%, which was increased by 134% to 236% compared with the wild type (0.53±0.04%).

[0086] (4) For C22:1:

[0087] like Figure 2 As shown, the percentage of C22:1 in dry seeds of Arabidopsis thaliana AtLPAT2-OE strain was 2.33±0.05% to 2.56±0.11%, which was increased by 21% to 33% compared with the wild type (1.92±0.04%).

[0088] The percentage of C22:1 in dry seeds of rapeseed BnaLPAT2-OE strain was 2.9±0.15% to 3.04±0.19%, which was increased by 43% to 50% compared with the wild type (2.03±0.05%).

[0089] The percentage of C22:1 in dry seeds of BraLPAT2-OE strain of Chinese cabbage was 2.62±0.1% to 2.85±0.04%, which was increased by 32% to 43% compared with the wild type (1.99±0.09%).

[0090] The percentage of C22:1 in dry seeds of the Brassica oleracea BolLPAT2-OE strain was 2.72±0.11% to 2.82±0.04%, which was 44% to 49% higher than that of the wild type (1.89±0.15%).

[0091] Another example Figure 3 As shown, the percentage of C22:1 in dry seeds of CsaLPAT2-OE strain was 2.43±0.12% to 2.66±0.17%, which was increased by 9% to 20% compared with the wild type (2.22±0.1%).

[0092] (V) Fatty acid composition analysis of phosphatidylcholine and phosphatidylethanolamine in overexpression lines

[0093] (1) Extraction of total oil

[0094] 1. Weigh 0.5 g of transgenic Arabidopsis T3 dry seeds, crush them in a grinder, add 3.75 ml of a mixture of chloroform and methanol (volume ratio 1:2), add 1 ml of 1 mM EDTA (0.15 M HAC as solvent), and mix thoroughly. Then add 1.25 ml of chloroform, wash in a crushing tube, and then transfer to a glass tube.

[0095] 2. Add 1.25 ml of 0.88% KCl and mix thoroughly; centrifuge at 3000 rpm for 3 minutes;

[0096] 3. Immediately transfer the lower organic phase into a new 10ml glass tube.

[0097] 4. Blow the organic phase dry under nitrogen, and the remaining portion at the bottom of the tube is the total oil.

[0098] (2) Separation of total oil on TLC

[0099] 1. Redissolve the extracted total oil in chloroform:methanol (1:2) and spot on a TLC plate;

[0100] 2. Separate on a TLC plate using chloroform:methanol:acetic acid:water = 90:15:10:3 (volume ratio, acetic acid concentration is 17.5 mol / L) as the developing solvent. Spray primulin and observe the bands under ultraviolet light.

[0101] 3. Scrape the bands representing phosphatidylcholine and phosphatidylethanolamine from the TLC plate and transfer them to a glass tube for methyl esterification (the method is the same as (IV) fatty acid analysis and expression analysis of the overexpression strain);

[0102] The results of the percentage of fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in mature seeds of transgenic lines and control groups showed that overexpression of LPAT2 significantly increased the accumulation of VLCFAs in phosphatidylcholine and phosphatidylethanolamine (e.g. Figure 4 ). VLCFAs accounted for 10.31±0.47% of the PC in the mature dry seeds of the overexpression strain, which was twice that of the wild control (5.64±0.19%); VLCFAs accounted for 6.08±0.28% of the PE in the mature dry seeds of the overexpression strain, which was 2.8 times that of the wild control (2.17±0.28%). Therefore, LPAT2 from the cruciferous family has the function of promoting the accumulation of VLCFAs in PC and PE, which is of great significance for breaking through the technical bottleneck in the field of VLCFA production. In addition, from Figure 4 It can be clearly seen that for non-very long-chain fatty acids, their content ratio is the same as or even decreased compared with the control group, which also confirms that the proportion of each fatty acid in the total fatty acids in plant seeds has no direct correlation with the proportion of each fatty acid in phosphatidylcholine and phosphatidylethanolamine.

[0103] The various reagents used in the above examples were purchased from commercial sources.

[0104] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a gene fragment in increasing the proportion of very long chain fatty acids in Arabidopsis seeds, characterized in that: The nucleotide sequence of the gene fragment is shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or its complementary sequence; The very long chain fatty acid is C20:0, C20:2, C20:3 or C22:

1.

2. A method for increasing the proportion of very long chain fatty acids in Arabidopsis seeds, characterized in that: The amino acid sequence of the protein is shown in any one of SEQ ID No. 6 to SEQ ID No. 10; The very long chain fatty acid is C20:0, C20:2, C20:3 or C22:

1.

3. Use of a recombinant vector in breeding improvement to increase the content of very long chain fatty acids in Arabidopsis seeds, characterized in that: The recombinant vector comprises a gene fragment, the nucleotide sequence of which is shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or its complementary sequence; The very long chain fatty acid is C20:0, C20:2, C20:3 or C22:

1.

4. A recombinant strain for use in breeding to increase the content of very long-chain fatty acids in Arabidopsis seeds, characterized in that: The recombinant strain comprises a gene fragment, the nucleotide sequence of which is shown in any one of SEQ ID No. 1 to SEQ ID No. 5 or a complementary sequence thereof; The very long chain fatty acid is C20:0, C20:2, C20:3 or C22:1.

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