method

Abstract

The present invention provides a method for increasing the sucrose ester content in a tobacco plant or a tobacco cell culture, the method comprising modifying the tobacco plant or the tobacco cell culture by inhibiting the activity or expression of a diterpene synthesis gene. The present invention also provides the use of a diterpene synthesis gene for increasing the sucrose ester content in a tobacco plant or a tobacco cell culture, and tobacco cells, tobacco plants, tobacco plant propagation materials, leaves harvested, processed tobacco or tobacco products obtainable according to the present invention.

Description

Field of the Invention

[0001] The present invention relates to improving the flavor of tobacco, and particularly to the sucrose ester content in tobacco plants. Specifically, the present invention relates to increasing the sucrose ester content in tobacco plants. More specifically, the present invention relates to methods and uses of inhibiting the activity or expression of diterpene synthesis genes and tobacco plants and downstream products thereof (e.g., propagation materials, leaves harvested, processed tobacco, tobacco products).

[0002] background

[0003] Plant trichomes are specialized structures containing epidermal protrusions that can be found on the aerial (ariel) surfaces of many plants. Their morphology is variable between species and even within species, including their position on plant organs, their size and density. They are considered to provide the first line of defense against insects, pathogenic microorganisms and herbivores, and provide protection against environmental conditions such as frost and windy conditions. Trichomes can be glandular and secrete products, and their main function can be to produce chemicals that interact with pests or pollinators that are stored or volatilized on the plant surface. The trichomes of such plants can serve as factories for the production of natural products with economic and commercial importance. Advantageously, the secreted compounds are only produced in the trichome glands and accumulate outside the glandular cells under the surrounding cuticle. Therefore, exudates can be simply and cleanly recovered by immersing the plant in a non-invasive solvent, because the exudate material is substantially accumulated outside the plant. Manipulation of plant trichome secondary exudate compositions has been used to enhance insect resistance (Wang et al. Nature Biotechnology 19, 371-374 (2001), incorporated herein by reference), and metabolic engineering of glandular trichomes has been used to enhance the quality of essential oils (Mahmoud and Croteau Proc Natl Acad Sci US A. 2001 Jul 17;98(15):8915-20. Epub 2001 Jun 26, incorporated herein by reference).

[0004] Many tobacco varieties have high biomass and high exudate accumulation potential. For example, experimental tobacco strain TI 1068 can produce up to 17% leaf dry weight as glandular trichome exudate (Wagner, GJ 1991 Plant Physiology 96, 675–679, which is incorporated herein by reference). Approximately 72% of the trichome gland exudate is diterpenoids, and approximately 24% is sucrose ester.

[0005] The most abundant natural source of sucrose ester seems to be tobacco plant.Sugar ester is a complex mixture of compounds, because many possible acyl groups and the combination of acyl groups can be esterified into sugar (sucrose or glucose). These compounds are mainly responsible for the sticky texture of their tobacco leaves (Kandra, L. and GJ Wagner, Archives Biochem. Biophys. 265 (1988) 425–432, which is incorporated herein by reference). Sucrose ester is the precursor (Kandra, L., et al. Eur. J. Biochem. 188 (1990) 385–391, which is incorporated herein by reference) of 3-methylvaleric acid and 3-methylbutyric acid (which is an important flavor component of Turkish tobacco smoke), and in addition, it is said that 3-methylvaleric acid provides "Turkish impression (Turkishnote)" for tobacco.

[0006] TI 1068 trichome exudate contains mainly cembrenoid and labdanoid diterpenes. CBT diol (α and β positional isomers), cis-aberyl and labdenediol account for approximately 60%, 10% and 0.6% of the exudate weight, respectively. The general pathway for the synthesis of the major diterpene components of TI 1068 trichome exudate diterpenes and CBT-diol is shown in Figure 2 The methyl-erythritol-phosphate (MEP) pathway of the chloroplast provides geranylgeranyl pyrophosphate (GGPP) for diterpene synthesis.

[0007] The organoleptic or sensory properties of different tobacco types vary widely and are affected by a complexity of factors including genetic differences. The antifungal and organoleptic properties of tobacco are believed to be related to the sucrose ester content. The unique aroma and flavor profile of tobacco is the result of the unique flavor of aromatic compounds or precursors of these compounds, which are present at specific levels in the curing leaves and may include sucrose ester content.

[0008] Turkish tobacco (sometimes referred to as oriental tobacco) has the organoleptic properties of expectation; It is a kind of highly aromatic, small leaf variety of tobacco, and its taste is gentle, and contains less nicotine than other tobacco varieties. It has been determined that the sucrose ester of measurable amount is retained after tobacco modulation / aging process, and therefore occurs as the overall composition of commercial Turkish (or oriental) tobacco (M. Ashraf-Khorassani, et al. Contributions to Tobacco Research Vol 21:7; October 2005, it is incorporated herein by reference). In addition, sucrose ester is the precursor of aromatic carboxylic acid in Turkish tobacco smoke, and therefore is the important flavor component (Arrendale, RF, et al., J. Agric. Food Chem. 38 (1990) 75–85, it is incorporated herein by reference) of Turkish tobacco.

[0009] Due to the distribution of the degree of esterification and the ester functional groups within the molecule, naturally occurring sucrose esters present challenging analytical problems. In the sucrose molecule, there are four potential acyl sites (e.g., one primary site, three secondary sites) on the glucose (GLU) ring; and another four sites (e.g., two primary sites, two secondary sites) can be found on the fructose (FRU) ring. So far, GC-MS (gas chromatography-mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry) of sucrose methyl / butyl esters or sucrose trimethylsilyl (TMS) derivatives have been used to identify the ester acid part (see Slocombe et al. Plant Physiol. 148, 1830-1846, which is incorporated herein by reference). In order to try to cover higher molecular weight carbohydrate derivatives, high temperature GC (gas chromatography) has been studied but with little success (Karrer, R., et al., J. High Resolu. Chromatogr. 15 (1992) 585–589, which is incorporated herein by reference). Inferences regarding acyl substitution patterns on sucrose esters from data obtained by acid hydrolysis of the esters and rederivatization of the liberated carboxylic acids have been reported, but this approach is open to different interpretations. SUMMARY OF THE INVENTION

[0011] Surprisingly found, by suppressing the activity or expression of the diterpene synthesis gene taught herein, can produce unique local flavor (and / or aroma and / or taste) characteristics, such as those found in Turkish tobacco, by modifying tobacco to produce the tobacco with increased sucrose ester content by tobacco plants with commercial desired proterties.Therefore, can produce the tobacco product with the excellent local flavor (and / or aroma and / or taste) characteristics that the consumer of tobacco product pursues.

[0012] The present inventors investigated the regulation and pathways of carbon flow in specialized trichome gland cells that produce specific secondary compounds. One objective of the present inventors was to optimize trichome gland diterpene metabolism to modify trichome exudate chemistry to provide surprising diterpene content. Plant strain cell lines were generated that unexpectedly exhibited increased sucrose ester content in exudates compared to their wild-type plant counterparts grown under the same conditions. The plant strains targeted enzymes that catalyze the formation of CBTol (synthase) and terpene synthase 3-8. In some embodiments, terpene synthase 3-8 may be referred to as cis-abirol synthase.

[0013] The inventors have surprisingly determined a method for increasing the sucrose ester content of tobacco plants by inhibiting the activity or expression of diterpene synthesis genes. Prior to the present invention, it was not known that inhibition of the activity or expression of diterpene synthesis genes could be used to increase sucrose ester content, particularly to improve yield and other commercially desirable traits simultaneously.

[0014] According to one aspect, the present invention provides a method for increasing the sucrose ester content in a tobacco plant or part thereof or a tobacco cell culture, the method comprising modifying the tobacco plant or tobacco cell culture by inhibiting the activity or expression of a diterpene synthesis gene.

[0015] In another aspect, there is provided use of a diterpene synthesis gene for increasing the sucrose ester content in a tobacco plant or part thereof or a tobacco cell culture.

[0016] In another aspect, the present invention provides a method for producing a tobacco plant or part thereof, tobacco plant propagation material, tobacco leaves, cut harvested tobacco leaves, processed tobacco leaves or cut and processed tobacco leaves or tobacco cell culture having an increased sucrose ester content, the method comprising modifying the tobacco to inhibit the activity or expression of a diterpene synthesis gene.

[0017] In another aspect, there is provided a method or use according to the invention, wherein the sucrose ester content is increased compared to a tobacco plant or tobacco cell culture which has not been modified to inhibit the activity or expression of a diterpene synthesis gene.

[0018] In another aspect, a tobacco plant or part thereof or a tobacco cell culture is provided, which is modified to achieve an increase in sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene.

[0019] In one aspect, tobacco plant propagation material (eg, plant seeds) obtainable from a tobacco plant or tobacco cell culture according to the invention is provided.

[0020] In another aspect, the present invention provides a method or use, a tobacco plant or tobacco plant propagation material or a tobacco cell culture, wherein the diterpene synthesis gene is a cyclase 2 gene (CYC2), a CBTol cyclase or a terpene synthase 3-8.

[0021] In one aspect, the present invention provides a method or use, a tobacco plant or tobacco plant propagation material, wherein the diterpene synthesis gene is a cyclase 2 gene (CYC2).

[0022] In one aspect, the present invention provides a method or use, a tobacco plant or tobacco plant propagation material, wherein the diterpene synthesis gene is CBTol cyclase.

[0023] In one aspect, the invention provides a method or use, a tobacco plant or tobacco plant propagation material, wherein the diterpene synthesis gene is a terpene synthase 3-8.

[0024] In another aspect, the invention provides a method or use, a tobacco plant or part thereof or tobacco plant propagation material or a tobacco cell culture according to the invention, wherein the expression of a diterpene synthesis gene is inhibited using RNA interference (RNAi).

[0025] In one aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture is provided, wherein the expression of the cyclase 2 gene (CYC2) is inhibited using a ddRNAi DNA construct comprising at least a portion of exon 1, at least a portion of exon 2 and at least a portion of exon 3 of the cyclase 2 gene (CYC2).

[0026] In one aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture is provided, wherein the expression of a cyclase 2 gene (CYC2) is inhibited using a dsRNA, the dsRNA being processed by an endogenous pathway in the cell into a single-stranded short interfering RNA (siRNA), which guides an RNA-induced silencing complex (RISC) to bind to an mRNA corresponding to at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of the cyclase 2 gene (CYC2). In one aspect, the single-stranded siRNA produces an antisense (or guide) strand having a complementary sequence to an mRNA corresponding to at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of the cyclase 2 gene (CYC2).

[0027] In one aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention is provided, wherein the expression of the CBTol cyclase gene is inhibited using a ddRNAi DNA construct comprising at least part of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and at least part of exon 7 of the CBTol cyclase gene.

[0028] In another aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention is provided, wherein the expression of the CBTol cyclase gene is inhibited using a dsRNA, the dsRNA being processed by an endogenous pathway in the cell into a single-stranded siRNA, which guides the RNA-induced silencing complex (RISC) to bind to an mRNA corresponding to at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and at least a portion of exon 7 of the CBTol cyclase. In one aspect, the single-stranded siRNA produces an antisense (or guide) strand having a complementary sequence to an mRNA corresponding to at least a portion of exon 4, exon 5, exon 6 and at least a portion of exon 7 of the CBTol cyclase gene.

[0029] In one aspect, there is provided a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention, wherein the expression of the terpene synthase 3-8 gene is inhibited using a ddRNAi DNA construct comprising a sequence corresponding to at least nucleotides 1497 to 1517 of SEQ ID No. 3.

[0030] In another aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention is provided, wherein the expression of the terpene synthase 3-8 gene is inhibited using an artificial micro RNAi (amiRNAi) comprising a sequence corresponding to at least nucleotides 1497 to 1517 of SEQ ID No. 3.

[0031] In one aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention is provided, wherein the expression of the terpene synthase 3-8 gene is inhibited using a ddRNAi DNA construct comprising a sequence corresponding to at least nucleotides 884 to 904 of SEQ ID No. 3.

[0032] In another aspect, a method, a use, a tobacco plant or a tobacco plant propagation material or a tobacco cell culture according to the invention is provided, wherein the expression of the terpene synthase 3-8 gene is inhibited using an artificial micro RNAi (amiRNAi) comprising a sequence corresponding to at least nucleotides 884 to 904 of SEQ ID No. 3.

[0033] In one aspect, a method or use of the invention, a tobacco plant of the invention or a part thereof, or a tobacco plant propagation material or a tobacco cell culture of the invention is provided, wherein the sucrose ester content of the tobacco plant in the modified tobacco plant or tobacco cell culture is at least 2 times (suitably at least 3 times) compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of diterpene synthesis genes.

[0034] In one aspect, there is provided use of a tobacco plant of the invention or a part thereof for growing tobacco plants.

[0035] In another aspect, the present invention provides use of the tobacco plant or part thereof or tobacco cell culture of the present invention for producing tobacco industry products.

[0036] In another aspect, there is provided use of a tobacco plant of the invention or a part thereof for growing crops.

[0037] In one aspect, the invention provides a cured tobacco material made from a plant or part thereof or an extract thereof according to the invention or a tobacco cell culture according to the invention.

[0038] In another aspect, there is provided a tobacco blend comprising the cured tobacco material according to the invention.

[0039] In one aspect, there is provided use of a tobacco plant of the invention, or a part thereof, for producing tobacco leaves, such as processed (preferably cured) tobacco leaves.

[0040] In another aspect, there is provided a harvested leaf of a tobacco plant of the invention or a harvested leaf of a tobacco plant obtainable from a tobacco plant propagated from the propagation material of the invention or obtainable from a tobacco plant obtained by using the invention.

[0041] In one aspect, there is provided a harvested leaf of a tobacco plant of the present invention, wherein the harvested leaf of the tobacco plant is a cut harvested leaf.

[0042] In another aspect, the present invention provides processed tobacco leaves (preferably non-viable processed tobacco leaves) which:

[0043] i) obtainable from a tobacco plant obtainable using the present invention;

[0044] ii) obtainable by processing the tobacco plant of the present invention;

[0045] iii) a tobacco plant propagated from the tobacco plant propagation material of the invention is obtainable; or

[0046] iv) can be obtained by processing the harvested leaves of the tobacco plant of the present invention.

[0047] In one aspect, there is provided a processed tobacco leaf of the present invention, wherein the tobacco is processed by curing, fermenting, pasteurizing, or a combination thereof.

[0048] In one aspect, there is provided a processed tobacco leaf of the present invention, wherein the processed tobacco leaf is a cut processed tobacco leaf.

[0049] In another aspect, the present invention provides a tobacco industry product prepared by:

[0050] i) a tobacco plant of the present invention or a part thereof or a tobacco cell culture of the present invention;

[0051] ii) a tobacco plant or part thereof propagated from the tobacco plant propagation material of the present invention;

[0052] iii) harvested leaves of the tobacco plant of the present invention;

[0053] iv) The processed tobacco leaves of the present invention.

[0054] In one aspect, there is provided a tobacco industry product of the present invention, wherein the tobacco product is a smoking article.

[0055] In another aspect, there is provided a tobacco industry product of the present invention, wherein the tobacco product is a smokeless tobacco product.

[0056] In one aspect, there is provided a tobacco industry product of the present invention, wherein the tobacco product is a tobacco heating device, such as an aerosol generating device.

[0057] In one aspect, a smoking article, a smokeless tobacco product or a tobacco heating device is provided, comprising a plant or part thereof or an extract thereof (e.g., a tobacco extract) according to the invention or a tobacco cell culture according to the invention; or a cured tobacco material according to the invention; or a tobacco blend according to the invention.

[0058] In one aspect, there is provided a method, tobacco leaf, tobacco plant, tobacco plant propagation material, harvested leaf, processed tobacco, tobacco product, use or combination thereof substantially as described herein with reference to the specification and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0061] Figure 1 The general structure of sucrose esters is shown.

[0062] Figure 2 Schematic diagram showing the diterpene synthesis pathway in plastids.

[0063] Figure 3 A graph showing green leaf analysis of field grown control versus transgenic lines is shown. Individual phytochemicals are arranged in ascending order of quantity.

[0064] Figure 4 Shown is a graph demonstrating total sucrose ester content in control compared to transgenic lines.

[0065] Figure 5 Shown is a graph demonstrating the enrichment of sucrose esters in the GW-3 line compared to the control. The sucrose ester content is expressed as a percentage of the total major exudate compounds.

[0066] Figure 6 Shown is a graph demonstrating the enrichment of cis-abieol in the GW-3 line compared to the control. The cis-abieol content is expressed as a percentage of the total major exudate compounds.

[0067] Figure 7 SEQ ID No. 1 is shown as follows.

[0068] Figure 8 SEQ ID No. 2 is shown as follows.

[0069] Fig. 9 SEQ ID No. 3 is shown as follows.

[0070] Fig.10 SEQ ID No. 4 is shown as follows.

[0071] Fig.11 SEQ ID No. 5 is shown as follows.

[0072] Fig.12 SEQ ID No. 6 is shown as follows.

[0073] Fig.13 SEQ ID No. 7 is shown as follows.

[0074] Fig.14 SEQ ID No. 8 is shown as follows.

[0075] Fig.15 SEQ ID No. 9 is shown as follows.

[0076] Fig.16 SEQ ID No. 10 is shown as follows.

[0077] Fig.17 SEQ ID No. 11 is shown as follows.

[0078] Sequence Listing

[0079] A summary of sequence identifiers used throughout this specification and the corresponding sequence listing is provided, wherein:

[0080] SEQ ID No. 1 corresponds to the nucleotide sequence encoding the CBTol cyclase gene. The nucleotide sequence is annotated in Genbank: AY049090.

[0081] SEQ ID No. 2 corresponds to the amino acid sequence of the polypeptide CBTol cyclase.

[0082] SEQ ID No.3 corresponds to the cDNA gene sequence from Nicotiana tabacum ( Nicotiana tabacum ) of the terpene synthase 3-8 gene. The full-length cDNA gene sequence is annotated in Genbank: AY528645.

[0083] SEQ ID No. 4 corresponds to the amino acid sequence of the polypeptide terpene synthase 3-8 from Nicotiana tabacum.

[0084] SEQ ID No.5 comprises, in order, the 5'-sense fragment from the 54th to the 716th nucleotides of the sequence AF401234 (which is the complete coding sequence of CYC2 mRNA and corresponds to the nucleotide numbering in SEQ ID No.9); a partial GUS A fragment as a hairpin loop (from the 787th to the 1812th nucleotides); and the reverse complement of the sense fragment-3'. In this figure, the underlined sequence corresponds to the GUS A fragment (AF502128) from the 787th to the 1812th nucleotides. The nucleotide sequence from 54 to 716 in the sense orientation from CYC2 mRNA (AF401234 corresponds to SEQ ID No. 9) corresponds to the following genomic sequence from AY495694 (AY495694 is the genomic sequence of the CYC2 gene and corresponds to SEQ ID No. 8): nucleotides 1 to 25, exon 1 (nucleotides 26 to 271), exon 2 (nucleotides 1253 to 1529), and the first 115 nucleotides of exon 3 (nucleotides 2366 to 2480), wherein the nucleotide numbering corresponds to that in SEQ ID No. 8. SEQ ID No. 5 encodes a dsRNA and is used in a ddRNAi DNA construct (sometimes referred to herein as GW1) to produce a dsRNA.

[0085] SEQ ID No. 6 comprises the sequence from the CBTol cyclase gene of Nicotiana tabacum, which comprises a partial sequence of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and a portion of exon 7 in sense and antisense directions, which are separated by a GUS spacer. The elements of the partial gene are shown in the following order in the figure: common tobacco cyclase gene (AY049090), partial exon 4 - from nucleotide 2854 to nucleotide 4175, forward direction, partial exon 4, intron 4 (bold), exon 5, intron 5 (bold), exon 6, intron 6 (bold), partial exon 7 (corresponding to nucleotides 2854 to 4175 of SEQ ID No.1); followed by GUS loop-partial GUS A gene (Genbank accession number AF502128)-from 786 to 1816 (underlined); followed by the same sequence of the common tobacco cyclase gene (AY049090), but in the reverse complement (reverse complement is shown in the shade). This sequence encodes dsRNA and is used in ddRNAi DNA constructs (sometimes referred to as GW3 herein) to produce dsRNA.

[0086] SEQ ID No.7 comprises 21 nucleotide sequences of terpene synthase 3-8 genes from common tobacco and a sequence matching with Arabidopsis miRNA168 sequence. The sequence corresponds to Arabidopsis miRNA168a, and the first and second italic sequences correspond to 21 nucleotide sequences (1497 to 1517nt) from terpene synthase 3-8 gene AY528645. The first italic sequence (prominent) is in the reverse complementary direction. The second italic sequence (underline) is in the forward direction and has three modifications (bold). The sequence encodes amiRNAi and is used in ddRNAi DNA constructs (sometimes referred to as GW2 herein) to produce amiRNAi.

[0087] SEQ ID No. 8 corresponds to the genomic sequence of the cyclase 2 gene (CYC2). The nucleotide sequence is annotated in Genbank: AY495694.

[0088] SEQ ID No. 9 corresponds to the complete coding sequence of the mRNA of the cyclase 2 gene (CYC2) annotated in Genbank: (AF401234).

[0089] SEQ ID No. 10 corresponds to the amino acid sequence of the polypeptide encoded by the cyclase 2 gene (CYC2) SEQ ID No. 9.

[0090] SEQ ID No.11 comprises 21 nucleotide sequences of terpene synthase 3-8 genes from common tobacco and a sequence matched with Arabidopsis miRNA168a sequence. The sequence corresponds to the Arabidopsis miRNA168a sequence, and the first and second italic sequences correspond to 21 nucleotide sequences (884 to 904 nt) of the reverse complement of terpene synthase 3-8 gene AY528645. The first italic sequence (prominent) is in the reverse complementary direction. The second italic sequence (underline) is in the forward direction and has modification (bold). The sequence encodes amiRNAi and is used in ddRNAi DNA constructs (sometimes referred to as GW5 herein) to produce amiRNAi.

[0091] Details

[0092] The inventors have shown for the first time that the sucrose ester content of tobacco plants can be increased by inhibiting the activity or expression of diterpene synthesis genes in tobacco. Without wishing to be bound by theory, it is believed that inhibition of diterpene synthesis genes results in reduced carbon utilization to prepare diterpenes and thus enhances sucrose ester production.

[0093] In one embodiment, the present invention provides a method for increasing the sucrose ester content of a tobacco plant, the method comprising modifying the tobacco plant by inhibiting the activity or expression of a diterpene biosynthesis gene.

[0094] Suitably, the diterpene synthesis gene used in the present invention can be a cyclase 2 gene (CYC2). Suitably, the diterpene synthesis gene used in the present invention can be a CBT11 cyclase gene. Suitably, the diterpene synthesis gene used in the present invention can be a terpene synthase 3-8 gene.

[0095] The term "increased" is used herein to mean that the concentration and / or total content of sucrose esters in the product (e.g., plant, part (e.g., leaf), processed leaf or tobacco product) of the invention is higher compared to a comparable product not modified according to the invention.

[0096] " comparable product " as defined herein will be the product that is never modified according to the present invention but wherein all other related features are identical tobacco plant (for example, plant species, growth conditions, method for processing tobacco etc.) derived.Comparable products according to the present invention can mean that can be obtained from or be obtained from tobacco plant or its part, such as tobacco leaf, the leaf of results, the leaf of cutting results, the tobacco leaf or tobacco plant propagation material of processing, or tobacco product or its combination that can be obtained from or be obtained from the tobacco plant that is not modified to suppress the activity of diterpene synthesis gene or expression.Comparable product also can be called contrast.In one embodiment, comparable product is the product that does not comprise its activity or expression the diterpene synthesis gene that has been suppressed.

[0097] The term "unmodified plant" as defined herein would be a tobacco plant that has not been modified according to the present invention to inhibit the activity or expression of a diterpene synthesis gene, and wherein all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing the tobacco, etc.). In one embodiment, an unmodified plant is a plant that does not contain a diterpene synthesis gene whose activity or expression has been inhibited.

[0098] In a further aspect, the sucrose ester content is measured from green leaves. In a further aspect, the sucrose ester content is measured from cured leaves, such as air-cured, flue-cured, fire-cured or sun-cured leaves. In a further aspect, the sucrose ester content is measured from flue-cured leaves. In a further aspect, the sucrose ester content is measured from air-cured leaves.

[0099] The term "sucrose ester content" is used herein to refer to the concentration and / or total content of the entire group of compounds classified as sucrose esters. Sucrose esters typically present in tobacco can be Figure 1 The formula shown in represents.

[0100] Any method known in the art for determining the concentration and / or total content of sucrose esters may be used. A preferred method for analyzing sucrose esters (SE) involves analysis of the acyl groups released from sucrose by saponification followed by analysis of their butyl esters by GC-MS. Analysis by this method provides strong quantification of the amount of sucrose esters. An alternative method involves measuring the amount of sucrose esters by ... 2 Sucrose esters can be analyzed by measuring the sucrose on the surface of the leaves. The determination of sucrose ester content can be evaluated in tobacco leaves. A suitable method for analyzing the sucrose ester content may include a step of saponification, for example placing the gum sample in 80% methanol at 22°C overnight to saponify. The sample can then be partitioned between hexane and water. To partition the sample, the sample can first be dried, for example in N 2 After drying, n-BuOH and H 2 SO 4 The sample can be heated to 110° for 1 hour before pulse vortexing. Extraction can be performed and the dried sample dissolved in CHCl 3 Transfer to another tube and place in N 2 The sample can be dissolved in dimethylformamide (DMF) and bis(trimethylsilyl)trifluoroacetamide. The sample can be derivatized, for example, at 70° and then cooled to room temperature. The derivatized sample can then be analyzed by GC-MS. The eluted compound can be identified by comparison of retention time and MS profile with a standard.

[0101] In one embodiment, provide for producing the tobacco plant obtainable or obtained by the tobacco plant of the present invention with the sucrose ester content of increase, tobacco plant propagation material, tobacco leaf, the leaf of results, the leaf of cutting results, the tobacco leaf of processing, the tobacco leaf of cutting and processing, the method for tobacco product or its combination, said method comprises modifying said tobacco to suppress the activity or expression of diterpene synthesis gene.The sucrose ester content of increase can be measured by comparing tobacco plant, tobacco plant propagation material, tobacco leaf, the leaf of results, the leaf of cutting results, the tobacco leaf of processing, the tobacco leaf of cutting and processing, tobacco product or its combination in the sucrose ester content and comparable product.

[0102] Suitably, sucrose ester content can be increased in tobacco plants, for example, tobacco plants of modification. Suitably, sucrose ester content can be increased in tobacco leaves (for example, tobacco leaves from the tobacco plants of modification). Suitably, sucrose ester content can be increased in leaves of harvest (for example, tobacco leaves of the tobacco plants of modification). Suitably, sucrose ester content can be increased in leaves of cutting harvest (for example, tobacco leaves of cutting harvest from the tobacco plants of modification). Suitably, sucrose ester content can be increased in tobacco leaves of processing (for example, tobacco leaves of the tobacco plants of modification). Suitably, sucrose ester content can be increased in tobacco leaves of cutting and processing (for example, tobacco leaves of cutting and processing from the tobacco plants of modification). Suitably, sucrose ester content can be increased in the trichome exudate of tobacco leaves of modulation (for example, tobacco leaves from the tobacco plants of modification). Suitably, sucrose ester content can be increased in the extract of green tobacco leaves (for example, tobacco leaves from the tobacco plants of modification). Suitably, the sucrose ester content may be increased in a tobacco product (eg a tobacco product produced from a modified tobacco plant or part thereof). Suitably, the sucrose ester content may be increased in any one or a combination of the above products.

[0103] In one embodiment, sucrose ester content is increased in trichome exudate of cured tobacco leaves (eg, tobacco leaves from modified tobacco plants).

[0104] In one embodiment, the sucrose ester content is increased in an extract of green tobacco leaves (eg, tobacco leaves from a modified tobacco plant).

[0105] In one embodiment, when compared with the sucrose ester content of a tobacco plant or part thereof that is not modified to inhibit the activity or expression of a diterpene biosynthesis gene grown under similar growth conditions, the sucrose ester content of a tobacco plant or part thereof can be increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times, respectively. Suitably, the sucrose ester content can be increased by about 2 times to about 10 times, preferably about 3 times to about 10 times, suitably about 3 times to about 5 times.

[0106] In one embodiment, the method or use results in an increase in sucrose ester content compared to a tobacco plant or part thereof that has not been modified to inhibit the activity or expression of a diterpene synthesis gene, and more specifically compared to or relative to the expression of a tobacco plant in which the inhibition has not been introduced.

[0107] In one embodiment, the tobacco plant or part thereof has been modified to achieve an increase in sucrose ester content compared to a respective tobacco plant or part thereof that has not been modified to inhibit the activity or expression of a diterpene synthesis gene.

[0108] The term "modification" or "modified" as used herein means a tobacco plant that has been changed or altered. The present invention includes the use of techniques for genetically modifying plants or non-genetically modifying plants to modify plants. Such methods are well known in the art, and examples of genetic modification techniques include transformation, transgenics, cisgenics, and gene editing methods. Examples of non-genetic modification techniques include fast neutron mutagenesis, chemical mutagenesis, and modern population analysis methods. In one embodiment, the term "modification" refers to the selection of natural variants of diterpene synthesis genes with inhibition, and breeding the trait or gene to a second plant with a commercially desirable trait.

[0109] As used herein, the term "inhibit" (e.g., inhibit the activity or expression of a diterpene synthesis gene) means that the activity or expression of a diterpene synthesis gene is reduced or decreased, or the amount or activity of a protein produced by a diterpene synthesis gene is reduced, compared to the gene activity or gene expression in a comparable product.

[0110] In one embodiment, the term "inhibit" as used herein (eg, inhibiting the activity or expression of a diterpene synthesis gene) means that the activity or expression of a diterpene synthesis gene is reduced compared to the gene activity or gene expression in a comparable product.

[0111] The "activity" or "function" of a diterpene synthesis gene relates to its ability to function in the biosynthesis of diterpenes. The activity or function of a diterpene synthesis gene can be determined by measuring the direct product of diterpene synthesis, i.e., by measuring the level of diterpenes. The exudate components can be measured by washing leaves or leaf disks with acetonitrile. The washings are concentrated via rotor evaporation to obtain an oily residue. The residue can then be derivatized to form trimethylsilyl (TMS) esters (as described in Severson et al. 1985. J. Agric. Food Chem. 33, 870-875 (which is incorporated herein by reference)). TMS derivatives can then be separated and analyzed by gas chromatography (GC-MS) coupled to a mass spectrometer. The eluted compounds can be identified by comparison of retention time and MS profile with standards, as described in Wang et al. 2001 (ibid., which is incorporated herein by reference). In one embodiment, the direct product of the diterpene synthesis gene may be cis-abieol, α-CBT-ol, β-CBT-ol, α-CBT-diol, β-CBT-diol or Labdenediol.

[0112] The activity of a specific diterpene synthesis gene can be measured by measuring the transcription of the gene. Methods for measuring transcription are well known in the art, and particularly include northern blots, RNA-Seq, in situ hybridization, DNA microarrays, and RT-PCR. Alternatively, the activity of a gene can be measured indirectly by measuring the level of a gene product (e.g., a protein encoded by the gene).

[0113] The activity or expression of a gene can refer to the level of activity of a protein encoded by a diterpene synthesis gene, such as transcription, translation (i.e. protein expression), or the like. According to one aspect of the invention, gene expression can be inhibited by suppressing transcription and / or translation. In one embodiment, the activity or expression of a gene can refer to the level of transcription (i.e. the amount of the mRNA produced) or the level of translation (i.e. the level or amount of the protein produced).

[0114] Inhibit, reduce or prevent expression and / or function

[0115] Any method known in the art for inhibiting or reducing or preventing the expression or function of diterpene synthesis genes can be used in the present method.

[0116] By way of example, the method may include:

[0117] • providing a mutation in a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11 or an amino acid sequence having at least 70% sequence identity thereto;

[0118] • mutations in regulatory regions (e.g., promoters and enhancers) that contribute to controlling the expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 11, or an amino acid sequence having at least 70% sequence identity thereto are provided;

[0119] • Provided is an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.11 or an amino acid sequence having at least 70% sequence identity thereto.

[0120] Each of the above methods results in reducing or preventing the expression or function of a protein comprising an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11 or an amino acid sequence having at least 70% sequence identity thereto.

[0121] As used herein, the term "mutation" includes natural genetic variants and engineered variants. Specifically, the term "mutation" refers to a change in a nucleotide sequence encoding an amino acid sequence or an amino acid sequence compared to a sequence shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.11 or an amino acid sequence having at least 70% sequence identity thereto, which reduces the expression or function of the protein.

[0122] In a preferred embodiment, each copy of the nucleic acid sequence encoding a protein comprising a sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11 or a sequence having at least 70% sequence identity thereto present in the plant is mutated as defined herein (e.g., each genomic copy of the gene encoding the protein in the plant is mutated). For example, common tobacco ( N. tabacum ) Every copy of a gene in an allotetraploid genome can be mutated.

[0123] In a preferred embodiment, the plant or plant cell according to the invention is homozygous for said mutation.

[0124] In one embodiment, preferably, the plant or plant cell according to the present invention only expresses the mutated nucleic acid. In other words, in some embodiments, no endogenous (or endogenous and functional) protein is present in the plant according to the present invention. In other words, if any endogenous protein is present, it is preferably in an inactive and / or truncated form.

[0125] In one embodiment, the method may comprise providing a mutation in a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 8, or a nucleic acid sequence having at least 70% identity thereto.

[0126] The mutation can change the plant genome so that the nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.11 or an amino acid sequence having at least 70% sequence identity thereto is completely or partially deleted or otherwise becomes non-functional.

[0127] The mutation may interrupt the nucleic acid sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11 or an amino acid sequence having at least 70% sequence identity thereto.

[0128] Interruption may result in the nucleic acid sequence not being transcribed and / or translated. For example, the nucleic acid sequence may be interrupted by deleting or otherwise modifying the ATG start codon of the nucleic acid sequence, thereby reducing or preventing the translation of the protein.

[0129] Nucleotide sequence can comprise one or more nucleotide changes that reduce or prevent protein expression or influence protein transport.For example, can reduce or prevent protein expression by introducing one or more premature termination codons, frameshift, splice mutant or non-acceptable amino acid substitution in open reading frame.Premature termination codon refers to the sudden change that termination codon is introduced into open reading frame and prevents whole amino acid sequence translation.Premature termination codon can be TAG (" amber "), TAA (" ochre ") or TGA (" opal " or " brown earth ") codon.

[0130] A frameshift mutation (also called a frame error or reading frame shift) is a mutation caused by an insertion or deletion (insertion or deletion) of multiple nucleotides in a nucleic acid sequence that is not divisible by 3. Due to the triplet nature of gene expression through codons, insertions or deletions can change the reading frame, resulting in a translation that is completely different from the original translation. Frameshift mutations often result in the codon after the mutation being read to encode a different amino acid. Frameshift mutations often result in the introduction of a premature stop codon.

[0131] Splicing mutants insert, delete or change multiple nucleotides in a specific site where splicing occurs during the processing of the precursor messenger RNA into the mature messenger RNA. The deletion of the splice site results in one or more introns being retained in the mature mRNA and may lead to the production of abnormal proteins.

[0132] Non-acceptable amino acid substitution refers to a sudden change that causes a non-synonymous amino acid substitution in a protein, which causes the protein function to be reduced or eliminated. Any method known in the art for providing a sudden change in a nucleotide sequence can be used in this method. For example, homologous recombination can be used to produce a vector in which the relevant nucleic acid sequence is mutated and used to transform a plant or plant cell. Then a recombinant plant or plant cell expressing the mutant sequence can be selected.

[0133] In one embodiment, the mutation introduces an unacceptable amino acid substitution in a protein comprising the amino acid sequence shown as SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 11, or a sequence having at least 70% sequence identity thereto.

[0134] In one embodiment, the mutation reduces the activity of the protein relative to the protein set forth in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11, or a sequence having at least 70% sequence identity thereto.

[0135] In one embodiment, the mutation does not alter the level or expression of the protein but reduces the activity of the protein relative to the protein set forth in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 11 or a sequence having at least 70% sequence identity thereto.

[0136] The nucleotide sequence may be deleted in whole or in part. The deletion may be continuous or may include multiple sequence portions. The deletion preferably removes a sufficient amount of nucleotide sequence so that the nucleotide sequence no longer encodes a functional protein. For example, the deletion may remove at least 50%, 60%, 70%, 80% or 90% of the nucleotide sequence encoding portion.

[0137] The deletion may remove one or more domains of a diterpene biosynthesis gene. The deletion may be total, in which case 100% of the coding portion of the nucleic acid sequence is absent, when compared to the corresponding genome of a comparable unmodified plant.

[0138] Methods for deleting nucleotide sequences in plants are known in the art. For example, homologous recombination can be used to produce a vector in which the relevant nucleic acid sequence is deleted and used to transform a plant or plant cell. The recombinant plant or plant cell that expresses the new part of the sequence can then be selected.

[0139] In some embodiments, the activity or expression of a diterpene synthesis gene may be inhibited or reduced by at least about 10%, 20%, 30% or 40%, suitably at least about 50%, 60%, 70%, more suitably at least about 80%, 90%, 95% or 100% when compared to the activity or expression of a diterpene synthesis gene in a tobacco plant not modified according to the present invention.

[0140] In a preferred embodiment, the diterpene synthesis gene may have substantially no activity or expression, meaning that the plant may contain less than about 1% (suitably less than about 0.1%) activity or expression, preferably when compared to a plant which has not been modified to inhibit the activity or expression of the diterpene synthesis gene.

[0141] As used herein, diterpene synthesis gene refers to any gene involved in the production of diterpene. Suitably, the diterpene synthesis gene is a plant diterpene synthesis gene. Such genes can directly be a part of the biosynthetic pathway for producing diterpene, or can be indirectly supplied to the biosynthetic pathway, for example, by providing metabolic precursors. Diterpene synthesis gene can participate in special (secondary) metabolism or general (primary) metabolism. In one embodiment, as used herein, diterpene synthesis gene refers to the gene involved in special (secondary) metabolism. Suitably, as used in the present invention, the diterpene synthesis gene can be selected from the group of diterpene synthesis genes composed of cyclase 2 gene (CYC2), CBTol cyclase and terpene synthase 3-8, or any one homology in cyclase 2 gene (CYC2), CBTol cyclase or terpene synthase 3-8. Suitably, as used in the present invention, the diterpene synthesis gene can be selected from the group of diterpene synthesis genes composed of cyclase 2 gene (CYC2), CBTol cyclase and terpene synthase 3-8.

[0142] Suitably, the diterpene synthesis gene as used in the present invention is a cyclase 2 gene (CYC2). Suitably, the diterpene synthesis gene as used in the present invention is a CBTol cyclase. Suitably, the diterpene synthesis gene as used in the present invention is a terpene synthase 3-8.

[0143] In one embodiment, the diterpene synthesis gene referred to herein is a cyclase 2 gene (CYC2), and may be encoded by a polynucleotide sequence comprising:

[0144] i) the polynucleotide sequence shown herein as SEQ ID No. 8 or SEQ ID No. 9, or

[0145] ii) a functional fragment of the polynucleotide sequence shown in i), which encodes a diterpene biosynthesis gene, or

[0146] iii) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown herein as SEQ ID No. 10, or

[0147] iv) a polynucleotide sequence that can hybridize to the polynucleotide taught in i), ii) or iii) above under highly stringent conditions, or

[0148] v) a polynucleotide sequence having at least 70% (preferably 85%, more preferably 90%) identity with the polynucleotide shown in i), ii) or iii) above, or

[0149] vi) a polynucleotide sequence which differs from the polynucleotide shown in i), ii) or iii) due to the degeneracy of the genetic code.

[0150] In one embodiment, the diterpene synthesis gene referred to herein is CBTol cyclase, and may be encoded by a polynucleotide sequence comprising:

[0151] i) the polynucleotide sequence shown herein as SEQ ID No. 1, or

[0152] ii) a functional fragment of the polynucleotide sequence shown in i), which encodes a diterpene biosynthesis gene, or

[0153] iii) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown herein as SEQ ID No. 2, or

[0154] iv) a polynucleotide sequence that can hybridize to the polynucleotide taught in i), ii) or iii) above under highly stringent conditions, or

[0155] v) a polynucleotide sequence having at least 70% (preferably 85%, more preferably 90%) identity with the polynucleotide shown in i), ii) or iii) above, or

[0156] vi) a polynucleotide sequence which differs from the polynucleotide shown in i), ii) or iii) due to the degeneracy of the genetic code.

[0157] In one embodiment, the diterpene synthesis gene referred to herein is terpene synthase 3-8, and may be encoded by a polynucleotide sequence comprising:

[0158] i) the polynucleotide sequence shown herein as SEQ ID No. 3, or

[0159] ii) a functional fragment of the polynucleotide sequence shown in i), which encodes a diterpene biosynthesis gene, or

[0160] iii) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown herein as SEQ ID No. 4, or

[0161] iv) a polynucleotide sequence that can hybridize to the polynucleotide taught in i), ii) or iii) above under highly stringent conditions, or

[0162] v) a polynucleotide sequence having at least 70% (preferably 85%, more preferably 90%) identity with the polynucleotide shown in i), ii) or iii) above, or

[0163] vi) a polynucleotide sequence which differs from the polynucleotide shown in i), ii) or iii) due to the degeneracy of the genetic code.

[0164] In one embodiment, the diterpene synthesis genes used according to the present invention may be endogenous to the tobacco plant.

[0165] Reference herein to an "endogenous" gene refers not only to the gene in question being found in its native form in the plant (i.e., in the absence of any human intervention), but also to the fact that the same gene (or substantially homologous nucleic acid / gene) has subsequently been (re)introduced into the plant in isolated form (transgenic). For example, transgenic plants containing such a transgene may experience a substantial reduction in transgene expression and / or a substantial reduction in expression of the endogenous gene. An isolated gene may be isolated from an organism or may be artificially prepared, for example, by chemical synthesis.

[0166] In another embodiment, the diterpene synthesis genes used according to the present invention may be foreign to the tobacco plant.

[0167] In order to determine whether a gene is a diterpene synthesis gene used according to the present invention, the technician can determine whether the gene is capable of producing diterpenes. The content of diterpenes can be measured as described above. In short, the method for measuring diterpene content can involve collecting the exudate of one or more leaves by washing leaves or leaf disks with acetonitrile. The residue from the washings can then be derivatized to form trimethylsilyl (TMS) esters (as described in Severson et al., 1985 supra (which is incorporated herein by reference)). TMS derivatives can then be separated and analyzed by gas chromatography (GC-MS) coupled to a mass spectrometer. The eluted compound can be identified by comparison of its retention time and MS profile with a standard.

[0168] The present invention also provides the use of the diterpene synthesis gene for increasing the sucrose ester content of plants.

[0169] Methods for reducing the expression of genes or gene products are well documented in the art.

[0170] In one embodiment, the activity or expression of diterpene synthesis genes can be suppressed by any method known in the art. Suitably, the activity or expression of diterpene synthesis genes selected from the group of diterpene synthesis genes consisting of cyclase 2 gene (CYC2), CBTol cyclase and terpene synthase 3-8 can be suppressed by any method known in the art. Suitably, the activity or expression of cyclase 2 gene (CYC2) can be suppressed by any method known in the art. Suitably, the activity or expression of CBTol cyclase can be suppressed by any method known in the art. Suitably, the activity or expression of terpene synthase 3-8 can be suppressed by any method known in the art.

[0171] Methods for inhibiting the activity or expression of diterpene synthesis genes can include RNA interference, antisense or sense co-suppression (see Wang and Wagner 2003, Planta Volume 216, Issue 4, pp 686–691, which are incorporated herein by reference), gene editing or directed mutagenesis. In one embodiment, inhibition of the activity or expression of diterpene synthesis genes can be achieved by using gene editing. Gene editing can be implemented using any method known in the art. Several non-limiting examples are presented herein.

[0172] In one embodiment, inhibition of the activity or expression of diterpene synthesis genes can be achieved using gene editing methods including CRISPR (including using the CRISPR-Cas9 system). CRISPR / Cas9 genome editing tools are commercially available, such as "Guide-it" from Clontech (Avenue du President Kennedy 78100 Saint-Germain-en-Laye, France).

[0173] Another gene editing method includes the use of TALEN (transcription activator-like effector nuclease) technology, which is commercially available (e.g., from Addgene, 1 Kendall Sq. Ste. B7102, Cambridge, MA 02139, USA). In one embodiment, TALEN can be used to achieve inhibition of the activity or expression of diterpene synthesis genes.

[0174] In another embodiment, the method may include the use of zinc finger nucleases, such as the CompoZr® zinc finger nuclease technology available from Sigma-Aldrich. Another embodiment may include the use of the meganucleases (or another method) described in Silva et al. Curr Gene Ther. Feb 2011; 11(1): 11–27 (the teachings of which are incorporated herein by reference).

[0175] In one embodiment, the method for suppressing the activity or expression of diterpene synthesis genes can be directed mutagenesis. Any method of targeted mutagenesis can be used. In one embodiment, the method can be oligonucleotide directed mutagenesis (ODM), such as KeyBase® available from Keygene (Agro Business Park 90, 6708 PW Wageningen, The Netherlands). In another embodiment, the activity or expression of diterpene synthesis genes can be suppressed by using constructs or vectors (e.g., plasmids).

[0176] The genetic construct of the present invention can be in the form of an expression cassette, which can be suitable for inhibiting the activity or expression of the diterpene synthesis gene in the host cell. The genetic construct can be introduced into the host cell without being introduced into a vector. For example, the genetic construct that can be a nucleic acid molecule can be incorporated into a liposome or a viral particle. Alternatively, the purified nucleic acid molecule (e.g., histone-free DNA or naked DNA) can be directly inserted into the host cell by a suitable means (e.g., direct endocytosis uptake). The genetic construct can be directly introduced into the cell of the host subject (e.g., plant) by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, a particle gun can be used to directly introduce the genetic construct of the present invention into the host cell.

[0177] Alternatively, the genetic construct may comprise or be carried in a recombinant vector for expression in a suitable host cell. The recombinant vector may be a plasmid, a cosmid or a bacteriophage. Such recombinant vectors are very useful for transforming host cells with the genetic construct of the present invention, and for replicating the expression cassette therein. The skilled person will appreciate that the genetic construct of the present invention can be combined with many types of backbone vectors for expression purposes. The backbone vector may be a binary vector, such as a vector that can be replicated in both Escherichia coli and Agrobacterium tumefaciens. For example, a suitable vector may be a pBIN plasmid, such as pBIN19 (Bevan M., 1984, Nucleic Acids Research 12:8711-21).

[0178] In addition to suppressing the activity of diterpene synthesis gene or the sequence of expression, recombinant vector can include a variety of other functional elements. For example, the vector can include a promoter. In addition, the recombinant vector can be designed so that it replicates autonomously in the cytosol of the host cell. In this case, the element of inducing or regulating DNA replication may be needed in the recombinant vector. Alternatively, the recombinant vector can be designed so that it is integrated into the genome of the host cell. In this case, it is envisioned that the DNA sequence that is conducive to targeted integration (for example, by homologous recombination) is.

[0179] The recombinant vector may also contain DNA encoding a gene that can be used as a selectable marker during the cloning process, i.e., to enable selection of cells that have been transfected or transformed, and to enable selection of cells carrying the vector into which the heterologous DNA has been incorporated. The vector may also contain DNA that is involved in regulating the expression of the coding sequence or for targeting the expressed polypeptide to a specific part of the host cell (e.g., to the trichomes or glandular trichomes). Thus, the vector may contain at least one additional element selected from: a selectable marker gene (e.g., an antibiotic resistance gene); a polypeptide termination signal; and a protein targeting sequence (e.g., a transit peptide).

[0180] In one embodiment, the method or use may include using RNAi to inhibit the activity or expression of diterpene synthesis genes. In one embodiment, the RNAi method uses miRNA, for example, artificial micro-RNA (amiRNA). In one embodiment, the RNAi method uses siRNA. In one embodiment, the RNAi method uses dsRNA.

[0181] In one embodiment, the method or purposes can include using an interfering oligonucleotide to suppress the activity or expression of a diterpene synthesis gene. In one embodiment, the oligonucleotide is based on RNA. In one embodiment, the oligonucleotide is RNA interference (RNAi), such as dsRNAi. In one embodiment, the method can include transforming a cell of a tobacco plant with an RNAi molecule (such as dsRNAi) that suppresses the activity or expression of a diterpene synthesis gene.

[0182] In one embodiment, tobacco plants and tobacco plant propagation materials are provided, wherein the expression of diterpene synthesis genes is inhibited using RNAi (eg, dsRNAi). The method may include regenerating tobacco plants from transformed cells.

[0183] RNAi (e.g., dsRNAi) molecules can reduce the activity or expression of diterpene synthesis genes in transformed plants by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more or 100% compared to the concentration of polypeptides in wild-type plants not transformed with RNAi. In one embodiment, the RNAi molecules can reduce the activity or expression of diterpene synthesis genes in transformed plants by 30% to 100%, preferably 40% to 100%, more preferably 90% to 100%.

[0184] The activity or expression of diterpene synthesis genes can be suppressed by any method known in the art. Suitably, the activity or expression of diterpene synthesis genes selected from the group of diterpene synthesis genes consisting of cyclase 2 gene (CYC2), CBTol cyclase and terpene synthase 3-8 can be suppressed by any method known in the art.

[0185] Suitably, the method may comprise transforming cells of a tobacco plant with a ddRNAi DNA construct encoding an RNA that forms a hairpin structure which is processed into a small or short interfering RNA (siRNA) by endogenous pathways in the cell.

[0186] Suitably, the activity or expression of the cyclase 2 gene (CYC2) can be inhibited by any method known in the art. In one embodiment, the sequence of the cyclase 2 gene (CYC2) is as shown in SEQ ID No.8. In one embodiment, the amino acid sequence of the cyclase 2 gene (CYC2) is as shown in SEQ ID No.10. The expression of the cyclase 2 gene (CYC2) can be inhibited by any method, including gene editing methods including CRISPR, including the use of the CRISPR-Cas9 system, RNA interference (RNAi), antisense or sense co-suppression, gene editing or directed mutagenesis. In one embodiment, the activity or expression of the cyclase 2 gene (CYC2) can be inhibited by RNAi. The activity or expression of the cyclase 2 gene (CYC2) can be inhibited by RNAi using miRNA, siRNA, dsRNA or shRNA.

[0187] In one embodiment, the method of inhibiting the activity or expression of the cyclase 2 gene (CYC2) targets at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of the cyclase 2 gene (CYC2). In a preferred embodiment, the method targets at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of the cyclase 2 gene (CYC2). In one embodiment, the method targets at least a portion of exon 1, at least a portion of exon 2, and the first 115 nucleotides of the third exon of the cyclase 2 gene (CYC2). In one embodiment, the method targets nucleotides 1-25, nucleotides 26-271 (exon 1), nucleotides 1253-1529 (exon 2), and the first 115 nucleotides of the third exon (nucleotides 2366-2480) of the cyclase 2 gene (CYC2), wherein the numbering is determined by comparison with SEQ ID No. 8.

[0188] In any of the foregoing embodiments, the activity or expression of the cyclase 2 gene (CYC2) can be inhibited using a gene editing method including CRISPR (including using a CRISPR-Cas9 system). In any of the foregoing embodiments, the activity or expression of the cyclase 2 gene (CYC2) can be inhibited using an RNAi method.

[0189] Suitably, the activity or expression of the CBTol cyclase gene can be suppressed by any method known in the art. In one embodiment, the sequence of the CBTol cyclase gene is as shown in SEQ ID No.1. In one embodiment, the amino acid sequence of the CBTol cyclase is as shown in SEQ ID No.2. The expression of the CBTol cyclase gene can be suppressed by any method, including the gene editing method including CRISPR, including the use of CRISPR-Cas9 system, RNA interference (RNAi), antisense or sense co-suppression, gene editing or directed mutagenesis. In one embodiment, the activity or expression of the CBTol cyclase gene can be suppressed by RNAi. The activity or expression of the CBTol cyclase gene can be suppressed by RNAi using miRNA, siRNA, dsRNA or shRNA.

[0190] In another embodiment, the method of inhibiting the activity or expression of the CBTol cyclase gene targets at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and at least a portion of exon 7 of the CBTol cyclase. In a preferred embodiment, the method targets at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and at least a portion of exon 7 of the CBTol cyclase gene. In one embodiment, the method targets nucleotides 5'-2854-4175-3' of the CBTol cyclase gene, wherein the numbering is determined by alignment with SEQ ID No.1.

[0191] In any of the aforementioned embodiments, the activity or expression of the CBTol cyclase gene can be suppressed using the gene editing method including CRISPR (including using the CRISPR-Cas9 system).In any of the aforementioned embodiments, the activity or expression of the CBTol cyclase gene can be suppressed by using the RNAi method.

[0192] Suitably, the activity or expression of the terpene synthase 3-8 gene can be inhibited by any method known in the art. In one embodiment, the sequence of the terpene synthase 3-8 gene is as shown in SEQ ID No.3. In one embodiment, the amino acid sequence of terpene synthase 3-8 is as shown in SEQ ID No.4. The expression of the terpene synthase 3-8 gene can be inhibited by any method, including a gene editing method including CRISPR, including the use of a CRISPR-Cas9 system, RNA interference (RNAi), antisense or sense co-suppression, gene editing or directed mutagenesis. In one embodiment, the activity or expression of the terpene synthase 3-8 gene can be inhibited by RNAi. The activity or expression of the terpene synthase 3-8 gene can be inhibited by RNAi using miRNA, siRNA, dsRNA or shRNA. In one embodiment, the method for inhibiting the activity or expression of the terpene synthase 3-8 gene targets at least nucleotides 1497 to 1517 of the terpene synthase 3-8 gene, wherein the numbering is determined by comparing with SEQ ID No.3. In one embodiment, the method of inhibiting the activity or expression of the terpene synthase 3-8 gene targets at least nucleotides 884 to 904 of the terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No. 3. In a preferred embodiment, the RNAi method targets nucleotides 884 to 904 of the terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No. 3.

[0193] In any of the foregoing embodiments, the activity or expression of the terpene synthase 3-8 gene can be inhibited using a gene editing method including CRISPR (including using a CRISPR-Cas9 system). In any of the foregoing embodiments, the activity or expression of the terpene synthase 3-8 gene can be inhibited using an RNAi method.

[0194] In one embodiment, the method can include transforming cells of a tobacco plant with a DNA-directed RNA interference (ddRNAi) construct packaged in a delivery vector, the construct comprising a nucleotide sequence encoding a dsRNA or a nucleotide sequence encoding an amiRNA.

[0195] In one embodiment, the RNAi molecule is a dsRNA encoded by a ddRNAi DNA construct.

[0196] In one embodiment, the RNAi molecule is an amiRNA encoded by a ddRNAi DNA construct. In one embodiment, the present invention provides a construct comprising a ddRNAi DNA sequence designed to encode a dsRNA that inhibits the expression of a diterpene biosynthesis gene.

[0197] In another embodiment, the present invention provides a construct comprising a ddRNAi DNA sequence designed to encode an amiRNA that inhibits the expression of a diterpene biosynthesis gene.

[0198] The construct may be comprised in a vector. Suitably, the vector may be a plasmid.

[0199] In one embodiment, the vector used in the present invention is based on the plasmid pKYLX71:35S2 of agrobacterium. This plasmid is based on the pGA471 plasmid described by An et al. (An, G. et al. 1985 EMBO J. 4, 277-284, which is incorporated herein by reference). In another embodiment, the vector used in the present invention is pCAMBIA2300. The pCAMBIA vector backbone is derived from the pPZP vector.

[0200] As used herein, term "DNA-guided RNA interference (ddRNAi)" means a DNA construct for endogenous RNA interference (RNAi) pathways for activating cells. Suitably, these constructs are designed to express self-complementary RNA, for example, double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) or short hairpin RNA (shRNA) or microRNA (such as artificial microRNA-amiRNA), which once processed, causes the silencing of one or more target genes. Advantageously, the use of ddRNAi means that RNA (such as dsRNA or amiRNA) expressed is continuously produced, and thus the long-term silencing of targeted genes can be provided. On the contrary, the small interfering RNA (siRNA) (for example, not continuously expressed from the ddRNAi DNA construct according to the present invention) directly applied to cells is transformed in cells and only transiently silences genes.

[0201] In one embodiment, the method or use may comprise inhibiting the activity or expression of a diterpene synthesis gene using dsRNA expressed from a ddRNAi DNA construct.

[0202] In another embodiment, the method or use may comprise inhibiting the activity or expression of a diterpene synthesis gene using an amiRNA expressed from a ddRNAi DNA construct.

[0203] Therefore, in one embodiment, provide tobacco plant and tobacco plant propagation material, tobacco leaf, cutting the leaf of harvest, the tobacco leaf of processing or cutting and processing tobacco leaf, wherein use ddRNAi DNA construct to suppress the expression of diterpene synthesis gene.Suitably, ddRNAi DNA construct can be incorporated in the genomic DNA of plant.Tobacco plant, tobacco plant propagation material, tobacco leaf, cutting the leaf of harvest, the tobacco leaf of processing or cutting and processing tobacco leaf can comprise ddRNAi DNA construct, and its expression suppresses the activity of diterpene synthesis gene or the dsRNA or amiRNA of expression.Compared with tobacco plant, tobacco plant propagation material, tobacco leaf, cutting the leaf of harvest, the tobacco leaf of processing or cutting and processing tobacco leaf that do not comprise ddRNAi DNA construct, tobacco plant, tobacco plant propagation material, tobacco leaf, cutting the leaf of harvest, the tobacco leaf of processing or cutting and processing tobacco leaf that comprises the ddRNAi DNA construct of the dsRNA or amiRNA of expression suppressing the activity of diterpene synthesis gene or expression can have increased sucrose ester content.

[0204] The ddRNAi DNA construct can include all or part of a diterpene synthesis gene. The construct can include exons and / or introns of a diterpene synthesis gene. The ddRNAi DNA construct can include a partial gene sequence that produces a hairpin RNA structure when transcribed. The RNA encoded by the ddRNAi DNA construct can have an intron-hairpin or a GUS-hairpin structure, or can be a single full-length RNA. As described herein, the inventor has demonstrated that the RNAi molecules that inhibit the activity or expression of diterpene synthesis genes are used to increase the surprising effectiveness of the sucrose ester content in tobacco plants.

[0205] The term "exon" as used herein means a portion of a gene that encodes the final mature RNA produced by the gene after removal of introns by RNA splicing.

[0206] The term "intron" as used herein means a nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product.

[0207] As used herein, the term "at least a portion of" or "partial sequence" means a sequence comprising at least 5, 10, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 400, or at least 500 consecutive nucleotides. For example, a ddRNAi DNA construct may comprise at least a portion of an exon, wherein the construct comprises 100 consecutive nucleotides from said exon.

[0208] As used herein, the term "mRNA corresponding to..." means that the RNA has the same sequence as the DNA, that is, the nucleotide sequences in the mRNA and DNA sequences are identical, except that in the RNA, thymine (T) is replaced by uracil (U), and deoxyribose is replaced by ribose.

[0209] The ddRNAi DNA construct can be used to inhibit the expression of the cyclase 2 gene (CYC2). In one embodiment, the sequence of the cyclase 2 gene (CYC2) is shown in SEQ ID No. 8. In one embodiment, the amino acid sequence of the polypeptide encoded by the cyclase 2 gene (CYC2) is shown in SEQ ID No. 10. Suitably, the ddRNAi DNA construct may comprise all or part of the cyclase 2 gene (CYC2).

[0210] In one embodiment, the ddRNAi DNA construct may comprise at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of a cyclase 2 gene (CYC2). Suitably, the ddRNAi DNA construct may comprise at least a portion of exon 1, at least a portion of exon 2, and the first 115 nucleotides of exon 3 of a cyclase 2 gene (CYC2). Suitably, the ddRNAi DNA construct may comprise nucleotides 1-25, nucleotides 26-271 (exon 1), nucleotides 1253-1529 (exon 2), and the first 115 nucleotides of exon 3 (nucleotides 2366-2480) of a cyclase 2 gene (CYC2), wherein the numbering is determined by comparison with SEQ ID No. 8. Suitably, the ddRNAi DNA construct may comprise the sequence shown in SEQ ID No. 5.

[0211] The ddRNAi DNA construct can be used to inhibit the expression of the CBTol cyclase gene. In one embodiment, the sequence of the CBTol cyclase gene is as shown in SEQ ID No.1. In one embodiment, the amino acid sequence of the CBTol cyclase is as shown in SEQ ID No.2. Suitably, the ddRNAi DNA construct can include all or part of the CBTol cyclase gene.

[0212] In another embodiment, the ddRNAi DNA construct may comprise at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and exon 7 of the CBTol cyclase gene. Suitably, the ddRNAi DNA construct may comprise nucleotides 5'-2854-4175-3' of the CBTol cyclase gene, wherein the numbering is determined by alignment with SEQ ID No. 1. Suitably, the ddRNAi DNA construct may comprise the sequence shown in SEQ ID No. 6.

[0213] The ddRNAi DNA construct can be used to inhibit the expression of the terpene synthase 3-8 gene. Suitably, the ddRNAi DNA construct can include all or part of the terpene synthase 3-8 gene. Suitably, the ddRNAi DNA construct can include at least nucleotides 1497 to 1517 of the terpene synthase 3-8 gene, wherein the numbering is determined by comparing with SEQ ID No.3. In one embodiment, the sequence of the terpene synthase 3-8 gene is as shown in SEQ ID No.3. In one embodiment, the amino acid sequence of terpene synthase 3-8 is as shown in SEQ ID No.4. Suitably, the ddRNAi DNA construct can include the sequence shown in SEQ ID No.7. Suitably, the ddRNAi DNA construct can include at least nucleotides 884 to 904 of the terpene synthase 3-8 gene, wherein the numbering is determined by comparing with SEQ ID No.3. Suitably, the ddRNAi DNA construct can include the sequence shown in SEQ ID No.11.

[0214] In one embodiment, the sequence of the ddRNAi DNA construct encoding the dsRNA may comprise the sequence shown in SEQ ID No. 7. The dsRNA expressed from the ddRNAi DNA construct comprising SEQ ID No. 7 can be used to inhibit the activity or expression of a diterpene synthesis gene or down-regulate a diterpene synthesis gene, wherein the diterpene synthesis gene is a cyclase 2 gene (CYC2).

[0215] In another embodiment, the sequence of the ddRNAi DNA construct encoding the dsRNA may comprise the sequence as shown in SEQ ID No. 8. The dsRNA expressed from the ddRNAi DNA construct comprising SEQ ID No. 8 can be used to inhibit the activity or expression of a diterpene synthesis gene or down-regulate a diterpene synthesis gene, wherein the diterpene synthesis gene is a CBTol cyclase gene.

[0216] In one embodiment, the sequence of the ddRNAi DNA construct encoding the amiRNA may comprise a sequence as shown in SEQ ID No. 9. The amiRNA expressed from the ddRNAi DNA construct comprising SEQ ID No. 9 can be used to inhibit the activity or expression of a diterpene synthesis gene or downregulate a diterpene synthesis gene, wherein the diterpene synthesis gene is a terpene synthase 3-8 gene.

[0217] As used herein, the term "GW1" or "GW-1" may mean a ddRNAi DNA construct comprising at least a portion of exon 1, at least a portion of exon 2, and the first 115 nucleotides of exon 3 of the cyclase 2 gene (CYC2).

[0218] The term "GW2" or "GW-2" as used herein may mean a ddRNAi DNA construct comprising at least nucleotides 1497 to 1517 of the terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No.3.

[0219] The term "GW3" or "GW-3" as used herein may mean a ddRNAi DNA construct comprising at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and at least a portion of exon 7 of the CBTol cyclase gene.

[0220] The term "GW5" or "GW-5" as used herein may mean a ddRNAi DNA construct comprising at least nucleotides 884 to 904 of a terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No.3.

[0221] Therefore, the ddRNAi DNA construct of the present invention may comprise a nucleotide sequence encoding a dsRNA, wherein the dsRNA encoding sequence is a nucleotide sequence substantially as shown in SEQ ID No. 5 or 8, or a functional variant or fragment thereof.

[0222] Suitably, the ddRNAi DNA construct of the present invention may comprise a nucleotide sequence encoding a dsRNA, wherein the dsRNA encoding sequence is substantially the nucleotide sequence as shown in SEQ ID No. 5 or a functional variant or fragment thereof.

[0223] Suitably, the ddRNAi DNA construct of the present invention may comprise a nucleotide sequence encoding a dsRNA, wherein the dsRNA encoding sequence is a nucleotide sequence substantially as shown in SEQ ID No. 6 or a functional variant or fragment thereof.

[0224] In another embodiment, the ddRNAi DNA construct of the present invention may comprise a nucleotide sequence encoding an amiRNA, wherein the amiRNA encoding sequence is a nucleotide sequence substantially as shown in SEQ ID No.9.

[0225] It should be understood that it is relatively simple for the technician to modify the sequence encoding dsRNA (e.g., SEQ ID No.7) or amiRNA (e.g., SEQ ID No.9) to produce a variant or fragment of a ddRNAi DNA construct, which will still function to inhibit or lower the expression of diterpene synthesis genes, thereby increasing the sucrose content of tobacco plants. Functional variants and fragments of ddRNAi DNA constructs can be easily identified by using standard laboratory techniques to determine whether the level of mRNA encoded by terpene synthesis genes has been reduced to a level lower than the same diterpene synthesis gene mRNA in the corresponding wild-type plant cells grown under the same conditions. An example of this technology is polymerase chain reaction (PCR). The skilled person will understand that by using standard techniques, such as enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting, Western blotting or chromatin immunoprecipitation (ChIP), the concentration of the polypeptide encoded by diterpene synthesis genes can be directly measured in wild-type and transgenic plants.

[0226] In another embodiment, tobacco cell, tobacco plant or its part and / or plant propagation material can comprise the construct of suppressing the activity of diterpene synthesis gene or expression.In a preferred embodiment, described construct is ddRNAiDNA construct, more preferably, described ddRNAiDNA construct comprises the RNAi module of suppressing the activity of cyclase 2 gene (CYC2), CBTol cyclase gene or terpene synthase 3-8 gene or expression.

[0227] In a further embodiment, the tobacco cell, tobacco plant or part thereof and / or plant propagation material may comprise:

[0228] i) the polynucleotide sequence shown herein as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.11, or

[0229] ii) a fragment of the polynucleotide sequence shown in i), wherein the functional fragment inhibits the activity or expression of a diterpene biosynthesis gene, or

[0230] iii) a polynucleotide sequence having at least 70% (preferably 85%, more preferably 90%) identity with the polynucleotide shown in i) or ii) above.

[0231] In one embodiment, the polynucleotide sequence may be at least 80% identical to SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 11. Suitably, the polynucleotide sequence may be at least 90% identical to SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 11. Suitably, the polynucleotide sequence may be at least 95% identical to SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 11 (more suitably at least 99% identical).

[0232] In a favourable embodiment, the activity of diterpene synthetic gene or the inhibition of expression can cause the sucrose ester content of tobacco plant to increase, and cause the change of one or more other flavor components of tobacco plant simultaneously.The activity of diterpene synthetic gene or the inhibition of expression can cause the increase of sucrose ester content and the change of diterpene content.The increase of sucrose ester content can be accompanied by one or more increases that are selected from following flavor compound content: cis-abirol, labdenediol and CBT-ol.

[0233] The increase in sucrose ester content can be accompanied by an increase in cis-abirol content and an increase in labdenediol content. In another embodiment, the increase in sucrose ester content can be accompanied by an increase in labdenediol content. The increase in sucrose ester content can be accompanied by an increase in CBT-ol and high cis-abirol content.

[0234] In one embodiment, the methods of the invention produce burley or flue-cured leaves having turkey-like exudate chemistry. Preferably, the burley or flue-cured leaves have chemistry more similar to turkey-like tobacco than comparable burley or flue-cured plants that do not have the activity or expression of inhibited diterpene synthesis genes.

[0235] In one embodiment, tobacco plant according to the present invention or its part is burley or flue-cured plant modified according to the present invention. In one embodiment, the present invention relates to burley or flue-cured plant modified according to the present invention with Turkey sample exudate chemical substance. In one embodiment, tobacco plant according to the present invention (for example modified tobacco plant) is not oriental or Türkiye tobacco plant.

[0236] In one embodiment, the tobacco plant or its part is cured. In one embodiment, the tobacco plant or its part is cured, such as air-cured, flue-cured, open flame-cured or sun-cured. In a further aspect, the tobacco plant or its part is flue-cured. In a further aspect, the tobacco plant or its part is air-cured.

[0237] Flue curing is well known in the art and refers to a method of curing tobacco with a flue, which is fed by a firebox or gas system. The process heat cures the tobacco without exposing it to smoke, slowly increasing the temperature during the curing process. The method produces tobacco that is high in sugars and has moderate to high levels of nicotine. Smith Tobacco Barn is an example of a traditional flue-cured tobacco barn.

[0238] Cured tobaccos include Burley, Maryland, and Dark tobaccos. The common factor is that curing is done primarily without artificial heat and humidity. Burley tobacco is light to dark brown in color, high in oils, and low in sugars. Burley tobacco is cured in barns. The main Burley growing countries are Argentina, Brazil, Italy, Malawi, and the United States. Burley tobacco plants include, for example, Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R711, R 712, NCBH 129, Bu 21xKy10, HB04P, Ky 14xL 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509.

[0239] Maryland tobacco is extremely fluffy, has good burning characteristics, low nicotine, and a neutral aroma. Major Maryland tobacco growing countries include the United States and Italy. The main difference between dark-cured tobacco and other types is its fermentation process, which gives dark-cured tobacco a medium to dark brown color and a unique aroma. Their leaves have a low sugar content but a high nicotine content. Dark-cured tobacco is mainly used to produce chewing tobacco and snuff. The main growing areas of dark fire-cured tobacco are Tennessee, Kentucky, and Virginia in the United States.

[0240] In a preferred embodiment, the tobacco of the present invention contains at least as much 3-methylvaleric acid acetylated sucrose ester as Turkish-like tobacco.

[0241] "Turkey-like exudate chemicals" means that the exudate produced by the plant of the present invention has similar exudate chemicals observed in Turkish tobacco. The chemical substances of Turkish (or Oriental) tobacco, such as common tobacco Samsun subspecies, have been characterized by R. Severson et al. 1985 (supra, which is incorporated herein by reference). It is known that Turkish tobacco contains low levels of CBT-diols, high levels of oxidation products of CBT and high levels of sucrose esters containing 3-methylvaleric acid. Therefore, the present invention advantageously provides a higher biomass tobacco plant with Turkish tobacco-like trichome exudates.

[0242] In one embodiment, provide tobacco plant, it comprises the 3-methyl valeric acid sucrose ester of increase.Compare with the tobacco plant not modified according to the present invention (that is, the plant of the inhibition of wherein not introducing diterpene synthesis gene), described tobacco plant can comprise the 3-methyl valeric acid sucrose ester of increase.Preferably, compare with the control tobacco plant of unmodified to suppress the activity of diterpene synthesis gene or expression, tobacco strain of the present invention contains at least twice the 3-methyl valeric acid sucrose ester.In a further aspect, tobacco plant of the present invention contains at least as many 3-methyl valeric acid esters as Turkish type tobacco.

[0243] Advantageously, tobacco plant of the present invention has normal growth in the field.In one embodiment, CBTol synthase strikes low plant and advantageously has low CBT diols, high cis-abirol, high sucrose ester containing 3-methylvaleric acid like Turkish tobacco, but different from Turkish type, CBTol synthase of the present invention strikes low plant and can produce the biomass higher than Turkish tobacco plant.For Turkish tobacco type, output is usually in the scope of 600 to 1100 pounds / acre.

[0244] The term "functional fragment" as used herein refers to a part of a polynucleotide that can function in the same manner as a polynucleotide. For example, if the polynucleotide is a diterpene synthesis gene, the functional fragment must be able to function as a diterpene synthesis gene, such as the functional fragment retains the activity of the diterpene synthesis gene. The functional fragment can have an activity level equal to or greater than the activity level of the full-length polynucleotide. If the polynucleotide encodes dsRNA or amiRNA, the functional fragment must be able to inhibit the activity or expression of the diterpene synthesis gene.

[0245] In one embodiment, the functional fragment can be a part of a diterpene synthesis gene as discussed herein, comprising at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 consecutive nucleotides. In some embodiments, the functional fragment can comprise at least 150 nucleotides of a diterpene synthesis gene as discussed herein. In one embodiment in which the functional fragment is a functional fragment of a ddRNAi DNA construct, the functional fragment can be a part of an RNAi as discussed herein, comprising at least 50, 75, 100, 150, 200 or 250 consecutive nucleotides.

[0246] As used herein, the term "degeneracy of the genetic code" refers to the redundancy of the codons encoding polypeptide sequences, which is manifested as the diversity of the three-codon combination of the specified amino acid. For example, in an mRNA molecule encoding a polypeptide having an isoleucine amino acid, the isoleucine can be encoded by AUU, AUC or AUA. This means that the DNA molecule encoding the RNA can have a variety of sequences, but the resulting polypeptide will have the same sequence. In other words, polymorphic nucleotide sequences can encode the same polypeptide product. This means that a nucleic acid sequence can contain a sequence with very low sequence identity to a second sequence, but encodes the same polypeptide sequence.

[0247] The methods and uses of the present invention include inhibiting at least one diterpene biosynthesis gene. Inhibition can be achieved by any means known to those skilled in the art.

[0248] In some embodiments of the present invention, a promoter may be provided. The promoter used in the present invention may be selected from one or more of the following: a constitutive promoter, an aging-specific promoter, a tissue-specific promoter, a developmentally regulated promoter, and an inducible promoter. In one embodiment, the promoter may be a constitutive promoter.

[0249] Constitutive promoters direct the expression of a gene continuously throughout various parts of the plant during plant development, although the gene may not be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with the cauliflower mosaic virus 35S transcript (Odell JT, Nagy F, Chua NH. (1985). Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313 810-2) and the rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991), which is incorporated herein by reference. Analysis of rice Act1 5'region activity in transgenic rice plants. (Plant Cell 3 1155-65, which is incorporated herein by reference) and the maize ubiquitin 1 gene (Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE. (1993). Activity of a maize ubiquitin promoter in transgenic rice. Plant Molec. Biol. 23 567-81 (which is incorporated herein by reference)). A constitutive promoter such as the carnation etched ring virus (CERV) promoter (Hull R, Sadler J, Longstaff M (1986, which is incorporated herein by reference) These sequences of carnation etched ring virus DNA: comparison with cauliflowermosaic virus and retroviruses. EMBO Journal, 5(2):3083-3090, which is incorporated herein by reference).

[0250] The constitutive promoter may be selected from: the Carnation Etch Ring Virus (CERV) promoter, the Cauliflower Mosaic Virus (CaMV35S promoter), the promoter from the rice actin 1 gene or the maize ubiquitin 1 gene. Suitably, the promoter is the CERV promoter.

[0251] Alternatively, in one embodiment, the promoter may not be the cauliflower mosaic virus (CaMV 35S promoter). In one embodiment, the promoter may be a senescence-specific promoter. A "senescence-specific promoter" (SAG) may be a promoter associated with controlling the expression of senescence-related genes. Thus, the promoter may substantially and exclusively restrict the expression of a coding sequence (i.e., a gene) to which it is operably linked to, to tissues that are senescent. Thus, a senescence-specific promoter may be a promoter that preferentially promotes gene expression in plant tissues in a developmentally regulated manner, such that expression of the 3' protein coding region occurs substantially only when plant tissues are subjected to senescence. It should be understood that senescence tends to occur in older parts of plants, such as older leaves, rather than in younger parts of plants, such as seeds.

[0252] An example of a plant known to express many aging-related genes is Arabidopsis. Therefore, promoters can be isolated from aging-related genes of Arabidopsis. Gepstein et al. (The Plant Journal, 2003, 36, 629-642), which is incorporated herein by reference, used Arabidopsis as a model for detailed studies of SAG and its promoter. The genetic construct can contain a promoter from any SAG disclosed in the document. For example, a suitable promoter can be selected from SAG12, SAG13, SAG101, SAG21 and SAG18 or a functional variant or functional fragment thereof.

[0253] In one embodiment, the promoter may be a SAG12 or SAG13 promoter. In one embodiment, the promoter may be a SAG12 promoter known to the skilled artisan, or a functional variant or functional fragment thereof (Gan & Amasino, 1997, Plant Physiology, 113: 313-319, which is incorporated herein by reference). Suitable promoters and their sequences may be found in WO2010 / 097623 (which is incorporated herein by reference).

[0254] In another embodiment, the promoter can be a tissue-specific promoter. A tissue-specific promoter is a promoter that directs the expression of a gene in one (or several) parts of a plant, typically during the entire life span of these plant parts. The types of tissue-specific promoters also generally include promoters whose specificity is not absolute, i.e., they can also direct lower levels of expression in tissues other than preferred tissues. Many tissue-specific promoters are known in the art and include those associated with the potato glycoprotein gene expressed in potato tubers and the high molecular weight glutenin gene expressed in wheat, barley or corn endosperm. Any of these promoters can be used in the present invention.

[0255] Suitably, the tissue-specific promoter may be a leaf-specific promoter. Suitably, the leaf-specific promoter may include ASYMMETRIC LEAVES 1 (AS1) In a particularly preferred embodiment, the tissue-specific promoter is not a root-specific promoter.

[0256] In another embodiment, the promoter can be a developmentally regulated promoter. A developmentally regulated promoter directs changes in gene expression in one or more parts of a plant at a specific time during plant development. The gene may be expressed in the plant part at other times at different (usually lower) levels, and may also be expressed in other plant parts.

[0257] In one embodiment, the promoter can be an inducible promoter. An inducible promoter can direct the expression of a gene in response to an inducer. In the absence of an inducer, the gene will not be expressed. The inducer can act directly on the promoter sequence, or can work by counteracting the effect of a repressor protein molecule. The inducer can be a chemical reagent such as a metabolite, an albumen, a growth regulator or a toxic element, an indirect result of a physiological stress such as heat, wound or osmotic pressure, or the effect of a pathogen or an insect. The developmentally regulated promoter can be described as a specific type of inducible promoter that reacts to an environmental stimulus at a specific time point in the endogenous inducer produced by a plant or a plant life cycle. Examples of known inducible promoters include those associated with wound response, such as described by Warner SA, Scott R, Draper J. (1993) (Isolation of an asparagus intracellular PR gene (AoPR1) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J. 3 191-201.), incorporated herein by reference, temperature response as disclosed by Benfey & Chua (1989) (Benfey, PN, and Chua, NH. (1989) Regulated genes in transgenic plants. Science 244 174-181), incorporated herein by reference, and chemical induction, such as described by Gatz (1995) (Gatz, C. (1995) Novel inducible / repressible gene expression systems. Methods in Cell Biol. 50 411-424), incorporated herein by reference.

[0258] Thus, in one embodiment, the promoter may be selected from the group consisting of: a CERV promoter, a cauliflower mosaic virus 35S promoter (complete or truncated), a rubisco promoter, a pea plastocyanin promoter, a nopaline synthase promoter, a chlorophyll r / b binding promoter, a high molecular weight glutenin promoter, an α, β-gliadin promoter, a hordein promoter, and a potato glycoprotein promoter.

[0259] In one embodiment, the promoter can be the CaMV 35S promoter or a modified 35S promoter having a duplicate enhancer region or a double enhancer region (R. Kay et al. Science. 1987 Jun 5;236(4806):1299-302, which is incorporated herein by reference).

[0260] The recombinant vector may also include DNA encoding a gene that can be used as a selectable marker during the cloning process, i.e., to enable selection of cells that have been transfected or transformed, and to enable selection of cells carrying a vector into which heterologous DNA has been incorporated. The vector may also include DNA that is involved in regulating the expression of the coding sequence or for targeting the expressed polypeptide to a specific part of the host cell (e.g., to the chloroplast). Thus, the vector may include at least one additional element selected from: a selectable marker gene (e.g., an antibiotic resistance gene); a polypeptide termination signal; and a protein targeting sequence (e.g., a chloroplast transit peptide).

[0261] Examples of suitable marker genes include antibiotic resistance genes, such as those that confer resistance to kanamycin, geneticin (G418) and hygromycin (npt-II, hyg-B); herbicide resistance genes, such as those that confer resistance to phosphinothricin and sulfonamide-based herbicides (bar and suI, respectively; EP-A-242246, EP-A-0249637); and screenable markers, such as beta-glucuronidase (GB2197653), luciferase and green fluorescent protein (GFP). The marker gene can be controlled by a second promoter, which allows expression in cells, which may or may not be in seeds, thereby allowing cells or tissues containing the marker to be selected at any stage of plant development. Suitable second promoters are promoters of the nopaline synthase gene of Agrobacterium and promoters of genes derived from encoding 35S cauliflower mosaic virus (CaMV) transcripts. However, any other suitable second promoter may be used.

[0262] Commercially desirable traits

[0263] The term "commercially desirable trait" will include traits such as yield, mature plant height, number of harvestable leaves, average node length, cut leaf length, cut leaf width, quality, abiotic (e.g. drought) stress tolerance, herbicide tolerance, and / or biotic (e.g. insect, bacterial or fungal) stress tolerance.

[0264] The term "commercially desirable trait" as taught herein means one or more traits selected from drought resistance, pest resistance, mature plant height, number of harvestable leaves, average node length, cut leaf length, cut leaf width, and yield, which are comparable to those said traits in the flue-cured parent of a comparable plant when grown under similar field conditions.

[0265] Unless otherwise indicated, tobacco yield as used herein refers to curing leaf yield, which is calculated based on the weight of curing leaf per acre under standard field conditions following standard agricultural and curing practices.

[0266] In one aspect, tobacco plants of the invention have 50% to 150%, 55% to 145%, 60% to 140%, 65% to 135%, 70% to 130%, 75% to 125%, 80% to 120%, 85% to 115%, 90% to 110%, 95% to 105% of the yield of comparable plants when grown under similar field conditions, 100%, 105% to 150%, 110% to 150%, 115% to 150%, 120% to 150%, 125% to 150%, 130% to 150%, 135% to 150%, 140% to 150% or 145% to 150% of the yield.

[0267] In another aspect, the tobacco plants of the invention yield approximately 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 times the yield of comparable plants when grown under similar field conditions.

[0268] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable plants that are flue-cured when grown under similar field conditions.

[0269] In one aspect, the tobacco plants of the invention provide a yield selected from the group consisting of about 1200 to 3500, 1300 to 3400, 1400 to 3300, 1500 to 3200, 1600 to 3100, 1700 to 3000, 1800 to 2900, 1900 to 2800, 2000 to 2700, 2100 to 2600, 2200 to 2500, and 2300 to 2400 pounds per acre.

[0270] In another aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1300 to 3500, 1400 to 3500, 1500 to 3500, 1600 to 3500, 1700 to 3500, 1800 to 3500, 1900 to 3500, 2000 to 3500, 2100 to 3500, 2200 to 3500, 2300 to 3500, 2400 to 3500, 2500 to 3500, 2600 to 3500, 2700 to 3500, 2800 to 3500, 2900 to 3500, 3000 to 3500, and 3100 to 3500 pounds per acre.

[0271] In a further aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1200 to 3400, 1200 to 3300, 1200 to 3200, 1200 to 3100, 1200 to 3000, 1200 to 2900, 1200 to 2800, 1200 to 2700, 1200 to 2600, 1200 to 2500, 1200 to 2400, 1200 to 2300, 1200 to 2200, 1200 to 2100, 1200 to 2000, 1200 to 1900, 1200 to 1800, 1200 to 1700, 1200 to 1600, 1200 to 1500, and 1200 to 1400 pounds per acre.

[0272] Tobacco Plant

[0273] The present invention provides methods and uses for tobacco plants as well as tobacco cells, tobacco plants and plant propagation materials.

[0274] As used herein, the term "tobacco" refers to the genus Nicotiana used to produce tobacco products. Nicotiana ) plants. Non-limiting examples of suitable "tobacco" plants include Nicotiana tabacum ( N. tabacum ) and Nicotiana rustica ( N. rustica ) (e.g., Nicotiana tabacum L., LA B21, LN KY171, Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).

[0275] In one embodiment, a suitable tobacco plant can be any Nicotiana tabacum species.

[0276] In another embodiment, a suitable tobacco plant may be a non-tobacco species.

[0277] Tobacco material can derive from or be obtained from the multiple varieties of common tobacco species, is commonly referred to as burley tobacco (Burley) variety, flue or light color (flue or bright) variety and dark (dark) variety.In some embodiments, tobacco material derives from burley tobacco, Virginia or dark tobacco plant.Tobacco plant can be selected from burley tobacco, rare tobacco, special tobacco, expansion tobacco etc.

[0278] The use of tobacco cultivars and premium tobacco cultivars is also contemplated herein. The tobacco plants used herein can therefore be tobacco cultivars or premium tobacco cultivars. Particularly useful common tobacco cultivars include burley type, dark type and flue-cured tobacco.

[0279] In some embodiments, the tobacco plant can be, for example, selected from one or more of the following varieties: Nicotiana tabacum L. cultivar TI 1068, Nicotiana tabacum AA 37-1, Nicotiana tabacum B 13P, Nicotiana tabacum Xanthi (Mitchell-Mor), Nicotiana tabacum KT D#3 Hybrid 107, Nicotiana tabacum Bel-W3, Nicotiana tabacum 79-615, Nicotiana tabacum SamsunHolmes NN, F4 from the cross of Nicotiana tabacum BU21 x Nicotiana tabacum Hoja Parado, line 97, Nicotiana tabacum KTRDC#2 Hybrid 49, Nicotiana tabacum KTRDC#4 Hybrid 1 10, Nicotiana tabacum Burley 21, Nicotiana tabacum PM016, Nicotiana tabacum KTRDC#5 KY 160 SI, Nicotiana tabacum KTRDC#7 FCA, Nicotiana tabacum KTRDC#6 TN 86 SI, Nicotiana tabacum PM021, Nicotiana tabacum K 149, Nicotiana tabacum K 326, Nicotiana tabacum K 346, Nicotiana tabacum K 358, Tobacco K 394, Tobacco K 399, Tobacco K 730, Tobacco KY 10, Tobacco KY 14, Tobacco KY160, Tobacco KY 17, Tobacco KY 8959, Tobacco KY 9, Tobacco KY 907, Tobacco MD 609, Tobacco McNair 373, Tobacco NC 2000, Tobacco PG 01, Tobacco PG 04, Tobacco P01, Tobacco P02, Tobacco P03, Tobacco RG 1 1, Tobacco RG 17, Tobacco RG 8, Tobacco Speight G-28, Tobacco TN 86, Tobacco TN 90, Tobacco VA 509, Tobacco AS44, Tobacco Banket A1, Tobacco Basma Drama B84 / 31, Tobacco Basma I Zichna ZP4 / B, Tobacco Basma Xanthi BX 2A, Tobacco Batek, Tobacco Besuki Jember, Tobacco C104, Tobacco Coker 319, Tobacco Coker 347, Tobacco Criollo Misionero, Tobacco PM092, Tobacco Delcrest, Tobacco Djebel 81, Tobacco DVH 405, Tobacco Galpao Comum, Tobacco HB04P, Tobacco Hicks Broadleaf, Tobacco Kabakulak Elassona, Tobacco PM102, Tobacco Kutsage E1, Tobacco KY 14xL8, Tobacco KY 171, Tobacco LABU 21, McNair944, NC 2326, NC 71, NC 297, NC 3, PVH 03, PVH 09, PVH 19, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, PM132, Wislica, Yayaldag, NC 4, TR Madole, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, Tobacco TI-1068, Tobacco KDH-960, Tobacco TI-1070, Tobacco TW136, Tobacco PM204, Tobacco PM205, Tobacco Basma, Tobacco TKF4028, Tobacco L8, Tobacco TKF 2002, Tobacco TN90, Tobacco GR141, Tobacco Basmaxanthi, Tobacco GR149, Tobacco GR153 and Tobacco Petit Havana.

[0280] Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF91 1, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, narrow leaf Madole, narrow leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD7302 LC, PD 7309 LC, PD 7312 LC 'Periq'e' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R 630, R 7-1 1, R 7-12, RG 17. RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, Tl1406, Tl 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TND950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B 13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, SamsunHolmes NN, KTRDC 2nd hybrid 49, Burley 21, KY 8959, KY 9, MD 609, PG 01, PG 04, P01, P02, P03, RG 1 1, RG 8, VA 509, AS44, Banket A1, Basma Drama B84 / 31, Basma IZichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, CriolloMisionero, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 21 10, RedRussian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, PrilepP23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, Tl-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi、GR149、GR153、Petit Even if not specifically indicated herein, the above-mentioned lowconverter subvarieties are also contemplated.

[0281] The tobacco plant may be Burley.

[0282] In one embodiment, the tobacco plant is Nicotiana tabacum L. In a most preferred embodiment, the tobacco plant is Nicotiana tabacum L cultivar TI1068.

[0283] In one embodiment, the plant propagation material may be obtainable from the tobacco plant of the present invention. "Plant propagation material" as used herein refers to any plant matter obtained from a plant from which further plants can be produced. Suitably, the plant propagation material may be a seed.

[0284] In one embodiment, tobacco cell of the present invention, tobacco plant and / or plant propagation material can comprise the diterpene synthetic gene of inhibition or downward regulation.In another embodiment, tobacco cell, tobacco plant and / or plant propagation material can comprise construct or carrier according to the present invention.In another embodiment, tobacco cell, tobacco plant and / or plant propagation material can be by obtainable (for example obtaining) according to method according to the present invention.

[0285] Suitably, the tobacco plant or part thereof according to the present invention may comprise reduced expression of at least one diterpene synthesis gene when compared to a tobacco plant or part thereof which has not been modified to inhibit expression of a diterpene synthesis gene.

[0286] In one embodiment, the tobacco plant or part thereof according to the invention comprises the tobacco cell of the invention.In another embodiment, the plant propagation material may be obtainable (eg obtained) from the tobacco plant of the invention.

[0287] In one embodiment, there is provided a use of a tobacco cell as provided in the preceding embodiments for producing a tobacco product. In addition, there is provided a use of a tobacco plant as described herein for cultivating a tobacco plant.

[0288] In another embodiment, the present invention also provides the tobacco plant of preceding embodiment for the purposes of producing tobacco product.In another embodiment, provide tobacco plant of the present invention for the purposes of growing crops.In one embodiment, can cause the change of the exudate chemical substance of tobacco plant according to the use of diterpene synthetic gene of the present invention.

[0289] In one embodiment, the use of the diterpene synthesis gene according to the present invention can result in an increase in the sucrose ester content of the tobacco plant. In another embodiment, the use of the diterpene synthesis gene (e.g., its inhibition) can result in an increase in the trichome exudate concentration of sucrose esters and an increase in the content of one or more of CBT-ol, cis-aberol, and labdenediol. Suitably, this situation can be observed when the plant shows reduced diterpene synthesis gene expression compared to the wild-type plant.

[0290] In one embodiment, the invention provides tobacco cell culture (for example in vitro culture).Described tobacco cell culture can be tobacco cell suspension culture.These tobacco cells of in vitro culture can be incorporated in the tobacco product, for example as the substitute of conventional tobacco particles, fragments, fine cutting or long cutting tobacco sheet, as additive composition or as substitute and additive.

[0291] In one embodiment, there is provided use of a tobacco cell culture, such as a harvested and / or processed tobacco cell culture, or an extract thereof, according to the invention for producing a tobacco product.

[0292] Tobacco cells harvested from in vitro culture can be dried, eg, freeze-dried, eg, to produce a powder.

[0293] The skilled person will appreciate the known methods for setting up the in vitro culture of tobacco cells. By way of example, only the following method can be used: collecting seeds from target tobacco plants and sterilizing their exterior to eliminate unwanted organisms, planting the seeds so that the target tobacco plants grow, removing tissue (e.g., from tobacco stems) from tobacco plants as explants, setting up callus cultures from tobacco explants, setting up cell suspension cultures from callus cultures, and gathering culture materials (e.g., comprising tobacco cells) to produce tobacco cell cultures.

[0294] Tobacco cells can be harvested by various methods including filtration, such as vacuum filtration. The sample can be washed in the filter by adding water and removing the remaining liquid by filtration, such as vacuum filtration.

[0295] The harvested tobacco cell culture may be further processed, for example, dried, such as air-dried and / or freeze-dried.The harvested tobacco cell culture or the dried harvested tobacco cell culture or extract thereof may be incorporated into a tobacco product according to the present invention.

[0296] product

[0297] The invention also provides products obtainable or obtained from tobacco according to the invention.Products obtainable or obtained from tobacco plants in which diterpene synthesis gene activity or expression has been inhibited and which comprise increased sucrose ester content are provided.

[0298] As used herein, the term "tobacco industry product" is intended to include combustible smoking articles, such as cigarettes, cigarillos, cigars, tobacco for pipes or for hand-rolled cigarettes (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokable materials), non-combustible aerosol delivery systems, such as heating products that release compounds from a matrix material without combustion, such as electronic cigarettes, tobacco heating products and hybrid systems that produce aerosols from a combination of matrix materials, for example hybrid systems containing liquid or gel or solid matrices, and aerosolizable matrix materials used in these aerosol delivery systems; and non-aerosol delivery products, such as lozenges, chewing gum, patches, products containing powders suitable for inhalation and smokeless tobacco products, such as snus and snuff, which may or may not deliver nicotine.

[0299] Suitably, the tobacco industry product may be prepared from (eg may comprise) a tobacco plant or part thereof according to the invention.

[0300] Suitably, the tobacco industry product may be prepared from the tobacco cell culture according to the present invention.

[0301] Suitably, the tobacco industry product may be prepared from (eg may comprise) a tobacco plant or part thereof propagated from tobacco plant propagation material according to the invention.

[0302] Suitably, the tobacco industry product may be prepared from (eg may comprise) harvested leaves of a tobacco plant according to the invention.

[0303] Suitably, the tobacco industry product may be prepared from (eg may comprise) processed tobacco leaves according to the invention.

[0304] Suitably, the tobacco industry product may be prepared from (eg may comprise) a cured tobacco material according to the invention.

[0305] Suitably, the tobacco industry product may be prepared from (eg may comprise) a tobacco blend according to the invention.

[0306] In one embodiment, the tobacco industry product is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0307] In one embodiment, the tobacco industry product comprises one or more components of a combustible smoking article, such as a filter, a filter plug, a filter plug segment, tobacco, a tobacco rod, a tobacco rod segment, an overflow, an additive release component such as a capsule, a thread, a bead, a paper such as a plug paper, a tipping paper or a cigarette paper.

[0308] In one embodiment, the tobacco industry product is a non-flammable aerosol delivery system.

[0309] In one embodiment, the tobacco industry product comprises one or more components of a non-combustible aerosol supply system, such as a heater and an aerosolizable substrate.

[0310] In one embodiment, the aerosol supply system is an electronic cigarette, also known as a vaping device.

[0311] In one embodiment, the electronic cigarette comprises a heater, a power source capable of powering the heater, an aerosolizable substrate such as a liquid or gel, a housing, and optionally a mouthpiece.

[0312] In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is combined with or comprises a heater.

[0313] In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating but not burning a matrix material. The matrix material is an aerosolizable material, which may be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0314] In one embodiment, the heated product is an electronic device.

[0315] In one embodiment, the tobacco heating product comprises a heater, a power source capable of powering the heater, and an aerosolizable substrate, such as a solid or gel material.

[0316] In one embodiment, the heating product is a non-electronic article.

[0317] In one embodiment, the heating product comprises an aerosolizable substrate, such as a solid or gel material; and a heat source capable of providing thermal energy to the aerosolizable substrate without any electronic means, such as by burning a combustion material, such as charcoal.

[0318] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0319] In some embodiments, the aerosolizable base material may include a vapor or aerosol generating agent or a humectant, such as glycerin, propylene glycol, triacetin, or diethylene glycol.

[0320] In one embodiment, the tobacco industry product is a hybrid system that produces aerosol by heating, but does not burn the combination of host material. Host material can include solid, liquid or gel, which may or may not contain nicotine. In one embodiment, the hybrid system includes liquid or gel matrix and solid matrix. Solid matrix can be tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the hybrid system includes liquid or gel matrix and tobacco.

[0321] In another embodiment, the product may comprise a ddRNAi DNA construct of the present invention, which inhibits diterpene synthesis gene activity or expression and increases sucrose ester content.

[0322] In one embodiment, tobacco plant of the present invention is provided for the purposes of producing tobacco leaves. Suitably, tobacco leaves can be subjected to downstream applications such as processing. Therefore, in one embodiment, the use of the aforementioned embodiments can provide processed tobacco leaves. Suitably, tobacco leaves can be subjected to modulation, fermentation, pasteurization or its combination.

[0323] In another embodiment, the tobacco leaves may be cut. In some embodiments, the tobacco leaves may be cut before or after being subjected to curing, fermentation, pasteurization, or a combination thereof.

[0324] In one embodiment, the invention provides the leaf of the results of tobacco plant of the present invention.In one embodiment, the leaf of the results can be available from tobacco plant, and described tobacco plant has the diterpene synthesis gene activity of inhibition or expression and the sucrose ester content of increase.In a further embodiment, the leaf of the results can be available from (for example obtained from) the tobacco plant bred from propagation material of the present invention.In another embodiment, the leaf of the results available from method of the present invention or purposes is provided.Suitably, the leaf of the results can be the leaf of cutting results.In some embodiments, the leaf of the results can comprise living tobacco cell.In some embodiments, the leaf of the results does not comprise living tobacco cell.In other embodiments, the leaf of the results can be further processed.

[0325] Some tobacco plants (such as Burley tobacco) can be harvested by cutting the stalks and harvesting all the leaves at the same time. Other tobacco plants (such as flue-cured tobacco) can be harvested in stages in a process such as infusion, where individual leaves are removed from the stalks as they mature.

[0326] Also provided is a processed tobacco leaf. The processed tobacco leaf can be available from tobacco plant of the present invention. Suitably, the processed tobacco leaf can be available from the tobacco plant obtained according to any method and / or purposes of the present invention. In one embodiment, the processed tobacco leaf can be available from a tobacco plant, and the tobacco plant preferably has the diterpene synthesis gene activity or expression of inhibition and the sucrose ester content of increase when compared with a control leaf, i.e. compared with the leaf from the tobacco plant not modified according to the present invention. The processed tobacco leaf can include a reduction in diterpene synthesis gene activity or expression and an increase in sucrose ester content.

[0327] In another embodiment, processed tobacco leaves may be obtainable from tobacco plants propagated from tobacco plant propagation material according to the invention.The processed tobacco leaves of the invention are obtainable by processing the harvested leaves of the invention.

[0328] As used herein, the term "processed tobacco leaf" refers to a tobacco leaf that has undergone one or more processing steps to which tobacco is subjected in the art."Processed tobacco leaf" contains no or substantially no living cells.

[0329] The term "living cell" refers to a cell that is capable of growth and / or metabolic activity. Thus, if a cell is not considered alive, also known as "non-viable," the cell does not display characteristics of a living cell.

[0330] The term "substantially free of viable cells" means that less than about 5% of all cells are viable. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of all cells are viable.

[0331] In one embodiment, the processed tobacco leaves may be processed by one or more of: curing, fermentation and / or pasteurization. Suitably, the processed tobacco leaves may be processed by curing. The tobacco leaves may be cured by any method known in the art. In one embodiment, the tobacco leaves may be cured by one or more of the following curing methods: air-curing, open flame curing, flue curing and sun-curing. Suitably, the tobacco leaves may be air-cured. Suitably, the tobacco leaves may be flue-cured.

[0332] In one embodiment the processed leaves according to the invention comprise an increased content of sucrose esters compared to a comparable product (eg processed leaves) which has not been modified according to the invention.

[0333] In one aspect, the air-cured leaves according to the invention comprise an increased content of sucrose esters compared to a comparable product (eg air-cured leaves) which has not been modified according to the invention.

[0334] Suitably, the sucrose ester content can be measured in the lower 1 / 3 leaves of the stalk-cut air-dried plants. Suitably, the sucrose ester content can be measured in the middle 1 / 3 leaves of the stalk-cut air-dried plants. Suitably, the sucrose ester content can be measured in the upper 1 / 3 leaves of the stalk-cut air-dried plants.

[0335] As used herein, "lower leaves" refers to the leaves in the lower third of a plant (e.g., leaves closest to the base of the plant), and "upper leaves" as used herein refers to the leaves in the upper third of a plant (e.g., leaves farthest from the base of the plant). As used herein, "middle leaves" refers to the central third of a plant between the lower and upper positions (e.g., leaves halfway between lower and upper leaves.

[0336] Suitably, the lower 1 / 3 of the leaves of the stalk-cut air-cured plant according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. the lower 1 / 3 of the leaves of the stalk-cut air-cured plant). Suitably, the middle 1 / 3 of the leaves of the stalk-cut air-cured plant according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. the middle 1 / 3 of the leaves of the stalk-cut air-cured plant). Suitably, the upper 1 / 3 of the leaves of the stalk-cut air-cured plant according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. the upper 1 / 3 of the leaves of the stalk-cut air-cured plant).

[0337] In one aspect, flue-cured leaves according to the invention comprise an increased content of sucrose esters compared to a comparable product (eg flue-cured leaves) which has not been modified according to the invention.

[0338] Suitably, the sucrose ester content may be measured in the first initiation of flue-cured plants. Suitably, the sucrose ester content may be measured in the second (or alternatively intermediate) initiation of flue-cured plants. Suitably, the sucrose ester content may be measured in the third (or alternatively last) initiation of flue-cured plants.

[0339] As used herein, "priming" refers to removing leaves from a tobacco plant. This can refer to removing mature or cooked leaves of flue-cured plants.

[0340] As used herein, "first initiator" refers to leaves first harvested from a tobacco plant (e.g., leaves from the lowest portion of a tobacco plant). As used herein, "second initiator (or intermediate initiator)" refers to leaves harvested from a tobacco plant after initial initiation (e.g., leaves from the middle portion of a tobacco plant). As used herein, "third (or final) initiator" refers to leaves harvested from a tobacco plant at last (e.g., leaves from the top portion of a tobacco plant).

[0341] Suitably, a first initiator of leaves of flue-cured plants according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. a first initiator of leaves of flue-cured plants). Suitably, a second (or intermediate) initiator of leaves of flue-cured plants according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. a second (or intermediate) initiator of leaves of flue-cured plants). Suitably, a third (or final) initiator of leaves of flue-cured plants according to the present invention may comprise an increased sucrose ester content compared to a comparable product not modified according to the present invention (e.g. a third (or final) initiator of leaves of flue-cured plants).

[0342] Typically, air-curing is achieved by hanging the leaves in a well-ventilated barn and allowing them to dry. This is usually done over a period of four to eight weeks. Air-curing is particularly suitable for Burley tobacco.

[0343] Suitably, the tobacco leaves may be open fire cured. Open fire curing is usually achieved by hanging the tobacco leaves in large barns where hardwood flames are maintained at a continuous or intermittent low smoulder depending on the process and tobacco and typically takes between three days and ten weeks.

[0344] In another embodiment, the tobacco leaves can be flue-cured. Flue-curing can include stringing the tobacco leaves onto tobacco stems and hanging them from tier-poles in a curing barn. The barn typically has a flue that enters from an externally fed firebox. Typically, this produces tobacco that has been heat-cured without being exposed to smoke. Typically, the temperature rises slowly during the curing process, and the entire process takes about 1 week.

[0345] Suitably, the tobacco leaves may be cured. This process generally involves exposing the uncovered tobacco to the sun.

[0346] Suitably, the tobacco leaf of processing can be processed by fermentation. Fermentation can be carried out in any manner known in the art. Usually, during the fermentation process, tobacco leaves are piled into flue-cured tobacco piles (pile) covered in, for example, sacks to retain moisture. The combination of residual moisture in the leaf and tobacco weight produces natural heat that makes tobacco mature. The temperature in the center of the pile is monitored every day. In some methods, the whole pile is opened every week. Then the leaves are moved to shake and moisten, and the pile is turned over so that the inner leaves are outside and the bottom leaves are placed on the top of the pile. This ensures the uniform fermentation of the whole pile. The extra moisture on the leaf plus the actual turning over of the leaf itself produces heat, releases the natural ammonia of tobacco and reduces nicotine, while also deepening the color and improving the aroma of tobacco. Usually, the fermentation process lasts up to 6 months, which depends on the variety of tobacco, the stalk position on the leaf, the thickness of the leaf and the intended use.

[0347] Suitably, the processed tobacco leaves may be processed by pasteurization. When the tobacco leaves are to be used in the manufacture of smokeless tobacco products (most preferably snus), pasteurization may be particularly preferred. Tobacco leaf pasteurization may be performed by any method known in the art. For example, pasteurization may be performed as described in detail in: J Foulds, L Ramstrom, M Burke, K Fagerstrom. Effect of smokeless tobacco (snus) on smoking and public health in Sweden. Tobacco Control (2003) 12 :349–359, the teachings of which are incorporated herein by reference.

[0348] During snus production, pasteurization is usually carried out by a process in which the tobacco is heat treated with steam for 24-36 hours, reaching a temperature of about 100° C. This produces a nearly sterile product, and without wishing to be bound by theory, one consequence of this is thought to be limiting further TSNA formation.

[0349] In one embodiment, the pasteurization may be steam pasteurization.

[0350] In some embodiments, the processed tobacco leaves can be cut. The processed tobacco leaves can be cut before or after processing. Suitably, the processed tobacco leaves can be cut after processing.

[0351] In some embodiments, tobacco plant, tobacco plant harvested leaves and / or processed tobacco leaves can be used to extract nicotine. The extraction of nicotine can be achieved using any method known in the art. For example, the method for extracting nicotine from tobacco is taught in US 2,162,738, which is incorporated herein by reference.

[0352] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention or from a tobacco cell culture according to the present invention.

[0353] In another aspect, the present invention provides a tobacco blend comprising a tobacco material made from a tobacco plant or part thereof according to the present invention or from a tobacco cell culture according to the present invention. In one aspect, the present invention provides a tobacco blend comprising a cured tobacco material according to the present invention.

[0354] Suitably, tobacco blends according to the present invention may include approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 10% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 20% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 30% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 40% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 50% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend may include approximately 60% of tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, the tobacco blend can comprise approximately 70% of tobacco from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, the tobacco blend can comprise approximately 80% of tobacco from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, the tobacco blend can comprise approximately 90% of tobacco from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention.

[0355] In one aspect, the tobacco blend products of the invention comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% by dry weight of tobacco cured from a tobacco plant or part thereof according to the invention or a tobacco cell culture according to the invention.

[0356] Suitably, the cured tobacco material may be air-cured. Suitably, the cured tobacco material may be flue-cured. Suitably, the cured tobacco material may be sun-cured.

[0357] Tobacco products or smoking articles according to the invention may comprise a tobacco material (eg, a cured tobacco material) according to the invention.

[0358] In yet another aspect, the invention provides tobacco products. Tobacco products according to the present invention can be blended tobacco products. In one embodiment, tobacco products can be prepared from tobacco plant of the present invention or its parts. In one embodiment, the tobacco products can be prepared from tobacco plants with diterpene synthesis gene activity or expression and the sucrose ester content of increase that suppress. The tobacco products can include the reduction of diterpene synthesis gene activity or expression and the increase of sucrose ester content. Suitably, tobacco plant or its parts can be bred from tobacco plant propagation materials according to the present invention.

[0359] The term "parts thereof" as used herein in the context of tobacco plants refers to parts of the tobacco plant. Preferably, the "parts thereof" are leaves of the tobacco plant.

[0360] In another embodiment, tobacco product can be prepared from the leaf of results of the present invention.In a further embodiment, tobacco product can be prepared from the tobacco leaf of processing of the present invention.Suitably, tobacco product can be prepared from the tobacco leaf of one or more processings in the following: modulation, fermentation and / or pasteurization.Suitably, tobacco product can comprise cut tobacco leaf, optionally, it is processed according to the aforementioned embodiment.

[0361] In one embodiment, the tobacco product can be a smoking article. As used herein, the term "smoking article" can include smokeable products such as cigarettes, cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes.

[0362] In another embodiment, the tobacco product can be a smokeless tobacco product. As used herein, the term "smokeless tobacco product" refers to a tobacco product that is not intended to be ignited and smoked and / or subjected to combustion. In one embodiment, the smokeless tobacco product can include snus, snuff, chewing tobacco, etc.

[0363] In a further embodiment, the tobacco product may be a tobacco heating device or a mixing device or an electronic cigarette, etc. Typically, in a heating device or a mixing device, an aerosol is generated by transferring heat from a heat source to a physically separated aerosol-forming substrate or material (which may be located inside, around, or downstream of the heat source). During smoking, volatile compounds are released from the aerosol-forming substrate by heat transfer from the heat source and are entrained in the air inhaled by the smoking article. When the released compounds cool, they condense to form an aerosol inhaled by the user.

[0364] Aerosol generating articles and devices for consuming or smoking tobacco heating devices are known in the art. They may include, for example, electrically heated aerosol generating devices, wherein an aerosol is generated by transferring heat from one or more electrical heating elements of the aerosol generating device to an aerosol-forming substrate of the tobacco heating device. Suitably, the tobacco heating device may be an aerosol generating device.

[0365] Preferably, the tobacco heating device may be a heat-not-burn device. Heat-not-burn devices are known in the art and release compounds by heating rather than burning tobacco. Examples of suitable heat-not-burn devices may be the devices taught in WO2013 / 034459 or GB2515502, the disclosures of which are incorporated herein by reference.

[0366] In one embodiment, the aerosol-forming substrate of the tobacco heating device may be a tobacco product according to the invention.

[0367] In one embodiment, the tobacco heating device may be a hybrid device.

[0368] Polynucleotide / polypeptide / construct

[0369] In certain embodiments of the invention, a construct that inhibits the activity or expression of a diterpene synthesis gene under the guidance of a promoter can be transformed into a plant cell. In certain embodiments of the invention, a ddRNAi DNA construct that expresses dsRNA or amiRNA to inhibit the activity or expression of a diterpene synthesis gene under the guidance of a promoter can be transformed into a plant cell.

[0370] The construct can be introduced into the plant according to the present invention by means of a suitable vector such as a plant transformation vector. The plant transformation vector can include an expression cassette, which includes in the transcription direction 5'-3': a promoter sequence, a ddRNAi DNA construct sequence targeting a diterpene synthesis gene, preferably targeting a cyclase 2 gene (CYC2), a CBTol cyclase or a terpene synthase 3-8 (optionally including an intron), and optionally a 3' non-translated terminator sequence, the terminator sequence including a termination signal for an RNA polymerase and a polyadenylation signal for a polyadenylation enzyme. The promoter sequence can exist in one or more copies, and such copies can be variants of the promoter sequence that is identical or as described above. The terminator sequence can be obtained from a plant, a bacterium or a viral gene. For example, a suitable terminator sequence is a pea rbcS E9 terminator sequence, a nos terminator sequence of a nopaline synthase gene derived from Agrobacterium tumefaciens, and a 35S terminator sequence from a cauliflower mosaic virus. Those skilled in the art will readily recognize other suitable terminator sequences.

[0371] The construct of the present invention may also include a gene expression enhancement mechanism that increases promoter strength. An example of such an enhancer element is an enhancer element derived from a portion of the pea plastocyanin gene promoter, and is the subject of International Patent Application No. WO 97 / 20056 (which is incorporated herein by reference). For example, suitable enhancer elements may be the nos enhancer element of the nopaline synthase gene derived from Agrobacterium tumefaciens and the 35S enhancer element from cauliflower mosaic virus.

[0372] These regulatory regions may be derived from the same gene as the promoter DNA sequence, or may be derived from a different gene, such as that from Nicotiana tabacum ( Nicotiana tabacum ) or other organisms, such as those from the Solanaceae family ( Solanaceae ) or from the subfamily Cinerariaceae ( Cestroideae ). All regulatory regions should be able to function in the cells of the tissue to be transformed.

[0373] The promoter DNA sequence may be derived from the same gene as the coding sequence of the target gene used in the present invention (e.g., the gene to which the promoter is to be directed, such as the gene encoding the deregulated nitrate reductase of the present invention), or may be derived from a different gene, the gene being derived from Nicotiana tabacum ( Nicotiana tabacum ) or other organisms, such as those from the Solanaceae family ( Solanaceae ) or from the subfamily Cinerariaceae ( Cestroideae ).

[0374] The expression cassette can be incorporated into a basic plant transformation vector, e.g. pBIN 19 Plus, pBI 101 , pKYLX71:35S2, pCAMBIA2300 or other suitable plant transformation vectors as known in the art.Except the expression cassette, the plant transformation vector will contain such sequence as required for the transformation process.These sequences may include agrobacterium vir genes, one or more T-DNA border sequences and selectable markers or other means for identifying transgenic plant cells.

[0375] The term "plant transformation vector" refers to a construct capable of in vivo or in vitro expression. Preferably, the expression vector is integrated into the genome of the organism. The term "integrated" preferably encompasses stable integration into the genome.

[0376] The technology for transforming plants is well known in the art and includes, for example, Agrobacterium-mediated transformation. The basic principle of the construction of genetically modified plants is to insert genetic information into the plant genome, thereby obtaining stable maintenance of the inserted genetic material. A review of general techniques can be found in the articles of Potrykus (Annu Rev Plant Physiol Plant Mol Biol

[1991] 42: 205-225) and Christon (AgroFood-Industry Hi-Tech March / April 1994 17-27) (which are incorporated herein by reference).

[0377] Typically, in Agrobacterium-mediated transformation, by co-cultivating Agrobacterium with the explant from the target plant, the binary vector (i.e., ddRNAi DNA construct) carrying the exogenous DNA of the target plant is transferred from a suitable Agrobacterium strain to the target plant. The plant tissue of the transformation is then regenerated on a selection medium, wherein the selection medium comprises a selectable marker and a plant growth hormone. The selectable method is a flower dip method (Clough & Bent, 1998 Plant J. 1998 Dec; 16 (6): 735-43, which is incorporated herein by reference), wherein the flower buds of a complete plant are contacted with a suspension of an Agrobacterium strain containing a mosaic gene, and after seed formation, the individual transformed is germinated and identified by growth on a selective medium. Direct infection of plant tissue by Agrobacterium is a simple technique, which has been widely adopted and is described in Butcher DN et al., (1980), Tissue Culture Methods for Plant Pathologists, Editors: DS Ingrams and JP Helgeson, 203-208, which are incorporated herein by reference.

[0378] Other suitable transformation methods include using polyethylene glycol or electroporation technology, particle bombardment, microinjection and, for example, using silicon carbide fibers to transfer genes directly into protoplasts. The use of ballistic transformation (ballistic transformation) including silicon carbide whisker technology to transform plants is taught in Frame BR, Drayton PR, Bagnaall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA & Wang K (1994), which is incorporated herein by reference. Transgenic corn plants fertile by silicon carbide whisker-mediated transformation are taught in The Plant Journal 6:941-948, which is incorporated herein by reference) and viral transformation techniques are taught in, for example, Meyer P, Heidmm I & Niedenhof I (1992). The use of cassava mosaic virus as a vector system for plants is taught in Gene 110:213-217, which is incorporated herein by reference. Further teachings on plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

[0379] In a further aspect, the present invention relates to a vector system that carries a ddRNAi DNA construct and introduces it into the genome of an organism such as a plant. The vector system can include a vector, but it can also include two vectors. In the case of two vectors, the vector system is generally referred to as a binary vector system. The binary vector system is described in more detail in Gynheung Anetal, (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19, which is incorporated herein by reference.

[0380] A widely used system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens or the Ri plasmid from Agrobacterium rhizogenes Anetal., (1986), Plant Physiol. 81, 301-305 and Butcher DN et al., (1980), Tissue Culture Methods for Plant Pathologists, eds. DS Ingrams and JP Helgeson, 203-208, which are incorporated herein by reference. After each method of introduction of the desired foreign gene in the plant according to the present invention, the presence and / or insertion of further DNA sequences may be necessary. The use of T-DNA for transformation of plant cells has been extensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B. B., Amsterdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4: 1-46; and Anetal., EMBO J (1985) 4: 277-284, which are incorporated herein by reference.

[0381] Plant cells transformed with a ddRNAi DNA construct expressing a dsRNA or amiRNA that inhibits the activity or expression of a diterpene biosynthesis gene can be grown and maintained according to well-known tissue culture methods, for example by culturing the cells in a suitable medium provided with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0382] The term "transgenic plant" in connection with the present invention includes any plant comprising a ddRNAi DNA construct according to the present invention. Thus, a transgenic plant is a plant that has been transformed with a ddRNAi DNA construct according to the present invention. Preferably, according to the present invention, the transgenic plant exhibits suppressed diterpene synthesis gene activity or expression and increased sucrose ester content. The term "transgenic plant" does not encompass a native nucleotide coding sequence in its natural environment when it is under the control of its native promoter (the promoter is also in its natural environment).

[0383] In one aspect, the ddRNAi DNA construct, plant transformation vector or plant cell according to the invention is in isolated form. The term "isolated" means that the sequence is at least substantially free of at least one other component with which the sequence is naturally associated in nature and as found in nature.

[0384] In one aspect, the ddRNAi DNA construct, plant transformation vector or plant cell according to the present invention is in a purified form. The term "purified" means in a relatively pure state, for example, at least about 90% pure, or at least about 95% pure, or at least about 98% pure.

[0385] As used herein, the term "nucleotide sequence" refers to an oligonucleotide sequence or a polynucleotide sequence and variants, homologues, fragments and derivatives (such as parts thereof) thereof. The nucleotide sequence may be of genomic or synthetic or recombinant origin, and may be double-stranded or single-stranded, whether representing the sense strand or the antisense strand.

[0386] The term "nucleotide sequence" in connection with the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it means DNA, more preferably a cDNA sequence encoding the present invention.

[0387] In a preferred embodiment, the nucleotide sequence, when relating to and covered by the scope of the present invention itself, i.e. the diterpene synthesis gene, when in its natural environment and when associated with a sequence naturally associated therewith (also in its natural environment), comprises a native nucleotide sequence. For ease of reference, we shall refer to this preferred embodiment as a "native nucleic acid sequence". In this regard, the term "native nucleotide sequence" means the entire nucleotide sequence in its natural environment and when operably linked to the entire promoter (which is naturally associated with the sequence), which promoter is also in its natural environment.

[0388] Typically, the RNAi molecules covered by the scope of the present invention are prepared using recombinant DNA technology (e.g., recombinant DNA). However, in an alternative embodiment of the present invention, the nucleotide sequence can be synthesized in whole or in part using chemical methods well known in the art (see Caruthers MH et al. , (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al. , (1980) Nuc Acids ResSymp Ser 225-232, which are incorporated herein by reference).

[0389] The nucleotide sequence of the protein with specific characteristics as the diterpene synthesis gene as defined herein or the protein suitable for modification can be identified and / or separated and / or purified from any cell or organism producing the protein. The method for identifying and / or separating and / or purifying nucleotide sequences is well known in the art. By way of example, once the suitable sequence has been identified and / or separated and / or purified, more sequences can be prepared using PCR amplification technology.

[0390] By way of further example, genomic DNA and / or cDNA library can be constructed using chromosomal DNA or messenger RNA from the organism that produces enzyme. If the amino acid sequence of enzyme is known, then the oligonucleotide probe of synthetic labeling can be used to identify the clone of codase from the genomic library of organism prepared. Or, the oligonucleotide probe containing the labeling of the sequence homologous to another known enzyme gene can be used to identify the clone of codase. In the latter case, hybridization and washing conditions of lower stringency are used.

[0391] In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, such as those described by Beucage SL et al. , (1981) Tetrahedron Letters 22, p 1859-1869 (which is incorporated herein by reference) or by the phosphoroamidite method described in Matthes et al. , (1984) EMBO J . 3, p 801-805 (which is incorporated herein by reference). In the phosphoramidite method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, annealed, ligated and cloned into an appropriate vector.

[0392] As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".

[0393] The present invention also covers the use of sequences having a certain degree of sequence identity or sequence homology with the amino acid sequence of a polypeptide having specific properties as defined herein or any nucleotide sequence (i.e., a diterpene synthesis gene encoding such a polypeptide) (hereinafter referred to as "homologous sequence"). Here, the term "homolog" means an entity having a certain homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" can be equivalent to "identity".

[0394] Homologous amino acid sequences and / or nucleotide sequences and / or fragments should provide and / or encode polypeptides that retain the functional activity of diterpene synthesis genes and / or enhance the activity of diterpene synthesis genes. Usually, homologous sequences will comprise the same active site as, for example, the subject amino acid sequence, or will encode the same active site. Although homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, homology is preferably represented by sequence identity.

[0395] In one embodiment, homologous sequences are comprised in amino acid sequences or nucleotide sequences which have one or several additions, deletions and / or substitutions compared to the subject sequence.

[0396] Homology or identity comparisons can be performed visually, or more generally with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the % homology between two or more sequences. % homology or % identity can be calculated on a continuous sequence, i.e. one sequence is aligned with another sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is referred to as an "ungapped" alignment. Typically, this ungapped alignment is performed only on a relatively small number of residues.

[0397] Although this is a very simple and consistent approach, it does not take into account that, for example, in an otherwise identical pair of sequences, an insertion or deletion may cause subsequent amino acid residues to not align, which may result in a greatly reduced % homology when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0398] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher affinity between the two compared sequences - will receive a higher score than a sequence alignment with many gaps. "Affine gap costs" are commonly used, which impose a relatively high cost on the existence of a gap and a smaller penalty on each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using these software for sequence comparisons, it is preferred to use the default values.

[0399] Therefore, calculation of maximum % homology first requires producing an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, for example, the BLAST package (see Ausubel et al. 1999 Short Protocols in Molecular Biology, 4th edition - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8 and tatiana@ncbi.nlm.nih.gov ), FASTA (Altschul et al. 1990 J. Mol. Biol. 403-410) and AlignX. At least BLAST, BLAST 2 and FASTA can be used for offline and online searching (see Ausubel et al. 1999, pages 7-58 to 7-60).

[0400] Although final % homology can be measured according to identity, the alignment process itself is usually not based on an all-or-nothing comparison. On the contrary, a scaled similarity score matrix is ​​usually used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of this matrix commonly used is the BLOSUM62 matrix - the default matrix of the BLAST program suite. The Vector NTI program usually uses public default values ​​or custom symbol comparison tables (if provided) (see user manual for more details). For some applications, the default values ​​of the Vector NTI software package are preferably used.

[0401] Alternatively, percent homology can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.) based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

[0402] Once the software has generated an optimal alignment, the % homology, preferably % sequence identity, can be calculated. The software typically does this as part of a sequence comparison and generates a numerical result.

[0403] If gap penalties are used when determining sequence identity, the following parameters are preferred for pairwise alignments:

[0404] For BLAST Open seats 0 Vacancy extension 0

[0405] For CLUSTAL DNA protein Character size 2 1 K triple Gap Penalty 15 10 Vacancy extension 6.66 0.1

[0406] In one embodiment, CLUSTAL can be used with gap penalty and gap extension as defined above. In some embodiments, the gap penalty for BLAST or CLUSTAL comparison can be different from those described in detail above. The technician will appreciate that the standard parameters for performing BLAST and CLUSTAL comparison can be changed regularly, and will be able to select appropriate parameters based on the standard parameters detailed for BLAST or CLUSTAL comparison algorithm at that time.

[0407] Suitably, the degree of identity of the nucleotide sequence is determined over at least 20 consecutive nucleotides, preferably at least 30 consecutive nucleotides, preferably at least 40 consecutive nucleotides, preferably at least 50 consecutive nucleotides, preferably at least 60 consecutive nucleotides, preferably at least 100 consecutive nucleotides; wherein the nucleotide sequence encodes a ddRNAi DNA construct. The degree of identity of the nucleotide sequence is determined over at least 10 consecutive nucleotides, preferably 20 consecutive nucleotides, preferably at least 30 consecutive nucleotides, preferably at least 40 consecutive nucleotides, preferably at least 50 consecutive nucleotides, preferably at least 60 consecutive nucleotides, preferably at least 100 consecutive nucleotides.

[0408] Suitably, the degree of identity with respect to nucleotide sequences may be determined over the entire sequence.

[0409] The sequences may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent substances. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0410] For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other:

[0411] .

[0412] The present invention also includes homologous substitutions (substitution and replacement are used herein to indicate the exchange of an existing amino acid residue with an alternative residue) that may occur, i.e., like-for-like substitutions (such as basic substitutions for basic, acid substitutions for acid, polar substitutions for polar, etc.). Non-homologous substitutions may also occur, i.e., from one class of residues to another, or alternatively involve the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridine alanine, thienyl alanine, naphthyl alanine and phenylglycine.

[0413] Substitution can also be made with unnatural amino acids, including: α* and α-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allylglycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α-aminoisobutyric acid*, L-ε-aminocaproic acid # , 7-aminoheptanoic acid*, L-methionine sulfone# * , L-norleucine*, L-valeric acid*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) ＃ 、L-Tyr(methyl)*、L-Phe(4-isopropyl)*、L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*、L-diaminopropionic acid # and L-Phe (4-benzyl)*. The symbol * has been used for the purposes of the above discussion (involving homologous or nonhomologous substitution) to indicate the hydrophobic nature of the derivatives, while # has been used to indicate the hydrophilic nature of the derivatives, # * Indicates amphipathic characteristics.

[0414] Variant amino acid sequences may include, in addition to amino acid spacers such as glycine or β-alanine residues, suitable spacer groups that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups. Other forms of variation involve the presence of one or more amino acid residues in a peptoid form, which will be fully understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues in which the α-carbon substituent is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for preparing peptides in peptoid form are known in the art, such as Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol.(1995)13(4), 132-134.

[0415] Nucleotide sequences used in the present invention may include synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonates and thiophosphate backbones and / or the addition of acridine or polylysine chains at the 3' end or 5' end of the molecule. For purposes of the present invention, it will be appreciated that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be implemented to enhance the in vivo activity and lifespan of the nucleotide sequences of the present invention.

[0416] The present invention also includes sequences complementary to the nucleic acid sequences of the present invention, or sequences capable of hybridizing to the sequences of the present invention or to sequences complementary thereto. The term "hybridization" as used herein shall include "a process by which a nucleic acid strand binds to a complementary strand through base pairing" and amplification processes such as those performed in polymerase chain reaction (PCR) technology.

[0417] The present invention also relates to nucleotide sequences that can hybridize with the nucleotide sequences of the present invention (including complementary sequences of those presented herein). Preferably, hybridization is determined under stringent conditions (e.g., 50° C. and 0.2×SSC {1×SSC = 0.15 M NaCl, 0.015 M trisodium citrate pH 7.0}). More preferably, hybridization is determined under high stringency conditions (e.g., 65° C. and 0.1×SSC {1×SSC = 0.15 M NaCl, 0.015 M trisodium citrate pH 7.0}).

[0418] In one aspect, the sequences used in the present invention are synthetic sequences - i.e., sequences prepared by in vitro chemical or enzymatic synthesis. They include, but are not limited to, sequences made with optimal codon usage for the host organism.

[0419] The term "expression vector" means a construct capable of in vivo or in vitro expression. In one embodiment, the vector of the invention expresses dsRNA. In one embodiment, the vector of the invention expresses amiRNA. Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably encompasses stable incorporation into the genome.

[0420] The nucleotide sequence of the present invention may be present in a vector, wherein the nucleotide sequence may be operably linked to a regulatory sequence that can provide expression of the nucleotide sequence by a suitable host organism. The ddRNAi DNA construct used in the present invention may be transformed into a suitable host cell described herein to provide expression of the polypeptide of the present invention. The selection of a vector such as a plasmid, a cosmid or a phage vector often depends on the host cell to be introduced into the vector. The vector may be used in vitro, for example, for producing RNA or for transfection, transformation, transduction or infection of a host cell.

[0421] Therefore, in a further embodiment, the invention provides a method for preparing the nucleotide sequence of the invention by introducing the nucleotide sequence of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that cause the vector to replicate. The vector may further comprise a nucleotide sequence that enables the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702.

[0422] In some applications, the nucleotide sequence used in the present invention may be operably linked to a regulatory sequence that can provide expression of the nucleotide sequence (such as by a selected host cell). By way of example, the present invention encompasses a vector comprising a nucleotide sequence of the present invention that is operably linked to such a regulatory sequence, i.e., the vector is an expression vector.

[0423] The term "operably linked" refers to a juxtaposition in which the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0424] The term "regulatory sequence" includes promoters and enhancers and other expression regulatory signals. The term "promoter" is used in the usual sense of the art, for example, an RNA polymerase binding site. The nucleotide sequence within the ddRNAi DNA construct encoding dsRNA or amiRNA can be operably linked to at least one promoter.

[0425] The term "construct" - which is synonymous with terms such as "cassette" or "vector" - comprises a nucleotide sequence for use in accordance with the present invention, which is directly or indirectly attached to a promoter.

[0426] An example of indirect attachment is to provide a suitable spacer such as an intron sequence, such as Sh1-intron or ADH intron, between the promoter and nucleotide sequence of the present invention. The same is true for the term "fusion" associated with the present invention, which includes direct or indirect attachment. In some cases, the term does not encompass the natural combination of nucleotide sequences of protein encoding proteins that are usually associated with wild-type gene promoters (and when they are all in their natural environment). ddRNAi DNA constructs can even contain or express markers that allow selection of gene constructs.

[0427] It can be found in Potrykus ( Annu Rev Plant Physiol Plant Mol Biol

[1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994 17-27) (which are incorporated herein by reference) for a review of general techniques for transforming plants. Further teachings on plant transformation can be found in EP-A-0449375 (which is incorporated herein by reference).

[0428] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al. , DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled artisan with a general dictionary of many of the terms used in this disclosure.

[0429] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

[0430] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which may be had by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0431] Amino acids are referred to herein using the name, three-letter abbreviation or single-letter abbreviation of the amino acid. As used herein, the term "protein" includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".

[0432] The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids is as defined by the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0433] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus may, of course, vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0434] In the case of providing a numerical range, it should be understood that, unless the context clearly specifies otherwise, each intermediate value between the upper and lower limits of the range to one-tenth of the lower limit unit is also specifically disclosed. Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges can be independently included in the range or excluded from the range, and each range wherein any limit, no limit or two limits are included in the smaller range is also included in the present disclosure, subject to any specifically excluded limit in the specified range. In the case where the specified range includes one or two limits, the scope excluding any one or two of these included limits is also included in the present disclosure.

[0435] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" or "nitrate reductase" includes a variety of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

[0436] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the claims appended hereto.

[0437] advantage

[0438] Advantageously, the activity of diterpene synthesis genes or the inhibition of expression increase the sucrose ester content of tobacco plants. Without wishing to be bound by theory, it is believed that reducing the carbon utilization of preparing diterpene leads to sucrose ester output enhancement. Advantageously, the method of the present invention allows the production of tobacco plants with preferred "Turkey sample" chemicals, which are high biomass plants. Advantageously, the invention provides one or more desirable smoke chemicals that can be used for heat-not-burn products. The present invention will now be described by way of example only with reference to the following drawings and Examples. Example

[0439] Example 1 Preparation of transgenic constructs GW1, GW2, GW3, and GW5

[0440] The inventors attempted to dissect plant trichome secondary metabolism and used a post-transcriptional gene silencing strategy (PTGS) to evaluate the effects of trichome expressed genes on sucrose ester content. RNAi was used to evaluate the function of the cyclase 2 gene (CYC2), CBTol cyclase and terpene synthase 3-8. Methods for construct preparation and plant regeneration are given in detail in Wang and Wagner 2003 (supra, which is incorporated herein by reference) together with conventional molecular cloning techniques (Sambrook et al., 1989 Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York).

[0441] Transgenic construct GW1 - inhibits the expression of the cyclase 2 gene (CYC2)

[0442] The ddRNAi DNA construct is designed based on the cyclase 2 gene (CYC2). The complete coding sequence of the mRNA of CYC2 is annotated as AF401234. The genomic sequence is annotated as AY495694. The construct for producing GW1 plants consists of: 5'-sense fragments from the 54th to the 716th nucleotides of the sequence AF401234; a partial GUS A fragment (from the 787th to the 1812th nucleotides) as a hairpin loop, the GUS A annotation is AF502128; and the reverse complement of the CYC2 sense fragment-3'. The construct comprises nucleotides 54 to 716 from CYC2 mRNA (AF401234) in the sense direction. This sequence corresponds to the nucleotide positions from the genomic sequence (AY495694): nucleotides 1 to 25, nucleotides 26 to 271 (exon 1), nucleotides 1253 to 1529 (exon 2) and the first 115 nucleotides of exon 3 (nucleotides 2366 to 2480). Plasmid pKYLX71-35S2 is a binary vector for gene constructs (Wang et al., 2001, supra, which is incorporated herein by reference). The sequence of the GW1 construct insert is listed in SEQ ID No. 5.

[0443] Transgenic construct GW2 - suppresses the expression of terpene synthase 3-8 genes

[0444] The ddRNAi DNA construct was designed based on the miRNA168 primary transcript (EU549055.1; GI: 171195398) (from 5'127- to 535-3'), wherein 196-216 was replaced by 21 nucleotides starting from nucleotide 1497 that were reverse complementary to the original sequence (Genbank: AY528645, corresponding to the nucleotide position as shown in SEQ ID NO.3), and nucleotides 279-299 were replaced by 21 nucleotides from the original chain in the forward direction, but wherein 3 bases were modified when region 1497-1517 was used. The module flanked by 5'-HindIII and 3'-EcoRI restriction site extension was inserted between the 2X35S promoter and the 35S terminator. The extension module containing 2X35S prom-miRNAi 3-8 and 35S terminator was introduced into the multiple cloning site (MCS) of the binary vector pCAMBIA2300. The recombinant vector (KmR) was inserted into Agrobacterium tumefaciens GV3101 (RifR, GmR). Colonies were selected on triple antibiotics (Km, Rif, Gen). Agrobacterium transformation was confirmed by plasmid PCR. The sequence of the GW2 construct insert is listed in SEQ ID No.7.

[0445] Transgenic construct GW3 - inhibits the expression of the CBTol cyclase gene

[0446] The ddRNAi DNA construct is designed based on the tobacco cyclase gene (AY049090). The double-stranded RNAi module is assembled in the following manner: -partial sequence from the tobacco cyclase gene (AY049090), in the forward and reverse directions, 5' 2854 to 4175- 3' of SEQ ID No.1, consisting of: partial exon 4, complete intron 4, exon 5, intron 5, exon 6, intron 6 and partial exon 7. The spacer between the forward and reverse segments is a partial GUS A gene, from 5'- 786-1816-3'. Plasmid pCAMBIA2300 is a binary vector for the gene construct. The sequence of the GW3 construct insert is listed in SEQ ID No.6.

[0447] Transgenic construct GW5 - suppresses the expression of terpene synthase 3-8 genes

[0448] The ddRNAi DNA construct was designed based on the miRNA168 primary transcript (EU549055.1; GI: 171195398) (from 5'127- to 535-3'), wherein 196-216 were replaced by 21 nucleotides starting from nucleotide 884 that were reverse complementary to the original sequence (Genbank: AY528645, corresponding to the nucleotide position as shown in SEQ ID NO.3), and nucleotides 279-299 were replaced by 21 nucleotides from the original chain in the forward direction, but wherein 4 bases of the sense chain were modified when region 884-904 was used. The module flanked by 5'-HindIII and 3'-EcoRI restriction site extension was inserted between the 2X35S promoter and the 35S terminator. The extension module containing 2X35S prom-miRNAi 3-8 and 35S terminator was introduced into the multiple cloning site (MCS) of the binary vector pCAMBIA2300. The recombinant vector (KmR) was inserted into Agrobacterium tumefaciens GV3101 (RifR, GmR). Colonies were selected on triple antibiotics (Km, Rif, Gen). Agrobacterium transformation was confirmed by plasmid PCR. The sequence of the GW5 construct insert is listed in SEQ ID No. 11.

[0449] Example 2 Agrobacterium transformation and regeneration of TI 1068

[0450] Common tobacco TI 1068 seed is obtained from KTRDC seed collection center, and with 70% EtOH surface sterilization 1 minute, then with 5% (v / v) Chlorox surface sterilization, and then with sterile water washing three times.Make plant in PLANTCON® container (MP Biomedicals, LLC) in vitro growth, and be used as the storage of explant for transformation.All constructs are introduced in tobacco cultivar TI 1068 by agrobacterium-mediated transformation, and confirm the existence of transgenic by PCR.Agrobacterium tumefaciens transformation is basically as people such as Horsch at Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227:1229–1231 (it is incorporated to this paper by reference and has made some modifications) described in and carries out.Make agrobacterium grow overnight (GW1, GW2,) on the LB substratum containing 50 mg / l Km, 50 mg / l Rif and 35 mg / l Gm. For GW3, Km was replaced by Hyg (55 mg / l).

[0451] Leaves (1 cm) cut from aseptically grown plants (45 days old) were 2 ) in the dark with bacteria (1x10 8 cfu / ml) for 2 days, then blotted and transferred to MS medium supplemented with Gamborg B-5 vitamins (Sigma-Aldrich Co.LTD, Irvine, UK), 3% sucrose, 1 mg / l BAP and 0.05 mg / l NAA and supplemented with 200 mg / l Km (or, for GW3, 55 mg / l Hyg) and 400 mg / l cefotaxime. After about a month, small plantlets formed at the edge of the piece, and after one passage on the same medium, the plantlets were transferred to the same medium, but hormones were removed and antibiotics were reduced by half. Rooted plants were transferred to fertilized Pro-Mix (PremierHorticulture Inc., Canada) in a growth chamber. Individual transformants were analyzed via GC-MS, and those with the desired changes were self-inoculated for further analysis of the T1 generation.

[0452] Example 3 Green leaf analysis of field-grown controls versus transgenic lines

[0453] Preparation of strains for field testing and field test design

[0454] Seeds of lines GW1 and GW2, generated from self-pollination of T3 or T2 plants, were surface sterilized and germinated in vitro on MS medium supplemented with Gamborg B-5 vitamins (Sigma-Aldrich Co. LTD, Irvine, UK), 3% sucrose, and Km (200 mg / l). GW3 seeds were surface sterilized and germinated on medium containing Hyg (55 mg / l). One month later, the plantlets were transferred to floating trays (fertilized Pro-Mix (Premier Horticulture Inc., Canada) in the greenhouse and grown for 7-8 weeks before being transplanted to the field. Standard field practices were used and irrigation was used as necessary. Harvesting and collection of flue-cured and air-cured (for burley tobacco) were completed after approximately 4 and a half months. Flue-curing included: 48 hours of coloring, 85 to 100°F, 94% RH; 24 hours of wilting, 100°F to 120°F, to 54% RH; 30 hours of leaf drying, 120°F to 135°F, 40% RH; 40 hours of stem drying, 135 to 168°F, to 22% RH.

[0455] Measurement of exudate components as TMS derivatives by GC-MS

[0456] For green leaf measurements, two leaf disks (2 cm diameter) were cut from the middle of the leaf blade of the middle leaf of each plant. The disks were washed with acetonitrile (5 ml) for 30 seconds. The washes were concentrated via vacuum rotor evaporation to give an oily residue. The residue was derivatized to form trimethylsilyl (TMS) esters as described by Severson et al., 1985 (supra, which is incorporated herein by reference) as follows: The acetonitrile washes were evaporated to dryness and dissolved in 1 ml CHCl 3 , transferred to 1.5 ml GC vials and dried at 40 °C under N2 flow. The derivatized samples were dissolved in 1 ml dimethylformamide (DMF), and 50 μl BSTFA [bis(trimethylsilyl)trifluoroacetamide] and 24 μg of coniferene as internal standard were added to each vial. The samples were derivatized at 70 °C for 45 minutes, cooled to room temperature and analyzed.

[0457] TMS derivatives were separated and analyzed by GC-MS (HP6890 GC equipped with HP5973 MS and autosampler injector) using a 30.0 m Agilent capillary column 19091J-413 with 0.25 μm film thickness and 0.25 mm diameter. Helium was the carrier gas with a constant flow of 1.8 ml / min and an injection temperature of 250°C. The oven program for running the TMS ester derivatives was as follows: initial temperature 180°C for 2.0 minutes; rate 8°C per minute until 280°C, held for 9.5 minutes. The total run time was 24.0 minutes. The eluted compounds were identified by comparison of their retention times and MS profiles with standards. Diterpenes eluted as follows: cis-abieol to βCBT-diol, 6 to 8 minutes; diterpene oxidation products, 8 to 13.5 minutes; sucrose esters, 17 to 22.5 minutes. For the composition measurement of flue-cured and air-cured tissue, a typical leaf 6.14 x 6.14 cm block (equivalent to 12 green tissue leaf disks) was cut from the dried tissue and washed with acetonitrile. 10 to 16 replicates were prepared for each strain, and the same leaves used for green leaf analysis (leaf punches) were used as controls for air-cured and flue-cured. The preparation of samples for chemical analysis as TMS derivatives was as described above, and the preparation of samples of acyl groups of sucrose esters was described below.

[0458] The integrated peak areas of all compounds and internal standards were recorded and the relative amount of each was calculated as % of the total. The many peaks of the sucrose ester type were combined to give a summary peak area.

[0459] The average number of leaves per plant, green weight of leaves per plant and total exudate weight per plant for the control versus transgenic lines are presented in Table 1 below. The data are the average of 3 plants per line 19 days after topping. (Topping refers to the removal of tobacco flowers)

[0460] Constructs GW2 and GW5 (T 0 ) and their T 1 and T 2 The GC-MS results of the generations were identical; both showed very high labdenediol and low CBT-diol and cis-abieol. To avoid redundancy, field work was performed using construct GW2 only.

[0461] Table 1.

[0462] .

[0463] Table 1 shows that the average number of leaves per plant, the green weight of leaves per plant were similar among the transgenic lines tested and similar to the control plants.

[0464] Green leaf analysis of field-grown controls versus transgenic lines

[0465] Green leaf exudate chemistry analysis was performed just after topping and showed that for each line, exudate chemistry in the field was similar to that observed in plants in the greenhouse, and plant-to-plant variability in chemistry was approximately 25-30% for each line in the control and in the field (see Figure 3 ). Each point on the graph represents data from one plant. Figure 3 The data presented in shows:

[0466] ● Strains GW1 and GW3 are rich in cis-abietanol

[0467] ● Strain GW2 is rich in labdene-diol.

[0468] Measurement of acyl composition of sucrose esters

[0469] The method used was modified from Severson et al. 1985 (supra, which is incorporated herein by reference). 0.5 ml of 80% KOH (1 M) in MeOH was added to 5 to 10 mg of gum sample in a 30 ml Corex glass tube and allowed to stand overnight at 22°C for saponification. The sample was then heated to 40 °C for 24 h at 37 °C. 2 Then 1 ml of n-BuOH and 3 drops of concentrated H 2 SO 4 The sample was then heated at 110°C for 1 hour, after which the components were partitioned between hexane and water (1.5 ml each) using a pulse vortex mixer (10 pulses, 1.5 seconds each). Six to seven extractions were performed (until the water was neutral with respect to litmus paper). The aqueous phase contained K 2 SO 4 , H 2 SO 4 and sugars, and the hexane phase contained the butyl esters of the acyl groups. The hexane was transferred to a 1.5 ml GC vial and analyzed by GC-MS using the same column and for TMS. Helium was the carrier gas with a constant flow of 1.8 ml / min and an injection temperature of 250°C. The oven program for running the butyl ester derivatives was as follows: initial temperature 90°C for 3.0 minutes; rate 3°C per minute until 160°C, hold for 2 minutes, rate 15°C per minute to 250°C, hold for 5 minutes. The total run time was 39.33 minutes.

[0470] Figure 4The data in clearly show that strains GW1 and GW3 (which contain high cis-abirol) also exhibit enhanced sucrose ester production when compared to the control. Each point on the graph represents data from one plant. The acyl composition is unchanged, so these strains resemble Turkish or Oriental tobacco in that there are high levels of cis-abirol and high 3-methylvaleric acid precursors, but advantageously, they are produced from relatively high biomass plants.

[0471] Figure 4 show:

[0472] • In the high cis-abieol lines (GW1 and GW3), sucrose esters were greatly enriched compared to the control.

[0473] ● In the GW2 strain (high labdenediol), sucrose esters were also elevated.

[0474] Example 4 Exudate Components in Green vs Flue-cured Field Tobacco and Green vs Air-cured Tobacco

[0475] RNAi lines were grown in the field, T2 or T3, and the traits were stable. The harvest and collection of flue-cured and air-cured (for burley tobacco) were completed after approximately 4 and a half months. Flue-curing included: 48 hours of coloring, 85 to 100°F, 94% RH; 24 hours of wilting, 100°F to 120°F, to 54% RH; 30 hours of leaf drying, 120°F to 135°F, 40% RH; 40 hours of stem drying, 135 to 168°F, to 22% RH, as described in Example 3.

[0476] Use the Figure 3 and 4 The same TMS chromatograms were generated, with the exudate components recalculated based on surface area and expressed as µg / cm in Tables 2 and 3. 2 (Green vs Flue and Green vs Air-cured). Calculations were based on comparison with known amounts of internal standard coniferene, assuming that the peak response for all compounds was identical. The amounts of compounds shown in Table 2 represent the mean values ​​of 20 green plants of the control, and 34 to 36 green plants and 10 to 14 modulated plants of strains GW1-3, ± their respective standard deviations.

[0477] Table 2. Exudate components from green vs flue-cured 2014 field tobacco

[0478]

[0479] * The abundance of cis-abieol from 2 leaf disc extracts is very low. Numbers are calculated from % of total green leaf extracts. ** The abundance of a- and b-CBT-diols from 2 leaf disc-greens is low. Here, numbers are calculated from % of total green leaf extracts.

[0480] The data presented in Table 2 show that flue curing does not increase the sucrose ester exudate components of control plants, but it does increase the sucrose ester content of transgenic plants containing constructs GW1, GW2 or GW3. Flue curing also increases the LD content of transgenic plants containing constructs GW1, GW2 or GW3. Flue curing increases the cis-abirol content of all tobaccos.

[0481] The data show that flue curing increases the α-CBT-diol content of the exudate fraction of GW2 transgenic plants compared to green tobacco. The α-CBT-diol content of the control plants was also increased by flue curing, but to a lesser extent. The data also show that flue curing increases the β-CBT-diol content of the exudate fraction of GW2 transgenic plants compared to green tobacco. The β-CBT-diol content of the control plants was not affected by flue curing.

[0482] Oxidative products were increased in the transgenic lines.

[0483] The compound amounts shown in Table 3 (below) present the mean values ​​of 20 green plants for the control, and 34 to 36 green plants and 8 to 16 cured plants for lines GW1-3, ± their respective standard deviations.

[0484] The data presented in Table 3 below show that air-curing does not increase the sucrose ester exudate component of the control plants, but it does slightly increase the sucrose ester content of the transgenic plants containing constructs GW1, GW2 or GW3. The sucrose ester content is stable in the air-cured transgenic lines. The data also show that flue-curing increases the α-CBT-diol content of the exudate component of the GW2 transgenic plants (which is a minor component in the transgenics) compared to green tobacco. The α-CBT-diol content of the control plants is not affected by flue curing.

[0485] The data show that air-curing increases the β-CBT-diol content of the exudate components of GW2 transgenic plants compared to green tobacco. The β-CBT-diol content of the control plants is also increased by flue curing, but to a lesser extent. Air-curing also increases the LD content of transgenic plants comprising constructs GW1, GW2 or GW3. Air-curing increases the cis-abirol content of the control and tobacco comprising GW1 or GW3.

[0486] Oxidative products were increased in the transgenic lines.

[0487] Table 3. Exudate components from green vs air-cured 2014 field tobacco

[0488]

[0489] * The abundance of cis-abieol from 2 leaf disc extracts is very low. Figures are calculated as % of total green leaf extracts. ** The abundance of a- and b-CBT-diols from 2 leaf disc-greens is low. Figures are calculated as % of total green leaf extracts.

[0490] Example 5 Analysis of diterpenoid composition and acyl composition of sucrose esters

[0491] The diterpenoid composition and sucrose ester acyl composition were measured as described in Example 3. The inventors compared the diterpenoid and sucrose ester compositions in the transgenic lines with the control TI 1068 and with sun-cured Turkish-type commercial tobacco. The sun-cured commercial tobacco was sent to the inventors as pressed leaves. The data presented for the transgenic tobacco lines present the average of 10 to 14 plants, while the TI 1068 control presents the average of more than 16 plants.

[0492] The following commercial Türkiye types were analyzed for comparison:

[0493] A = Oriental, Supplier: Socotab EOOD, Crop 2013

[0494] B = Türkiye-Samsun crop 2013, grade SMAL

[0495] C = Türkiye-IXMIR, Crop 2013, Grade YZAL

[0496] See Table 4 for exudate composition analysis of diterpenoid content.

[0497] The data in Table 4 (below) show that the flue-cured transgenic lines GW1, GW2 and GW3 had higher sucrose ester content compared to the control and / or turkey plants tested.

[0498] See Table 5 for exudate composition analysis of sucrose ester acyl compositions.

[0499] Data presented for transgenic tobacco lines present averages of 4 to 8 plants, while TI 1068 controls present averages of over 10 plants. The data in Table 5 (below) show that the sucrose ester acyl composition of transgenic tobacco lines GW1, GW2 and GW3 is similar to that of Turkish-type tobacco compositions, i.e., rich in 3-Me valeryl.

[0500]

[0501]

[0502] Example 6 Analysis of chemical substances on the surface of GW3

[0503] Analysis of green field, air-cured, and flue-cured samples was performed. Where possible, when leaf punches were taken to obtain green field data, all samples were taken from the same leaf by marking and numbering the leaf in the field. After air-curing or flue-curing, the same numbered leaves were sampled so that direct comparisons of green / air-cured and green / flue-cured could be made. The following table shows the results for green compared to flue-cured samples and green compared to air-cured samples. Data were normalized to micrograms / cm 2 leaf.

[0504] Table 6. GW3 surface chemical analysis of green field, air-cured, and flue-cured samples

[0505]

[0506] The number in the brackets after green or flue-cured or air-dried indicates how many independent samples were analyzed and used to derive the mean. For example, for the control, green (20) means 20 independent samples. The mean of these is shown with the standard deviation below, for example, for the control green (2), 6.695 ± 3.834.

[0507] The data for green to flue-cured are for the following lines: control and GW3-high cis-abietin. The data show that cis-abietin is greatly reduced from green to flue-cured, and for the GW3-high cis-abietin line, in the far right column, this cis may have been degraded to some "oxidation products". The data also show that in GW3, labdenediol increased from green to flue-cured. The changes are proportional, i.e., as cis-abietin decreases from green to flue-cured (µg / cm 2 ), labdenediol increased proportionally. Note that for GW3, the oxidation bar also increased significantly from green to flue-cured. In addition, CBT diol did not change much from green to flue-cured, and the amount of sucrose esters did not change much from green to flue-cured in the control; while it increased significantly (+4.7) in the GW3 strain. Separate analysis (not shown) showed that the acyl composition did not change qualitatively from green to flue-cured, as expected for stable sucrose esters. The data from green to air-cured show that the change from green to air-cured is smaller than the change observed for green to flue-cured. Only oxidation appears to increase significantly, probably due to degradation of cis-abietanol.

[0508] Figure 5 It was shown that sucrose ester content was enriched in the transgenic line GW-3 compared to the control. The sucrose ester content was expressed as a percentage of the total major exudate compounds. Figure 6 It was shown that cis-abieol was enriched in the transgenic line GW-3 compared to the control. The cis-abieol content was expressed as a percentage of the total major exudate compounds.

[0509] Example 7 Exudate Components of Flue-cured and Air-cured 2016 Field Tobacco

[0510] RNAi lines were grown in the field, T2 or T3, stabilized, and harvested. In 2016, flue-cured plants were harvested as 3 primings. In the 2016 trial, air-cured plants were stalk cut, air-cured, and divided into 1 / 3 lower leaves, 1 / 3 middle leaves, and 1 / 3 upper leaves before analysis. This was to mimic the general method used for commercial production.

[0511] Table 7. Exudate components from flue-cured 2016 field tobacco

[0512]

[0513] a) In 2016, unlike 2014, for flue-cured, 3 initiators were taken and analyzed separately.

[0514] b) Each value for each elicitor is the average of 3 replicates. Each replicate is the average of 20 plants.

[0515] c) In 2016, unlike in 2014, green tissue of plants in the field was not monitored.

[0516] in conclusion

[0517] The data in Table 7 show that in the 2016 flue-cured field tobacco, the GW1-3 line was enriched in sucrose esters compared to the control.

[0518] Table 8. Exudate components from air-cured 2016 field tobacco

[0519]

[0520] a) In 2016, unlike in 2014, the upper 1 / 3, middle 1 / 3 and lower 1 / 3 leaves of stem-cut, air-cured plants were combined and analyzed separately.

[0521] b) Each value for each level is the average of 20 leaves from 20 independent plants.

[0522] c) In 2016, unlike in 2014, green tissue of plants in the field was not monitored.

[0523] in conclusion

[0524] The data in Table 8 show that in the 2016 air-cured field tobacco, the GW1-3 line was enriched in sucrose esters compared to the control.

[0525] All publications mentioned in the above specification are incorporated herein by reference. Various changes and variations of the methods and systems described in the present invention will be apparent to those skilled in the art and will not depart from the scope and spirit of the present invention. Although the present invention has been described with respect to specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to such specific embodiments. In fact, various changes in the described modes for carrying out the present invention that are apparent to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for increasing the sucrose ester content in a tobacco plant or part thereof or a tobacco cell culture, the method comprising modifying the tobacco plant or tobacco cell culture by inhibiting the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: i) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or ii) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or iii) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto.

2. Use of a ddRNAi DNA construct for increasing the sucrose ester content in a tobacco plant or part thereof or a tobacco cell culture, wherein the use comprises inhibiting the activity or expression of a diterpene synthesis gene, wherein the ddRNAi DNA construct comprises: (a) at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of a cyclase 2 gene (CYC2), and wherein the diterpene synthesis gene is i) a cyclase 2 gene (CYC2) having a polynucleotide sequence shown in SEQ ID No. 8 or SEQ ID No. 9, or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence shown in SEQ ID No. 10, or a polynucleotide sequence having at least 90% sequence identity thereto; or (b) at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and at least a portion of exon 7 of the CBTol cyclase gene, and wherein the diterpene synthesis gene is ii) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No. 1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 2 or a polynucleotide sequence having at least 90% sequence identity thereto; or (c) a ddRNAi DNA construct of at least nucleotides 1497 to 1517 of a terpene synthase 3-8 gene inhibits the expression of a terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No. 3, or a ddRNAi DNA construct of at least nucleotides 884 to 904 of a terpene synthase 3-8 gene inhibits the expression of a terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No. 3, and wherein the diterpene synthesis gene is iii) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto.

3. A method for producing a tobacco plant or part thereof, or a tobacco cell culture, having an increased sucrose ester content, the method comprising modifying the tobacco to inhibit the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: i) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or ii) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or iii) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto.

4. The method according to claim 3, wherein the part of the tobacco plant is tobacco plant propagation material or tobacco leaves.

5. The method according to claim 4, wherein the tobacco leaf is: (a) cut harvested tobacco leaves; or (b) Processed tobacco leaves.

6. The method according to claim 4, wherein the tobacco leaves are cut and processed tobacco leaves.

7. The method of claim 1, wherein the sucrose ester content is increased compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

8. The use according to claim 2, wherein the sucrose ester content is increased compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

9. The method of claim 3, wherein the sucrose ester content is increased compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

10. The method according to claim 3, wherein RNA interference (RNAi) is used to inhibit the expression of diterpene biosynthesis genes.

11. The method according to claim 10, wherein: (a) inhibiting the expression of a cyclase 2 gene (CYC2) using a ddRNAi DNA construct comprising at least a portion of exon 1, at least a portion of exon 2, and at least a portion of exon 3 of a cyclase 2 gene (CYC2); or (b) inhibiting CBTol cyclase gene expression using a ddRNAi DNA construct comprising at least a portion of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and at least a portion of exon 7 of the CBTol cyclase gene; or (c) inhibiting the expression of a terpene synthase 3-8 gene using a ddRNAi DNA construct comprising at least nucleotides 1497 to 1517 of the terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No. 3; or (d) inhibiting the expression of the terpene synthase 3-8 gene using a ddRNAi DNA construct comprising at least nucleotides 884 to 904 of the terpene synthase 3-8 gene, wherein the numbering is determined by alignment with SEQ ID No.

3.

12. A tobacco plant part or tobacco cell culture, which has been modified to achieve an increase in sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is terpene synthase 3-8, wherein the gene of terpene synthase 3-8 has a polynucleotide sequence as shown in SEQ ID No.3 or a polynucleotide sequence having at least 90% sequence identity thereto, or has a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.4 or a polynucleotide sequence having at least 90% sequence identity thereto, and wherein the tobacco plant part is a non-fertile material.

13. A tobacco plant part or tobacco cell culture comprising a ddRNAi construct as defined in claim 11, and wherein the tobacco plant part is non-fertile material.

14. The method of any one of claims 1, 3-7 or 9-11, wherein the sucrose ester content of the tobacco plant in the modified tobacco plant or tobacco cell culture is at least 2-fold compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

15. The use according to claim 2 or 8, wherein the sucrose ester content of the tobacco plant in the modified tobacco plant or tobacco cell culture is at least twice as high as that of a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

16. The tobacco plant part or tobacco cell culture of claim 12 or 13, wherein the sucrose ester content of the tobacco plant in the modified tobacco plant or tobacco cell culture is at least 2-fold as compared to a tobacco plant or tobacco cell culture that has not been modified to inhibit the activity or expression of a diterpene biosynthesis gene.

17. Use of the tobacco cell culture according to claim 12 or 13 for growing tobacco plants.

18. Use of the following a) or b) for the production of tobacco industry products: a) a tobacco plant part or tobacco cell culture according to claim 12 or 13, or b) a tobacco plant or part thereof or a tobacco cell culture modified to achieve an increased sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: i) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or ii) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or iii) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto.

19. Use of the tobacco cell culture according to claim 12 or 13 for growing crops of tobacco plants.

20. Use of the tobacco plant part or tobacco cell culture according to claim 12 or 13 for producing tobacco leaves.

21. The use according to claim 20, wherein the use is for the production of processed tobacco leaves.

22. The use according to claim 21, wherein the processed tobacco leaf is a cured tobacco leaf.

23. Harvested leaves of a tobacco plant, which are: i) a tobacco plant part according to claim 12 or 13, or ii) harvested leaves obtainable from a tobacco plant obtainable by the use according to claim 17, or iii) harvested leaves of a tobacco plant modified to achieve an increased sucrose ester content compared to an unmodified plant, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is terpene synthase 3-8, wherein the gene of terpene synthase 3-8 has the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or has a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto, or iv) harvested leaves of a tobacco plant comprising a ddRNAi DNA construct as defined in claim 11; and The harvested leaves of the tobacco plant are non-fertile material.

24. The harvested leaves of the tobacco plant of claim 23, wherein the harvested leaves of the tobacco plant are cut harvested leaves.

25. Processed tobacco leaves: i) obtainable from a tobacco plant obtainable from the use according to claim 17; or ii) obtainable by processing a tobacco plant part according to claim 12 or 13; or iii) obtainable by processing a tobacco plant which has been modified to achieve an increase in the sucrose ester content compared to an unmodified plant or an unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: 1) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or 2) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or 3) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto; or iv) can be obtained by processing harvested leaves of the tobacco plant according to claim 23 or 24.

26. The processed tobacco leaf according to claim 25, wherein the processed tobacco leaf is a non-viable processed tobacco leaf.

27. The processed tobacco leaf of claim 26, wherein the tobacco is processed by curing, fermenting, pasteurizing, or a combination thereof.

28. The processed tobacco leaf of claim 26, wherein the processed tobacco leaf is a cut processed tobacco leaf.

29. Cured tobacco material, i) is made from a tobacco plant part or tobacco cell culture according to claim 12 or 13, or ii) prepared from harvested leaves of a tobacco plant according to claim 23 or 24, or iii) prepared from a tobacco plant or part thereof or a tobacco cell culture which has been modified to achieve an increase in the sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: 1) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or 2) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or 3) Terpene synthase 3-8, which has the polynucleotide sequence shown in SEQ ID No.3 or a polynucleotide sequence having at least 90% sequence identity thereto, or has a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.4 or a polynucleotide sequence having at least 90% sequence identity thereto.

30. A tobacco blend comprising the cured tobacco material of claim 29.

31. Tobacco industry products prepared from: i) a tobacco plant part or tobacco cell culture according to claim 12 or 13; or ii) a tobacco plant or part thereof or a tobacco cell culture, which has been modified to achieve an increase in the sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: 1) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or 2) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or 3) terpene synthase 3-8, which has the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or has a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto; iii) harvested leaves of the tobacco plant according to claim 23 or 24; or iv) a processed tobacco leaf according to any one of claims 25 to 28; or v) The tobacco blend according to claim 30.

32. The tobacco industry product according to claim 31, wherein the tobacco product is: (a) combustible smoking articles; (b) smokeless tobacco products; or (c) Non-flammable aerosol supply systems.

33. The tobacco industry product according to claim 32, wherein the non-combustible aerosol supply system is a tobacco heating device or an aerosol generating device.

34. A tobacco industry product prepared from the cured tobacco material according to claim 29.

35. The tobacco industry product according to claim 34, wherein the tobacco product is: (a) combustible smoking articles; or (b) smokeless tobacco products; or (c) Non-flammable aerosol supply systems.

36. The tobacco industry product according to claim 35, wherein the non-combustible aerosol supply system is a tobacco heating device or an aerosol generating device.

37. A combustible smoking article, a non-combustible aerosol delivery system, a smokeless tobacco product, or a tobacco heating device containing: 1) A tobacco plant part or tobacco cell culture according to claim 12 or 13; or 2) harvested leaves of the tobacco plant according to claim 23 or 24; or 3) A tobacco plant or part thereof or a tobacco cell culture, which has been modified to achieve an increase in the sucrose ester content compared to an unmodified plant or unmodified tobacco cell culture, wherein the modification is the inhibition of the activity or expression of a diterpene synthesis gene, wherein the diterpene synthesis gene is selected from: i) a cyclase 2 gene (CYC2) having a polynucleotide sequence as shown in SEQ ID No.8 or SEQ ID No.9 or a polynucleotide sequence having at least 90% sequence identity thereto, or a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No.10 or a polynucleotide sequence having at least 90% sequence identity thereto; or ii) CBTol cyclase having the polynucleotide sequence shown in SEQ ID No.1 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No.2 or a polynucleotide sequence having at least 90% sequence identity thereto; or iii) a terpene synthase 3-8 having the polynucleotide sequence shown in SEQ ID No. 3 or a polynucleotide sequence having at least 90% sequence identity thereto, or having a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4 or a polynucleotide sequence having at least 90% sequence identity thereto.

38. A combustible smoking article, a non-combustible aerosol delivery system, a smokeless tobacco product or a tobacco heating device comprising the prepared tobacco material according to claim 29.

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