Method for altering the alkaloid content in plants

Abstract

The present invention relates to a method for modulating the alkaloid content of a plant or part thereof, the method comprising modifying the plant by modulating the activity or expression of at least one gene encoding an RNA binding protein. The present invention also relates to a method for reducing the content of at least one tobacco-specific nitrosamine (TSNA) precursor in tobacco, the method comprising modulating the activity or expression of at least one gene encoding an RNA binding protein.

Description

Technical Field

[0001] The present invention relates to methods for regulating the alkaloid content of plants or parts thereof or cells or cell cultures. The present invention also extends to methods for regulating the expression and / or activity of polypeptides that regulate the alkaloid content in plants. On the other hand, the present invention provides methods for regulating the expression and / or activity of genes encoding polypeptides that regulate the alkaloid content in plants. The present invention also extends to constructs that can be used to regulate polypeptides. The present invention further relates to plant cells and plants that are modified to achieve regulation of alkaloid content. The present invention also relates to leaves processed and harvested from such regulated plants and their use in tobacco industry products, including combustible smoking products. Background Art

[0002] Alkaloids are a group of naturally occurring compounds that contain primarily basic nitrogen atoms and are produced by a wide variety of organisms including bacteria, fungi, plants and animals.

[0003] Alkaloids can be classified based on similarity of carbon skeletons such as indole, isoquinoline and pyridine. Pyridine derivatives are a class of monomeric alkaloids; this class includes simple pyridine derivatives, polycyclic condensed and non-condensed pyridine derivatives, and sesquiterpene pyridine derivatives. Examples are nicotine, nornicotine, pseudooxynicotine, anabasine, mithofamine and anatabine.

[0004] Most of the known biological functions of alkaloids are related to protection. Neuroactive molecules such as caffeine, cocaine, morphine and nicotine act as defense compounds against invading predators. The accumulation of these alkaloids is the result of a signal transduction cascade that monitors gene expression, enzyme activity and alkaloid concentration. The fine regulation of alkaloid content in plants involves negative feedback loops and degradation pathways.

[0005] Nicotine occurs naturally in several plant species, but is found at the highest levels in tobacco plants. Cultivated tobacco produces 2-4% of the alkaloid by dry weight. Nicotine is present in both wild and cultivated tobacco ( Nicotiana ) species and plays an important role in plant defense against herbivores and insects (Voelckel et al. (2001) Oecologia 127(2):274-280, incorporated herein by reference). It accounts for ~90% of the total alkaloid content. The remaining 10% of the alkaloid pool is mainly composed of the structurally related compounds nornicotine, anatabine, anabasine and pseudooxynicotine (PON).

[0006] The regulation of alkaloid content in tobacco is complex. Several factors include genotype, environment, fertilization and agronomic practices (e.g., topping) that affect alkaloid levels in tobacco plants. Some key regulators of nicotine biosynthesis are well characterized, such as putrescine N-methyltransferase (PMT), which plays a key role in this pathway, is activated by members of the ethylene response factor (ERF) superfamily, the largest transcription factor family in the tobacco genome (Rushton et al. (2008) Plant Physiol. 147(1): 280-295, incorporated herein by reference). Other transcription factors that induce alkaloid biosynthesis belong to the MYC2-like basic helix-loop-helix (bHLH) family. MYC2-like bHLHs directly regulate alkaloid levels through the binding and activation of Gbox-mediated alkaloid structural genes, and indirectly regulate alkaloid levels through the activation of ERFs.

[0007] Tobacco pyridine alkaloids are precursors of tobacco-specific nitrosamines (TSNAs) formed during the post-harvest leaf curing process. The four major TSNAs found in cured tobacco leaves are N'-nitrosonornicotine (NNN), N'-nitrosonatabine (NAT), N'-nitrosonatabine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). During the post-harvest leaf curing process, reactions between pyridine alkaloids and nitrosating species lead to the formation of tobacco-specific nitrosamines (TSNAs). PON may function as a direct precursor in the synthesis of the TSNA NNK (Bush et al., 2001, incorporated herein by reference). Reducing the production and accumulation of TSNAs is of great significance. The CYP82E family of nicotine demethylase genes is one of the major regulators of the conversion of nicotine to nornicotine, and altering its activity or accumulation may lead to reduced NNN levels. However, to date, the enzyme or gene responsible for the production of PON has not been identified.

[0008] As described in the examples, the inventors sought to investigate genes responsible for the synthesis of alkaloids and / or TSNA precursors with the aim of modulating the alkaloid content in plants, for example reducing the TSNA content in tobacco. Summary of the invention

[0009] It has surprisingly been found that by regulating the activity or expression of a gene encoding an RNA binding protein, the alkaloid content and / or the TSNA content or the content of a precursor of TSNA of a plant can be regulated. A gene encoding an RNA binding protein as taught herein, such as Nitab4.5_0013685g0010.2, is a regulator of the alkaloid and TSNA precursor content in cultivated tobacco. In particular, a gene as taught herein, such as Nitab4.5_0013685g0010.2, is a regulator of the alkaloid content in cultivated tobacco. Nitab4.5_0013685g0010.2 encodes an RNA binding protein according to the invention. Nitab4.5_0000308g0150.2, Nitab4.5_0003919g0010.2, Nitab4.5_0002978g0020.2, Nitab4.5_0001361g0220.2, Nitab4.5_0002978g0030.2, Nitab4.5_0001361g0225.2, Nitab4.5_0003978g0020.2, Nitab4.5_0000078g0240.2, Nitab4.5_0005079g0050.2, Nitab4.5_0008185g0030.2 are homologues of Nitab4.5_0013685g0010.2 according to the present invention.

[0010] The RNA binding protein according to the present invention may contain conserved domains called RNA binding domain (RBD) and RGG domain.

[0011] According to the present invention, tobacco industry products can be produced with regulated alkaloid content and commercially desirable traits sought by consumers of tobacco industry products. In some cases, consumers may desire products with low levels of alkaloid content. In some cases, consumers may desire products with low levels of TSNA precursors.

[0012] The present invention may be particularly useful in the field of plant molecular agriculture, where plants (e.g., tobacco and other Nicotiana species) are used to produce proteins, peptides and metabolites, e.g., for the production of therapeutic agents and drugs such as antibiotics, virus-like particles, or neutraceuticals or small molecules. In an EU-funded project called PharmPlant, tobacco has been used to develop HIV-neutralizing antibodies, and Medicago Inc. in Canada has been involved in a tobacco-based platform for the production of virus-like particles for influenza vaccine manufacturing.

[0013] Therefore, the plants according to the invention can be used in molecular agriculture to reduce or eliminate the presence of nicotine alkaloids. The use of low nicotine plants or rootsocks is beneficial in molecular agriculture and will reduce downstream processing costs associated with purification.

[0014] The present inventors have surprisingly identified a method for regulating (e.g. reducing) the alkaloid content of a plant (e.g. a tobacco plant) by regulating (e.g. reducing) the activity or expression of a gene encoding an RNA binding protein. The alkaloid content of a plant (e.g. a tobacco plant) (e.g. the content of one or more of nornicotine, PON, anabasine, anatabine or mithosamine, suitably the content of one or more of nornicotine, PON, anabasine or anatabine) can be reduced by reducing the activity or expression of a gene encoding an RNA binding protein, or can be increased by increasing the activity or expression of a gene encoding an RNA binding protein. Prior to the present invention, it was not known that regulating the activity or expression of a gene encoding an RNA binding protein as described herein could be used to regulate the alkaloid content or to regulate the TSNA precursor content, in particular, the nornicotine, PON, anabasine and / or anatabine content.

[0015] In one aspect, the present invention provides a method for regulating (e.g., reducing) the alkaloid content of a tobacco plant or part thereof or a tobacco cell or a tobacco cell culture, the method comprising modifying the plant or part thereof or cell by reducing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, or a sequence having at least 80% identity with SEQ ID NO. 1, or a homolog of SEQ ID NO. 1.

[0016] In one aspect, the present invention provides a method for regulating (e.g., reducing) the alkaloid content of a tobacco plant or part thereof or a tobacco cell or a tobacco cell culture, the method comprising modifying the plant or part thereof or cell by reducing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31.

[0017] In another aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamine (TSNA) precursors in tobacco plants or parts thereof or tobacco cells or tobacco cell cultures, the method comprising modifying the plant or part thereof or cell by reducing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, or a sequence having at least 80% identity with SEQ ID NO. 1, or a homolog of SEQ ID NO. 1.

[0018] In one aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant or part thereof or a tobacco cell or a tobacco cell culture, the method comprising modifying the plant or part thereof or cell by reducing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31.

[0019] In another aspect, the present invention provides the use of at least one gene encoding an RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31 for modulating (e.g., reducing) alkaloid content or reducing TSNA precursor content in tobacco cells or tobacco plants or parts thereof or tobacco cell cultures.

[0020] In a further aspect, a method is provided for producing a plant or part thereof, tobacco cell or tobacco cell culture, plant propagation material, leaves, cut harvested leaves, processed leaves or cut and processed leaves having a modulated (e.g., reduced) alkaloid content or reduced TSNA precursor content, the method comprising modifying the plant or part thereof or cell or cell culture or plant propagation material to reduce the activity or expression of at least one gene encoding an RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31.

[0021] Suitably, the alkaloid content or TSNA precursor content may be reduced compared to a plant or part thereof or a cell or a cell culture or a plant propagation material or a leaf which has not been modified to reduce the activity or expression of at least one gene encoding said RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31.

[0022] In another aspect, the present invention provides a plant or part thereof or a cell or a cell culture which has been modified to achieve modulation (e.g., reduction) in alkaloid content or reduction in TSNA precursor content compared to an unmodified plant or part thereof or an unmodified cell culture, wherein the modification is a reduction in the activity or expression of at least one gene encoding an RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31.

[0023] In one aspect, the invention provides plant propagation material obtainable (eg obtained) from a plant according to the invention or from a plant or cell culture produced by a method according to the invention.

[0024] Suitably, the TSNA precursor may be selected from nornicotine, PON, ananicotine and anatabine. Suitably, the TSNA precursor may be nornicotine. Suitably, the TSNA precursor may be PON. Suitably, the TSNA precursor may be anatabine. Suitably, the TSNA precursor may be anatabine.

[0025] In one aspect, the invention provides the use of a plant or part or cell culture thereof according to the invention, or a plant produced by a method according to the invention, for growing plants.

[0026] In another aspect, the present invention provides use of a plant or part or cell culture thereof according to the invention, or a plant produced by a method according to the invention, for producing a product.

[0027] In a further aspect, the present invention provides the use of a plant according to the invention or a part thereof, or a plant produced by a method according to the invention, for growing crops.

[0028] In another aspect, the present invention provides use of a plant according to the present invention or a part thereof, or a plant produced by a method according to the present invention, for producing leaves.

[0029] In a further aspect, the present invention provides harvested leaves of a plant according to the invention, or obtainable (e.g. obtained from) a plant propagated from propagation material according to the invention, or obtainable (e.g. obtained from) a plant obtained by use according to the invention, or obtainable (e.g. obtained from) a plant produced by a method according to the invention.

[0030] Suitably, the harvested leaves of the plant may be cut harvested leaves.

[0031] In another aspect, the present invention provides a processed leaf, preferably a processed tobacco leaf, preferably a non-viable processed tobacco leaf, which:

[0032] obtainable (e.g. obtained) from a plant obtainable (e.g. obtained) from the use according to the invention;

[0033] obtainable (e.g. obtained) by processing the plant according to the invention;

[0034] obtainable (eg obtained) from a plant propagated from the plant propagation material according to the invention; or

[0035] obtainable (e.g. obtained) by processing the harvested leaves according to the invention; or

[0036] Obtainable (eg obtained) from a plant produced by the method of the invention.

[0037] Suitably, the leaves may be processed by curing, fermenting, pasteurising or a combination thereof.

[0038] Suitably, the content of one or more TSNAs selected from N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanatabine (NAB) may be reduced, wherein preferably the content of NNN and / or NNK is reduced, wherein more preferably at least the content of NNN is reduced.

[0039] Suitably, the machining blade may be a cutting machining blade.

[0040] In a further aspect, the present invention provides a cured tobacco material made from a plant or part thereof or an extract thereof according to the invention or from a leaf or an extract thereof according to the invention.

[0041] In one aspect, the present invention provides a tobacco blend comprising a cured tobacco material according to the present invention.

[0042] In one aspect, the present invention provides a tobacco industry product prepared by:

[0043] A tobacco plant or part thereof or a tobacco cell or cell culture according to the invention;

[0044] Tobacco plants or parts thereof propagated from tobacco plant propagation material according to the invention;

[0045] Harvested leaves of a plant according to the invention;

[0046] Processing blades according to the invention;

[0047] Plants produced by the methods of the present invention;

[0048] Cured tobacco according to the present invention; or

[0049] Tobacco blends according to the present invention.

[0050] Suitably, the tobacco industry product may be a combustible smoking article.

[0051] Suitably, the tobacco industry product may be a smokeless tobacco product.

[0052] Suitably, the tobacco industry product may be a non-combustible gas aerosol delivery system, such as a tobacco heating device or an aerosol generating device.

[0053] In another aspect, the present invention provides a combustible smoking article, a non-combustible gas aerosol delivery system, a smokeless tobacco product or a tobacco heating device, which comprises a plant or part thereof according to the present invention, or a tobacco cell or cell culture according to the present invention; or a harvested leaf according to the present invention, or a processed leaf according to the present invention, or a cured tobacco material according to the present invention; or a tobacco blend according to the present invention.

[0054] In a further aspect, the present invention provides the use of a nucleotide sequence of at least one gene encoding an RNA binding protein in selecting plants having a modulated (e.g., reduced) alkaloid content and / or reduced tobacco-specific nitrosamine (TSNA) precursor content, wherein the sequence of the RNA binding protein is selected from SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or wherein the at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33 sequences with at least 80% identity.

[0055] In another aspect, the present invention provides a mutant of a plant carrying a heritable mutation in the nucleotide sequence of at least one gene encoding an RNA binding protein, wherein the gene is selected from SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33; wherein the heritable mutation reduces the activity or expression of the at least one gene encoding an RNA binding protein, and wherein the mutant plant has a modulated (e.g., reduced) alkaloid content and / or reduced tobacco-specific nitrosamine (TSNA) precursors relative to a comparable plant not carrying the heritable mutation.

[0056] In another aspect, the present invention provides progeny or seeds of a mutant plant carrying a heritable mutation according to the present invention.

[0057] In one aspect, the present invention provides harvested leaves, processed leaves or cured tobacco material produced from a plant comprising a modification in the nucleotide sequence of at least one gene encoding an RNA binding protein, wherein the at least one gene is selected from the group consisting of: SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33; or a modification of the nucleotide sequence of at least one gene encoding an RNA binding protein. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32 or 33 sequences having at least 80% identity; wherein the modification reduces the activity or expression of at least one gene encoding an RNA binding protein, and wherein the plant has a modulated (e.g., reduced) alkaloid content and / or a reduced tobacco-specific nitrosamine (TSNA) precursor content relative to a comparable plant that does not carry the modification in the at least one gene encoding the RNA binding protein.

[0058] In another aspect, the present invention provides a method, leaf, plant, plant propagation material, harvested leaf, processed tobacco, tobacco product, use or combination thereof as described herein with reference to the description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0060] Figure 1 Alkaloid content of 5-week-old tobacco leaves overexpressing Nitab4.5_0013685g0010.2 is shown. Alkaloid content is expressed relative to control and represents two biological replicates analyzed by one-way ANOVA and Tukey's multiple-comparison post-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P value ≤ 0.01.

[0061] Figure 2 Alkaloid content of 5 week-old tobacco leaves expressing artificial miRNA targeting Nitab4.5_0013685g0010.2 is shown. Alkaloid content is expressed relative to control, and comprises two biological replicates analyzed by single factor ANOVA and Tukey's multiple comparison post hoc test. Values ​​are shown as mean ± SEM. Asterisk indicates statistical significance of P value ≤ 0.01.

[0062] Figure 3Alkaloid content of 5 week-old tobacco leaves expressing Nitab4.5_0013685g0010.2_deltaRBD-RGG is shown. Alkaloid content is expressed relative to control, and represents two biological replicates analyzed by single-factor ANOVA and Tukey's multiple comparison post hoc test. Values ​​are shown as mean ± SEM. Asterisk indicates statistical significance of P value ≤ 0.05.

[0063] Figure 4 The amino acid sequence of Nitab4.5_0013685g0010.2 is shown - SEQ ID NO. 1 - from tobacco ( Nicotiana tabacum ) of protein.

[0064] Figure 5 The coding sequence of Nitab4.5_0013685g0010.2 - SEQ ID NO. 2 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0065] Figure 6 The genomic sequence of Nitab4.5_0013685g0010.2 - SEQ ID NO. 3 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0066] Figure 7 The amino acid sequence of Nitab4.5_0000308g0150.2 - SEQ ID NO. 4 - a protein from tobacco according to the invention is shown.

[0067] Figure 8 The coding sequence of Nitab4.5_0000308g0150.2 - SEQ ID NO. 5 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0068] Fig. 9 The genomic sequence of Nitab4.5_0000308g0150.2 - SEQ ID NO. 6 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0069] Fig.10 The amino acid sequence of Nitab4.5_0003919g0010.2 - SEQ ID NO. 7 - a protein from tobacco according to the invention is shown.

[0070] Fig.11 The coding sequence of Nitab4.5_0003919g0010.2 - SEQ ID NO. 8 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0071] Fig.12The genomic sequence of Nitab4.5_0003919g0010.2 - SEQ ID NO. 9 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0072] Fig.13 The amino acid sequence of Nitab4.5_0002978g0020.2 - SEQ ID NO. 10 - a protein from tobacco according to the invention is shown.

[0073] Fig.14 The coding sequence of Nitab4.5_0002978g0020.2 - SEQ ID NO. 11 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0074] Fig.15 The genotype sequence of Nitab4.5_0002978g0020.2 - SEQ ID NO. 12 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0075] Fig.16 The amino acid sequence of Nitab4.5_0001361g0220.2 - SEQ ID NO. 13 - a protein from tobacco according to the invention is shown.

[0076] Fig.17 The coding sequence of Nitab4.5_0001361g0220.2 - SEQ ID NO. 14 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0077] Fig.18 The genomic sequence of Nitab4.5_0001361g0220.2 - SEQ ID NO. 15 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0078] Fig.19 The amino acid sequence of Nitab4.5_0002978g0030.2 - SEQ ID NO. 16 - a protein from tobacco according to the invention is shown.

[0079] Fig. 20 The coding sequence of Nitab4.5_0002978g0030.2 - SEQ ID NO. 17 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0080] Fig.21 The genomic sequence of Nitab4.5_0002978g0030.2 - SEQ ID NO. 18 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0081] Fig. 22 The amino acid sequence of Nitab4.5_0001361g0225.2 - SEQ ID NO. 19 - a protein from tobacco according to the invention is shown.

[0082] Fig.23 The coding sequence of Nitab4.5_0001361g0225.2 - SEQ ID NO. 20 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0083] Fig.24 The genomic sequence of Nitab4.5_0001361g0225.2 - SEQ ID NO. 21 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0084] Fig.25 The amino acid sequence of Nitab4.5_0003978g0020.2 - SEQ ID NO. 22 - a protein from tobacco according to the invention is shown.

[0085] Fig.26 The coding sequence of Nitab4.5_0003978g0020.2 - SEQ ID NO. 23 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0086] Fig. 27 The genomic sequence of Nitab4.5_0003978g0020.2 - SEQ ID NO. 24 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0087] Fig.28 The amino acid sequence of Nitab4.5_0000078g0240.2 - SEQ ID NO. 25 - a protein from tobacco according to the invention is shown.

[0088] Fig.29 The coding sequence of Nitab4.5_0000078g0240.2 - SEQ ID NO. 26 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0089] Fig.30 The genomic sequence of Nitab4.5_0000078g0240.2 - SEQ ID NO. 27 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0090] Fig.31 The amino acid sequence of Nitab4.5_0005079g0050.2 - SEQ ID NO. 28 - a protein from tobacco according to the invention is shown.

[0091] Fig.32 The coding sequence of Nitab4.5_0005079g0050.2 - SEQ ID NO. 29 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0092] Fig.33 The genomic sequence of Nitab4.5_0005079g0050.2 - SEQ ID NO. 30 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0093] Fig.34 The amino acid sequence of Nitab4.5_0008185g0030.2 - SEQ ID NO. 31 - a protein from tobacco according to the invention is shown.

[0094] Fig.35 The coding sequence of Nitab4.5_0008185g0030.2 - SEQ ID NO. 32 - is shown encoding a protein from Nicotiana tabacum according to the present invention.

[0095] Fig.36 The genomic sequence of Nitab4.5_0008185g0030.2 - SEQ ID NO. 33 - encoding a protein from Nicotiana tabacum according to the present invention is shown.

[0096] Fig.37 SEQ ID NO. 34 corresponding to the nucleotide sequence of Nitab4.5_0013685g0010.2_deltaRBD-RGG as described in Example 3 is shown.

[0097] Fig.38 SEQ ID NO. 35 corresponding to the amino acid sequence of Nitab4.5_0013685g0010.2_deltaRBD-RGG as described in Example 3 is shown.

[0098] Fig.39 SEQ ID NO. 36 corresponding to the amino acid sequence of the RNA binding domain (RBD) (amino acid residues 6-84 of SEQ ID NO. 1) is shown.

[0099] Fig.40 SEQ ID NO. 37 corresponding to the RGG motif (amino acid residues 97-99 of SEQ ID NO. 1) is shown.

[0100] Fig.41SEQ ID NO. 38 corresponding to the amino acid sequence of Nitab4.5_0005487g0030.2, an interactor of Nitab4.5_0013685g0010.2, is shown.

[0101] Fig.42 SEQ ID NO. 39 corresponding to the coding sequence of the interacting factor Nitab4.5_0005487g0030.2 of Nitab4.5_0013685g0010.2 is shown.

[0102] Fig.43 SEQ ID NO. 40 of the genomic acid sequence corresponding to the interacting factor Nitab4.5_0005487g0030.2 of Nitab4.5_0013685g0010.2 is shown.

[0103] Fig.44 SEQ ID NO. 41 corresponding to the amino acid sequence of the interacting factor Nitab4.5_0007831g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0104] Fig.45 SEQ ID NO. 42 corresponding to the coding sequence of the interacting factor Nitab4.5_0007831g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0105] Fig.46 SEQ ID NO. 43 corresponding to the genomic sequence of the interacting factor Nitab4.5_0007831g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0106] Fig.47 SEQ ID NO. 44 corresponding to the amino acid sequence of the interacting factor Nitab4.5_0002978g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0107] Fig.48 SEQ ID NO. 45 corresponding to the coding sequence of the interacting factor Nitab4.5_0002978g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0108] Fig.49SEQ ID NO. 46 corresponding to the genomic sequence of the interacting factor Nitab4.5_0002978g0020.2 of Nitab4.5_0013685g0010.2 is shown.

[0109] Fig.50 SEQ ID NO. 47 corresponding to the amino acid sequence of the interacting factor Nitab4.5_0005552g0010.2 of Nitab4.5_0013685g0010.2 is shown.

[0110] Fig.51 SEQ ID NO. 48 corresponding to the coding sequence of the interacting factor Nitab4.5_0005552g0010.2 of Nitab4.5_0013685g0010.2 is shown.

[0111] Fig.52 SEQ ID NO. 49 corresponding to the genomic sequence of the interacting factor Nitab4.5_0005552g0010.2 of Nitab4.5_0013685g0010.2 is shown.

[0112] Fig.53 SEQ ID NO. 50 corresponding to the amino acid sequence of the interacting factor Nitab4.5_0003679g0060.2 of Nitab4.5_0013685g0010.2 is shown.

[0113] Fig.54 SEQ ID NO. 51 corresponding to the coding sequence of the interacting factor Nitab4.5_0003679g0060.2 of Nitab4.5_0013685g0010.2 is shown.

[0114] Fig.55 SEQ ID NO. 52 corresponding to the genomic sequence of the interacting factor Nitab4.5_0003679g0060.2 of Nitab4.5_0013685g0010.2 is shown.

[0115] Sequence Listing

[0116] A summary of sequence identifiers used throughout the subject specification and the corresponding sequence listing is provided, wherein:

[0117] SEQ ID NO. 1 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2.

[0118] SEQ ID NO. 2 corresponds to the coding sequence of Nitab4.5_0013685g0010.2.

[0119] SEQ ID NO. 3 corresponds to the genomic sequence of Nitab4.5_0013685g0010.2.

[0120] SEQ ID NO. 4 corresponds to the amino acid sequence of Nitab4.5_0000308g0150.2.

[0121] SEQ ID NO. 5 corresponds to the coding sequence of Nitab4.5_0000308g0150.2.

[0122] SEQ ID NO. 6 corresponds to the genomic sequence of Nitab4.5_0000308g0150.2.

[0123] SEQ ID NO. 7 corresponds to the amino acid sequence of Nitab4.5_0003919g0010.2.

[0124] SEQ ID NO. 8 corresponds to the coding sequence of Nitab4.5_0003919g0010.2.

[0125] SEQ ID NO. 9 corresponds to the genomic sequence of Nitab4.5_0003919g0010.2.

[0126] SEQ ID NO. 10 corresponds to the amino acid sequence of Nitab4.5_0002978g0020.2.

[0127] SEQ ID NO. 11 corresponds to the coding sequence of Nitab4.5_0002978g0020.2.

[0128] SEQ ID NO. 12 corresponds to the genomic sequence of Nitab4.5_0002978g0020.2.

[0129] SEQ ID NO. 13 corresponds to the amino acid sequence of Nitab4.5_0001361g0220.2.

[0130] SEQ ID NO. 14 corresponds to the coding sequence of Nitab4.5_0001361g0220.2.

[0131] SEQ ID NO. 15 corresponds to the genomic sequence of Nitab4.5_0001361g0220.2.

[0132] SEQ ID NO. 16 corresponds to the amino acid sequence of Nitab4.5_0002978g0030.2.

[0133] SEQ ID NO. 17 corresponds to the coding sequence of Nitab4.5_0002978g0030.2.

[0134] SEQ ID NO. 18 corresponds to the genomic sequence of Nitab4.5_0002978g0030.2.

[0135] SEQ ID NO. 19 corresponds to the amino acid sequence of Nitab4.5_0001361g0225.2.

[0136] SEQ ID NO. 20 corresponds to the coding sequence of Nitab4.5_0001361g0225.2.

[0137] SEQ ID NO. 21 corresponds to the genomic sequence of Nitab4.5_0001361g0225.2.

[0138] SEQ ID NO. 22 corresponds to the amino acid sequence of Nitab4.5_0003978g0020.2.

[0139] SEQ ID NO. 23 corresponds to the coding sequence of Nitab4.5_0003978g0020.2.

[0140] SEQ ID NO. 24 corresponds to the genomic sequence of Nitab4.5_0003978g0020.2.

[0141] SEQ ID NO. 25 corresponds to the amino acid sequence of Nitab4.5_0000078g0240.2.

[0142] SEQ ID NO. 26 corresponds to the coding sequence of Nitab4.5_0000078g0240.2.

[0143] SEQ ID NO. 27 corresponds to the genomic sequence of Nitab4.5_0000078g0240.2.

[0144] SEQ ID NO. 28 corresponds to the amino acid sequence of Nitab4.5_0005079g0050.2.

[0145] SEQ ID NO. 29 corresponds to the coding sequence of Nitab4.5_0005079g0050.2.

[0146] SEQ ID NO. 30 corresponds to the genomic sequence of Nitab4.5_0005079g0050.2.

[0147] SEQ ID NO. 31 corresponds to the amino acid sequence of Nitab4.5_0008185g0030.2.

[0148] SEQ ID NO. 32 corresponds to the coding sequence of Nitab4.5_0008185g0030.2.

[0149] SEQ ID NO. 33 corresponds to the genomic sequence of Nitab4.5_0008185g0030.2.

[0150] SEQ ID NO. 34 corresponds to the nucleotide sequence of Nitab4.5_0013685g0010.2_deltaRBD-RGG as described in Example 3.

[0151] SEQ ID NO. 35 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2_deltaRBD-RGG as described in Example 3.

[0152] SEQ ID NO. 36 corresponds to the amino acid sequence of the RNA binding domain (RBD) (amino acid residues 6-84 of SEQ ID NO. 1).

[0153] SEQ ID NO. 37 corresponds to the RGG motif (amino acid residues 97-99 of SEQ ID NO. 1).

[0154] SEQ ID NO. 38 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0005487g0030.2.

[0155] SEQ ID NO. 39 corresponds to the coding sequence of Nitab4.5_0005487g0030.2, the interacting factor of Nitab4.5_0013685g0010.2.

[0156] SEQ ID NO. 40 corresponds to the genomic sequence of Nitab4.5_0005487g0030.2, the interacting factor of Nitab4.5_0013685g0010.2.

[0157] SEQ ID NO. 41 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0007831g0020.2.

[0158] SEQ ID NO. 42 corresponds to the coding sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0007831g0020.2.

[0159] SEQ ID NO. 43 corresponds to the genomic sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0007831g0020.2.

[0160] SEQ ID NO. 44 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0002978g0020.2.

[0161] SEQ ID NO. 45 corresponds to the coding sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0002978g0020.2.

[0162] SEQ ID NO. 46 corresponds to the genomic sequence of Nitab4.5_0013685g0010.2's interacting factor Nitab4.5_0002978g0020.2.

[0163] SEQ ID NO. 47 corresponds to the amino acid sequence of Nitab4.5_0013685g0010.2, the interacting factor of Nitab4.5_0005552g0010.2.

[0164] SEQ ID NO. 48 corresponds to the coding sequence of Nitab4.5_0013685g0010.2, the interacting factor of Nitab4.5_0005552g0010.2.

[0165] SEQ ID NO. 49 corresponds to the genomic sequence of Nitab4.5_0013685g0010.2, the interacting factor of Nitab4.5_0005552g0010.2.

[0166] SEQ ID NO. 50 corresponds to the amino acid sequence of Nitab4.5_0003679g0060.2, the interacting factor of Nitab4.5_0013685g0010.2.

[0167] SEQ ID NO. 51 corresponds to the coding sequence of Nitab4.5_0003679g0060.2, the interacting factor of Nitab4.5_0013685g0010.2.

[0168] SEQ ID NO. 52 corresponds to the genomic sequence of Nitab4.5_0003679g0060.2, the interacting factor of Nitab4.5_0013685g0010.2.

[0169] Some sequences disclosed herein contain "X" or "N" in the nucleotide sequence. "X" or "N" can be a deletion or insertion of any nucleotide or one or more nucleotides. For example, in some cases, a string of "X" or "N" is shown. The number of "X" or "N" is not necessarily related to the actual number of nucleotides at the position. There may be more or fewer nucleotides than shown as "X" or "N" in the sequence. DETAILED DESCRIPTION

[0170] The inventors have shown for the first time that by modulating the activity or expression of at least one gene encoding an RNA binding protein in a plant (e.g. a tobacco plant) or cell (e.g. a tobacco cell), the alkaloid and / or TSNA precursor content of a plant (or a processed plant) or cell can be modulated.

[0171] The present invention provides a method for modulating (eg reducing) the alkaloid content of a plant or a part thereof, the method comprising modifying the plant by modulating (eg reducing) the activity or expression of at least one gene encoding an RNA binding protein.

[0172] Also provided is a method of modulating (e.g., reducing) the content of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant or a plant part thereof, the method comprising modifying the plant by modulating (e.g., reducing) the activity or expression of at least one gene encoding RNA binding.

[0173] The at least one gene encoding the RNA binding protein may be selected from at least one gene encoding an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding the RNA binding protein may comprise a nucleotide sequence as shown in SEQ ID NO. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30.

[0174] Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain. In one embodiment, at least two genes encoding RNA binding proteins are modified and selected from: a gene encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or a gene encoding an RNA binding protein comprising a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30.

[0175] In one embodiment, at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight, such as at least nine, such as ten genes encoding RNA binding proteins are regulated, wherein the genes are selected from: those encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30. Suitably, the protein may comprise an RNA binding protein and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain. In one aspect, at least one gene encoding an RNA binding protein encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2 or 3, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2 or 3, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2 or 3. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0176] In one aspect, the activity or expression of at least one further gene is modulated. Suitably, at least two (or at least three or at least four or at least five or at least six or at least seven or at least eight or at least nine) additional genes selected from SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0177] The "expression" of a gene encoding an RNA binding protein may refer to transcription, translation, or protein expression levels.

[0178] Measuring the level or amount of a gene product can be performed by any suitable method, such as comparison of mRNA transcript levels, protein or peptide levels and / or plant phenotype between modified plants and comparable plants that have not been modified according to the invention.

[0179] The term "comparable product" as defined herein will be a product derived from a plant (e.g., a tobacco plant) that has not been modified according to the present invention, but in which all other relevant characteristics are the same (e.g., plant species, growth conditions, methods for processing plants such as tobacco, etc.). Comparable products according to the present invention may mean a plant (e.g., a tobacco plant) or a part thereof, such as a leaf (e.g., a tobacco leaf), a leaf harvested (e.g., a tobacco leaf harvested), a leaf cut (e.g., a tobacco leaf harvested), a processed leaf (e.g., a processed tobacco leaf), or a plant propagation material (e.g., a tobacco plant propagation material), or a product comprising the plant or part thereof, such as a tobacco industry product or a combination thereof. In one embodiment, a comparable product is a product that does not comprise a gene encoding an RNA binding protein whose activity or expression has been regulated.

[0180] As used herein, the term "modification" or "modified" means a plant (e.g., a tobacco plant) or a nucleic acid sequence that has been altered or changed. The present invention encompasses plant modification using techniques for genetic modification of plants or non-genetic modification of 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 such as ethyl methanesulfonate (EMS) mutagenesis, and modern population analysis methods.

[0181] In one embodiment, a natural variant having a modified gene encoding an RNA binding protein is selected and the trait or gene is bred into a second plant that may have a commercially desirable trait.

[0182] In one embodiment, the plant according to the invention is a transgenic plant.In one embodiment, the plant according to the invention is a non-transgenic plant.

[0183] The term "unmodified plant" as defined herein will be a plant (e.g., a tobacco plant) that has not been modified according to the present invention, e.g., to modulate the activity or expression of a gene encoding an RNA binding protein or to modify the nucleic acid sequence of at least one gene encoding an RNA binding protein; and wherein all other relevant characteristics are the same (e.g., plant species, growth conditions, methods of processing tobacco, etc.). In one embodiment, an unmodified plant is a plant that does not contain a gene encoding an RNA binding protein whose activity or expression has been modulated. In one embodiment, an unmodified plant is a plant that does not contain a modified nucleic acid sequence encoding at least one gene, the at least one gene encoding an RNA binding protein.

[0184] RNA binding proteins

[0185] As used herein, "RNA binding protein" has its ordinary meaning in the art and refers to a protein capable of binding RNA. Methods for measuring binding between proteins and RNA are known in the art and include, for example, electrophoretic mobility shift assays.

[0186] An illustrative sequence of an RNA binding protein from tobacco is shown in SEQ ID NO.1.

[0187] As used herein, "RNA binding domain" has its ordinary meaning in the art, and refers to a common protein structural domain that binds single-stranded RNA.

[0188] The RNA binding domain facilitates RNA binding.

[0189] An illustrative sequence of an RNA binding domain is shown at amino acids 6-84 of SEQ ID NO. 1 and is also represented as SEQ ID NO. 35.

[0190] The RNA binding domain can be identified by comparing the protein under consideration to SEQ ID NO. 35 and / or SEQ ID NO. 1.

[0191] Suitably, the RNA binding domain as used herein may refer to the sequence shown in SEQ ID NO. 35 or a sequence having at least 80% identity thereto. Suitably, the RNA binding domain as used herein may refer to the sequence corresponding to amino acids 6-84 of SEQ ID NO. 1 when aligned with SEQ ID NO. 1.

[0192] As used herein, "RGG domain" or "RGG motif" has its usual meaning in the art and refers to the RGG sequence (SEQ ID NO. 36). The RGG domain or motif is a common feature of many RNA binding proteins.

[0193] An illustrative sequence of an RGG domain is shown at amino acids 97-99 of SEQ ID NO. 1 and is also represented as SEQ ID NO. 36.

[0194] The RGG domain may be identified by comparing the protein under consideration to SEQ ID NO. 36 and / or SEQ ID NO. 1.

[0195] Domain prediction software known in the art can be used to identify domains within the amino acid sequence of a protein.Domains are also described in protein databases such as UniprotKB.

[0196] Without wishing to be bound by theory, it is hypothesized that modulating the content of RNA binding proteins in plant cells or modulating the activity of RNA binding proteins in plants, such as RNA binding activity, will alter the metabolic pathways that produce alkaloids and TSNA precursors such as nornicotine and / or PON, thereby resulting in modulated alkaloid and / or TSNA precursor content.

[0197] In one embodiment, the RNA binding protein comprises an amino acid sequence as shown in SEQ ID NO.1 or a sequence having at least 80% identity thereto, or a homologue thereof. The protein comprises an RNA binding domain and an RGG domain. Suitably, a homologue of SEQ ID NO.1 may be selected from the group comprising: SEQ ID NO. 4, 7, 10, 13, 16, 19, 22 25 or 28, or a sequence having at least 80% identity thereto. Suitably, a homologue of SEQ ID NO.1 may be selected from the group comprising: SEQ ID NO. 4, 7, 10, 13, 16, 19, 22, 25 or 28, wherein the sequence comprises an RNA binding domain and an RGG domain, or a sequence having at least 80% identity to SEQ ID NO. 4, 7, 10, 13, 16, 19, 22, 25 or 28 and comprising an RNA binding domain and an RGG domain.

[0198] In one embodiment, the RNA binding protein comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22 25 or 28, or a sequence having at least 80% identity thereto (preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). In one embodiment, the RNA binding protein comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22 25 or 28, or a sequence having at least 80% identity thereto (preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto) and comprising an RNA binding domain and an RGG domain.

[0199] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO.1, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0200] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO.4, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0201] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO.7, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0202] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO.10, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0203] Suitably, the RNA binding protein according to the present invention may comprise an amino acid sequence as shown in SEQ ID NO. 13, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0204] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO. 16, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0205] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO. 19, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0206] Suitably, the RNA binding protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO. 22, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0207] Suitably, the RNA binding protein according to the present invention may comprise an amino acid sequence as shown in SEQ ID NO. 25, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0208] Suitably, the RNA binding protein according to the present invention may comprise an amino acid sequence as shown in SEQ ID NO. 28, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0209] In one embodiment, the RNA binding protein according to the present invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28.

[0210] Suitably, the protein may be derived from tobacco.

[0211] In one embodiment, at least one RNA binding protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; or a sequence having at least 80% sequence identity thereto. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0212] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 2, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0213] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 3, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0214] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 5, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0215] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 6, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0216] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 8, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0217] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 9, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0218] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 11, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0219] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 12, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0220] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 14, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0221] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 15, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0222] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 17, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0223] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 18, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0224] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 20, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0225] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 21, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0226] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 23, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0227] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 24, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0228] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 26, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0229] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 27, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0230] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 29, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0231] Suitably, the RNA binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 30, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0232] In one embodiment, at least the RNA binding protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) is selected from: SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0233] Suitably, the protein for use according to the present invention may be encoded by a polynucleotide sequence from Nicotiana tabacum.

[0234] In one aspect, the present invention provides a method for reducing the alkaloid content of a plant or a part or cell thereof (eg, a plant cell), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein.

[0235] In one aspect, the present invention provides a method for reducing the alkaloid content of a plant or a part thereof or a plant cell, the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding the RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0236] In one aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or a part thereof (e.g., leaves), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein.

[0237] In one aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or a part thereof (e.g., leaves), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0238] In one aspect, the present invention provides a method for reducing the TSNA content of processed leaves, such as cured leaves, the method comprising:

[0239] Modifying a plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein;

[0240] harvesting leaves from the plant;

[0241] and processing, e.g., conditioning, said harvested leaves.

[0242] Suitably, the method of reducing the TSNA content of processed leaves may comprise modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain.

[0243] As used herein, the term "reducing" or "inhibiting" (e.g., inhibiting the activity or expression of a gene encoding an RNA binding protein) means that the activity or expression of a gene encoding an RNA binding protein is lower or reduced compared to the activity or expression of the gene in a comparable product.

[0244] In one aspect, the present invention provides a method for increasing the alkaloid content in a plant or a part or cell thereof (eg, a plant cell), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding an RNA binding protein.

[0245] In one aspect, the present invention provides a method for increasing the alkaloid content in a plant or a part thereof or a plant cell, the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding the RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 have a nucleic acid sequence with at least 80% identity. Suitably, the protein may comprise an RNA binding domain and / or an RGG domain. Suitably, the protein may comprise an RNA binding domain and an RGG domain. In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or part thereof (e.g., leaves), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding an RNA binding protein.

[0246] In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or a part thereof (e.g., leaves), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0247] As used herein, the term "increase" or "enhance" (e.g., increase the activity or expression of a gene encoding an RNA binding protein) means that the activity or expression of a gene encoding an RNA binding protein is higher or increased compared to the activity or expression of the gene in a comparable product.

[0248] According to the invention, the activity or expression of a gene encoding an RNA binding protein is modulated.

[0249] In one aspect, the present invention provides a method for modulating (i.e. increasing or decreasing) the alkaloid content of a plant or a part or cell thereof (e.g. a plant cell), the method comprising modifying the plant by modulating (i.e. increasing or decreasing) the activity of at least one gene encoding an RNA binding protein.

[0250] The term "activity" refers to any functionality of the RNA binding protein encoded by the at least one gene. Examples of activities include the formation of protein: protein interactions, enzymatic activity, or localization of RNA binding proteins.

[0251] Suitably, the activity may be the ability of the RNA binding protein to interact with another molecule or molecules. In some embodiments, the invention provides a method of modulating (i.e. increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g. a plant cell), the method comprising modifying the plant by modulating (i.e. increasing or decreasing) the ability of the RNA binding protein to interact with another molecule.

[0252] Suitably, the ability of an RNA binding protein to interact with other molecules can be the ability to bind to other molecules. Another molecule can be a protein, such as another RNA binding protein. Suitably, another molecule can be more than one molecule, such as one or more molecules, such as two or more molecules, such as three or more molecules. Where another molecule is more than one molecule, the other molecules can be the same molecule or can be different molecules. Suitably, the other molecule can be RNA.

[0253] Suitably, the activity may be the ability of the RNA binding protein to bind RNA. In some embodiments, the invention provides a method of modulating (i.e. increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g. a plant cell), the method comprising modifying the plant by modulating (i.e. increasing or decreasing) the ability of the RNA binding protein to bind RNA.

[0254] Modulating the activity of a gene encoding an RNA binding protein may require increasing or decreasing the activity of the RNA binding protein.

[0255] Increasing the activity of an RNA binding protein refers to enhancing or improving the ability of the RNA binding protein to perform a specific function compared to the RNA binding protein in a plant that has not been modified according to the present invention.

[0256] Reducing the activity of a protein containing an RNA binding domain refers to reducing, inhibiting or destroying the ability of the RNA binding protein to perform a specific function compared to the RNA binding protein in a plant that has not been modified according to the present invention. The activity of a protein containing an RNA binding domain can be reduced to such an extent that the activity is prevented or eliminated.

[0257] In some embodiments, the activity of the RNA binding protein may be modulated (i.e., increased or decreased) 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%, compared to the activity of the gene encoding the RNA binding protein in a plant (e.g., a tobacco plant) that has not been modified according to the invention.

[0258] In some embodiments, the regulated RNA-binding protein exhibits an increased or decreased activity compared to the unmodified RNA-binding protein. The regulated RNA-binding protein may exhibit an activity that is increased or decreased by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% compared to the unmodified RNA-binding protein.

[0259] Techniques for measuring protein activity are known in the art. For example, assays for measuring the enzymatic activity of a protein are known, and microscopy techniques can be used to identify the localization of the protein.

[0260] In particular, the ability of RNA binding proteins to bind to another molecule can be measured using techniques known in the art. Examples of such techniques include immunoprecipitation, isothermal calorimetry, surface plasmon resonance, and microthermophoresis. For example, the ability of a regulated or mutated RNA binding protein to bind to other molecules can be determined, for example, by using a regulated or mutated RNA binding protein and a corresponding unmodified or unmutated RNA binding protein to perform a co-immunoprecipitation experiment. If the regulation or mutation in the RNA binding protein reduces, inhibits, or eliminates the ability of the RNA binding protein to bind to other molecules, co-immunoprecipitation will show that the regulated or mutated RNA binding protein binds to less other molecules.

[0261] The ability of RNA binding proteins to bind RNA can be measured using techniques known in the art. Examples of such techniques include techniques such as band shift analysis (e.g., RNA-electrophoretic mobility shift assay), RNA pull-down assays using affinity tags, RNA immunoprecipitation followed by RT-PCR, or non-specific assays such as poly(A) northern or oligonucleotide-targeted RNase H protection assays.

[0262] In one aspect, the present invention provides a method for modulating (i.e. increasing or decreasing) the alkaloid content of a plant or a part or cell thereof (e.g. a plant cell), the method comprising modifying the plant by modulating (i.e. increasing or decreasing) the expression of at least one gene encoding an RNA binding protein.

[0263] The "expression" of a gene refers to the degree to which the information encoded in the gene is converted into functionality. The expression level of a gene can be equated to the amount of the product of the gene present in a cell or organism. A modification that regulates (i.e., increases or decreases) gene expression is a modification that increases the amount of the gene product in a plant or cell compared to an unmodified plant or cell.

[0264] In some embodiments, expression of the RNA binding protein is modulated (ie, increased or decreased) compared to expression of a gene encoding the RNA binding protein in a plant (eg, a tobacco plant) that has not been modified according to the invention.

[0265] In some embodiments, the expression of the RNA binding protein may be modulated (i.e., increased or decreased) 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%, compared to the expression of the gene encoding the RNA binding protein in a plant (e.g., a tobacco plant) that has not been modified according to the invention.

[0266] In some embodiments, the RNA binding protein of regulation shows increased or decreased expression compared to the unmodified RNA binding protein. The RNA binding protein of regulation can show at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% increased or decreased expression compared to the unmodified RNA binding protein.

[0267] Typically, a gene is transcribed into mRNA, which is translated into protein, the final gene product. Proteins can be sequestered in cellular reservoirs and / or degraded. Gene expression can be regulated by regulating any or all of these steps. Thus, in some embodiments, the modification regulates expression of at least one gene encoding an RNA binding protein in one of the following ways:

[0268] modulates transcription from at least one gene encoding an RNA binding protein;

[0269] modulating translation of mRNA from at least one gene encoding an RNA binding protein;

[0270] Modulating the release of RNA binding proteins from intracellular storage; and / or

[0271] Regulates the degradation rate of RNA-binding proteins.

[0272] The expression of a specific gene encoding an RNA binding protein can be measured by measuring the transcription and / or translation of the gene. Methods for measuring transcription are well known in the art and include, inter alia, Northern blotting, RNA-Seq, in situ hybridization, DNA microarrays, and RT-PCR. On the other hand, the expression of a gene can be measured indirectly by measuring the level of a protein encoded by the gene product, for example, the gene. For example, the expression of an RNA binding protein can be determined by measuring the presence of the protein using an antibody specific to the RNA binding protein (e.g., an antibody specific to the RNA binding domain) by Western blotting.

[0273] Modification

[0274] Plants or cells can be modified in any manner that modulates the activity or expression of at least one gene encoding an RNA binding protein. The types of modifications of plants and cells that modulate gene activity or expression, as well as the techniques for achieving those modifications, are known in the art.

[0275] In some embodiments, the present invention provides a method of reducing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein as described herein.

[0276] In some embodiments, the present invention provides a method for reducing the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a tobacco plant or plant part thereof, the method comprising modifying the plant or cell culture by reducing the activity or expression of at least one gene encoding an RNA binding protein as described herein.

[0277] Any method known in the art for reducing or inhibiting gene activity or expression may be used in the methods according to the present invention.

[0278] Suitably, the activity or expression of the gene encoding the RNA binding protein may be reduced, partially inactivated, inhibited, eliminated, knocked out or lost, such that the protein activity, expression or function of the gene encoding the RNA binding protein may not be detectable.

[0279] In one aspect, at least one gene encoding an RNA binding protein is knocked out. In other words, the gene encoding the RNA binding protein has been rendered completely ineffective.

[0280] For example, the method may include:

[0281] ● providing a mutation in a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto;

[0282] ● providing a mutation in a regulatory region (e.g., a promoter or enhancer) that helps control the expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto;

[0283] ● Provide 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. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0284] Each of the above methods results in a reduction or prevention of the activity or expression of: a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or an amino acid sequence having at least 80% sequence identity thereto, or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 nucleic acid sequences that are at least 80% identical.

[0285] As used herein, the term "mutation" includes natural genetic variants or artificially engineered variants. In particular, the term "mutation" refers to a variation in a nucleotide sequence encoding an amino acid sequence or an amino acid sequence compared to a sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, preferably at least 98%, preferably at least 99%) sequence identity thereto.

[0286] In one embodiment, the mutation reduces the alkaloid content of the plant. In another embodiment, the mutation reduces the content of at least one TSNA precursor in the plant or part thereof, or leaves such as harvested or processed leaves. In one embodiment, the mutation reduces the content of one or more TSNAs selected from NNN, NNK, NAT, NAB, preferably in processed leaves the content of NNN and / or NNK is reduced. Suitably, the TSNA content is reduced relative to a comparable product.

[0287] In one embodiment, the method according to the invention may comprise providing a nucleic acid sequence to a plant or part thereof or a plant cell, wherein the nucleic acid results in a reduction or elimination of the activity or expression of at least one gene encoding an RNA binding protein.

[0288] In one embodiment, the method according to the invention may comprise providing a plant or part thereof or a plant cell with a nucleic acid sequence, wherein the nucleic acid results in the modification of a nucleic acid sequence of at least one gene encoding an RNA binding protein.

[0289] Suitably, the nucleic acid sequence may be introduced into a plant or part or cell thereof. Suitably, an endogenous nucleic acid sequence in a plant or part or cell thereof may be modified to encode a polypeptide according to the invention (e.g., by gene editing). For example, an endogenous nucleotide sequence may be modified to reduce the activity or expression of at least one gene encoding an RNA binding protein.

[0290] In a preferred embodiment, each copy of the nucleic acid sequence present in the plant is modified, e.g. mutated as defined herein (e.g., each genomic copy of the gene encoding the protein in the plant is mutated), the nucleic acid sequence encoding a protein comprising a sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto, or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or orthologue of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or orthologue of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 have a nucleic acid sequence that is at least 80% identical. For example, each copy of the gene in the tobacco allotetraploid genome can be mutated.

[0291] In preferred embodiments, some or all homologues of the RNA binding proteins described herein are modified, e.g., inhibited or mutated. Suitably, some or all of SEQ ID NO. NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or corresponding sequences having at least 80% sequence identity thereto are modified, e.g., inhibited or mutated.

[0292] In some embodiments, the plant or plant cell according to the invention is homozygous. Suitably, the plant or plant cell may be homozygous for a modification such as suppression or mutation.

[0293] In some embodiments, the plant or plant cell according to the invention expresses only a modified, e.g., mutated, nucleic acid encoding an RNA binding protein. In other words, in some embodiments, no endogenous (or endogenous and functional protein) is present in the plant according to the invention. In other words, if any endogenous protein is present, it is preferably in an inactive form.

[0294] In one embodiment, the method can include providing a mutation in a nucleic acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence having at least 80% identity thereto.

[0295] The mutation can change the plant genome so that the nucleic acid sequence encoding the protein is completely or partially deleted or otherwise modified to inhibit or eliminate the ability of the RNA binding protein to bind to RNA, the protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the mutation does not change the level or expression of the protein but reduces the ability of the RNA binding protein to bind to RNA.

[0296] Suitably, the mutation may be in the RNA binding domain of the RNA binding protein. Suitably, the mutation may be in the RGG domain of the RNA binding protein. Suitably, the RNA binding protein may comprise a plurality of mutations, each in a different domain, for example, at least one mutation in the RNA binding domain and at least one mutation in the RGG domain. In some embodiments, the mutation changes the ability of the RNA binding protein to bind RNA. Suitably, the mutation may prevent or reduce RNA binding. Suitably, the mutation may prevent or reduce RNA binding and reduce the activity of the RNA binding protein.

[0297] The mutation can be in one or more domains of the RNA binding protein, such as in the RNA binding domain and / or the RGG domain. In some embodiments, one or more domains of the RNA binding protein, such as the RNA binding domain and / or the RGG domain, can be mutated, thereby changing the ability of the protein to bind RNA. In some embodiments, one or more domains, such as the RNA binding domain and / or the RGG domain, are deleted from the RNA binding protein.

[0298] The mutation may interrupt a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0299] The interruption may result in the nucleic acid sequence not being transcribed and / or translated.

[0300] The nucleic acid sequence can be interrupted, for example, by deleting or otherwise modifying the ATG start codon of the nucleic acid sequence, such that translation of the protein is reduced or prevented.

[0301] The nucleotide sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or influence protein transport. For example, the expression of protein may be reduced or prevented by introducing one or more premature stop codons, frameshifts, splicing mutations or non-tolerant amino acid substitutions in the open reading frame.

[0302] A premature stop codon refers to a mutation that introduces a stop codon into the open reading frame and prevents translation of the entire amino acid sequence. The premature stop codon can be a TAG ("amber"), TAA ("ochre"), or TGA ("cream" or "umber") codon.

[0303] A frameshift mutation (also called a framing error or frame shift) is a mutation caused by the insertion / deletion (insertion or deletion) of multiple nucleotides in a nucleic acid sequence that cannot be divided by three. 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. Frameshift mutations will often cause the codon after the mutation to be read to encode a different amino acid. Frameshift mutations will usually result in the introduction of premature stop codons.

[0304] Splicing mutations insert, delete or change many nucleotides at specific sites where splicing occurs during the processing of precursor messenger RNA into mature messenger RNA. The loss of splicing sites results in one or more introns remaining in mature mRNA and may lead to the production of abnormal proteins.

[0305] Non-tolerant amino acid substitutions refer to mutations that cause non-synonymous amino acid substitutions in a protein, which result in reduced or eliminated function of the protein.

[0306] Any method known in the art for providing a mutation in a nucleotide sequence can be used in the method according to the present invention. For example, homologous recombination can be used to produce a vector in which one or more of the nucleotide sequences involved are mutated and used to transform a plant or plant cell. The recombinant plant or plant cell expressing the mutated sequence can then be selected.

[0307] In one embodiment, the mutation introduces a non-tolerant amino acid substitution in a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto.

[0308] In some embodiments, the RNA binding domain may contain a mutation that reduces expression of at least one gene encoding an RNA binding protein.

[0309] In some embodiments, the RGG domain may contain a mutation that reduces expression of at least one gene encoding an RNA binding protein.

[0310] The mutation may be a deletion, a splice mutant, or a codon encoding a non-tolerant amino acid substitution.

[0311] In one embodiment, the nucleic acid sequence encoding the RNA binding protein can be completely or partially deleted. The deletion can be continuous, or can comprise multiple sections of the sequence. The deletion preferably removes a sufficient amount of nucleotide sequence so that the nucleic acid sequence no longer encodes a functional RNA binding protein. When compared with the corresponding genome of a comparable unmodified plant, the deletion can be complete, in which case 100% of the coding portion of the nucleic acid sequence does not exist. The deletion can for example remove at least 50, 60, 70, 80 or 90% of the coding portion of the nucleic acid sequence. Suitably, at least a portion of the protein can be deleted. The deletion can for example remove at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the coding portion of the protein.

[0312] The deletion may remove at least 10 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids) from the C-terminus of the RNA binding protein. Suitably, the deletion may remove at least 10 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids) corresponding to the C-terminus of the RNA binding protein, wherein the sequence of the RNA binding protein is aligned with SEQ ID NO.1. Suitably, the deletion may remove at least 10 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids) from the C-terminus of the RNA binding protein, wherein the RNA binding protein before the deletion comprises the amino acid sequence shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto. Suitably, the deletion may remove at least 10 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids) from the C-terminus of the RNA binding protein, wherein the RNA binding protein before the deletion comprises the amino acid sequence shown in SEQ ID NO.1, or a sequence having at least 80% sequence identity thereto.

[0313] Suitably, the protein for use according to the invention may comprise a truncated RNA binding protein. Suitably, the truncated protein may be a truncated form of the amino acid sequence shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto. Suitably, the truncated protein lacks at least 10 amino acids (e.g. at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids) from the C-terminus of the RNA binding protein.

[0314] Deletion can remove at least a portion of the RNA binding domain. Deletion can, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the RNA binding domain. Suitably, deletion can remove at least 5 amino acids, at least 10 amino acids, at least 15, at least 20, at least 25, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids of the RNA binding domain. Suitably, deletion can remove 5 amino acids, 10 amino acids, 15, 20 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids of the RNA binding domain.

[0315] The deletion may remove at least a portion of the RGG motif. The deletion may, for example, remove at least one or at least two amino acids from the RGG domain. Suitably, the RGG domain may be deleted entirely.

[0316] In one embodiment, the RNA binding protein is encoded by a nucleotide sequence comprising or consisting of the sequence shown in SEQ ID NO. 34 or a sequence having at least 80% identity thereto (eg, at least 85%, 90%, 95% identity).

[0317] In one embodiment, the RNA binding protein comprises or consists of a sequence as shown in SEQ ID NO. 35, or a sequence having at least 80% identity thereto (eg, at least 85%, 90%, 95% identity thereto).

[0318] 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 one or more of the nucleotide sequences involved are lost and used to transform plants or plant cells. Recombinant plants or plant cells expressing the new sequence portion can then be selected.

[0319] Plant cells transformed with the vectors described herein can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins and the like.

[0320] Modification of nucleic acid sequences can be performed using directed mutagenesis methods, also known as targeted nucleotide exchange (TNE) or oligo-directed mutagenesis (ODM). Directed mutagenesis methods include, but are not limited to, those using zinc finger nucleases, TALENs (see WO2011 / 072246 and WO2010 / 079430), Cas9-like, Cas9 / crRNA / tracrRNA, Cas9 / gRNA or other CRISPR systems (see WO 2014 / 071006 and WO2014 / 093622), meganucleases (see WO2007 / 047859 and WO2009 / 059195), or directed mutagenesis methods using mutagenic oligonucleotides, which may contain chemically modified nucleotides having sequence complementarity to the gene for enhanced mutagenesis that are introduced into plant protoplasts (e.g., KeyBase® or TALENs).

[0321] On the other hand, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al. (2000) Nat Biotech 18:455, and McCallum et al. (2000) Plant Physiol. 123, 439-442, both of which are incorporated herein by reference) can be used to generate plant lines containing genes encoding proteins with mutations. TILLING uses traditional chemical mutagenesis (e.g., ethyl methanesulfonate (EMS) mutagenesis to produce random mutations), followed by high-throughput screening of mutations. Therefore, plants, seeds, cells and tissues containing genes with desired mutations can be obtained.

[0322] The method can include the following steps: mutagenesis of plant seeds (for example EMS mutagenesis), merging of plant individuals or DNA, pcr amplification of the region of interest, heteroduplex formation and high-flow detection, identification of mutant plants, sequencing of mutant pcr products. It should be understood that other mutagenesis and selection methods can be used equally to generate the plant of this modification. Seeds can for example be irradiated or chemically treated, and can screen plants with regard to the phenotype of modification.

[0323] Fast neutron deletion mutagenesis can be used in a reverse genetics sense (i.e., using PCR) to identify plant lines carrying deletions in endogenous genes. See, e.g., Ohshima et al. (1998) Virology 213:472-481; Okubara et al. (1994) Genetics 137:867-874; and Quesada et al. (2000) Genetics 154:421-4315, which are incorporated herein by reference.

[0324] In another approach, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of duplicated gene loci. See, e.g., Kusaba et al. (2003) Plant Cell 15: 1455-1467 (incorporated herein by reference).

[0325] The modified plants can be distinguished from unmodified plants, i.e., wild-type plants, by molecular methods, such as one or more mutations present in the DNA, and by the phenotypic characteristics of the modification. The modified plants can be homozygous or heterozygous for the modification. Preferably, the modified plants are homozygous for the modification.

[0326] In one embodiment, the method of reducing or preventing the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, does not include treating the plant with chemicals (e.g., agrochemicals).

[0327] Other ways to reduce or prevent expression will be apparent to those skilled in the art and include the use of virus-induced gene silencing (VIGs), microRNA silencing, RNAi, antisense, tDNA insertion, or dominant negative constructs (or antimorphic mutations).

[0328] In one embodiment, expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by virus-induced gene silencing.

[0329] In one embodiment, the expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by microRNAs.

[0330] In one embodiment, the expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by RNAi.

[0331] In one embodiment, expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by antisense inhibition.

[0332] In one embodiment, expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by sense suppression.

[0333] In one embodiment, expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by tDNA insertion.

[0334] In one embodiment, expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a dominant negative construct (or a reverse allele mutation).

[0335] In one embodiment, the expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a system based on site-directed mutagenesis.

[0336] In one embodiment, the expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by gene editing, such as a CRISPR-based system.

[0337] In one embodiment, the expression of a gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by zinc finger nucleases, TALENs, meganucleases, mutagenic oligonucleotides or TILLING.

[0338] In some embodiments, the present invention provides a method for increasing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding an RNA binding protein.

[0339] Any method known in the art for increasing or enhancing gene activity or expression may be used in the methods according to the present invention.

[0340] In some embodiments, the method may include overexpressing at least one gene encoding an RNA binding protein. Suitably, the method may include expressing one or more additional copies of at least one gene encoding an RNA binding protein in a plant or cell. Suitably, the method may include modifying an endogenous copy of at least one gene encoding an RNA binding protein so that its expression is increased. The method may include mutating the coding sequence of at least one gene encoding an RNA binding protein. The method may include mutating a regulatory sequence that regulates the expression of at least one gene encoding an RNA binding protein.

[0341] Suitably, the method may comprise transforming a cell of a plant (e.g., a tobacco plant) with a genetic construct encoding at least one RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding the RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 functional variants or functional fragments or orthologs, or with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 having at least 80% identity of the nucleic acid sequence; or comprising a nucleotide sequence encoding a protein capable of promoting or increasing at least one endogenous RNA binding protein. It will be understood that each of these options will result in increased activity and expression of the polypeptide encoded by the at least one RNA binding protein. The method may comprise regenerating a plant from the transformed cell. Provided is the use of a genetic construct capable of increasing the activity and / or expression of a polypeptide encoded by at least one gene encoding an RNA binding protein for increasing the alkaloid content (e.g. nicotine content) in a plant or part or cell thereof transformed with the construct.

[0342] The genetic construct may encode a polypeptide comprising the amino acid SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0343] In another embodiment, the present invention relates to a method for increasing the alkaloid content of a plant or a part or cell thereof, comprising modifying said plant or cell by increasing the activity of at least one gene encoding an RNA binding protein.

[0344] In one embodiment, the activity of at least one gene encoding an RNA binding protein may be increased by introducing (or providing) a mutation to at least one gene encoding an RNA binding protein.

[0345] Suitably, the activity of at least one gene encoding an RNA binding protein may be increased by introducing a mutation into: at least one gene encoding an RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0346] In some embodiments, the modification that increases the activity or expression of at least one gene encoding an RNA binding protein and thereby increases the alkaloid content is by one of the following:

[0347] modulates transcription from at least one gene encoding an RNA binding protein;

[0348] modulating translation of mRNA from at least one gene encoding an RNA binding protein;

[0349] Modulating the release of RNA binding proteins from intracellular storage; and / or

[0350] Regulates the degradation rate of RNA-binding proteins.

[0351] In one aspect, the activity and or expression of an RNA binding protein as defined herein may be modulated, e.g. reduced, by altering the activity or expression of an interactor protein.

[0352] Without wishing to be bound by theory, in one aspect, an interactor protein can reduce or block the activity and / or expression of the RNA binding protein. For example, an interactor protein can bind to and / or isolate an RNA binding protein; thus increasing the activity and / or expression of an interactor protein can reduce the activity and / or expression of an RNA binding protein.

[0353] In alternative aspects, the interacting factor protein can increase or enhance the activity and / or expression of an RNA binding protein as defined herein. For example, the interacting factor protein can serve as a cofactor of an RNA binding protein; thus reducing the activity and / or expression of the interacting factor protein can reduce the activity and / or expression of the RNA binding protein.

[0354] In one aspect, the activity and / or expression of an RNA binding protein can be modulated (eg, decreased) by altering the activity and / or expression of an interactor protein.

[0355] Suitably, the interactor protein may be selected from SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 50 or a variant thereof having at least 80% identity thereto (e.g. at least 85% identity, at least 90% identity, at least 95%, at least 97% identity, at least 98% identity).

[0356] Suitably, the interactor protein may have a coding sequence selected from SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 45, SEQ ID NO. 48, SEQ ID NO. 51 or a variant thereof having at least 80% identity thereto (e.g. at least 85% identity, at least 90% identity, at least 95%, at least 97% identity, at least 98% identity).

[0357] Suitably, the interactor protein may be encoded by a nucleotide sequence selected from SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52, or a variant thereof having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95%, at least 97% identity, at least 98% identity).

[0358] Alkaloid content

[0359] In one embodiment, the invention provides a method of modulating the alkaloid content of a plant (eg, a tobacco plant) or a part thereof, the method comprising modifying the plant by modulating the activity or expression of at least one gene encoding an RNA binding protein.

[0360] The term "modulate" is used herein to mean to increase or decrease.

[0361] The term "increased alkaloid content" is used herein to mean that the alkaloid content in the product of the invention (e.g. a plant, a part thereof (e.g. leaves), processed leaves or a product prepared from the plant (e.g. a tobacco industry product)) is higher than in a comparable product which has not been modified according to the invention.

[0362] The term "reduced alkaloid content" is used herein to mean that the alkaloid content in the product of the invention (e.g. a plant, a part thereof (e.g. leaves), processed leaves or a product prepared from a plant (e.g. a tobacco industry product)) is lower than in a comparable product which has not been modified according to the invention.

[0363] In some embodiments, modulation of alkaloid content refers to an increase in alkaloid content, wherein the activity or expression of at least one gene encoding a protein containing an RNA binding protein domain is increased (or in other words the protein is overexpressed).

[0364] In some embodiments, modulation of alkaloid content refers to a decrease in alkaloid content, wherein the expression of at least one gene encoding an RNA binding protein is decreased or inhibited or eliminated.

[0365] In a further aspect, the alkaloid content is measured from leaves. In one aspect, the alkaloid content is measured from green leaves. In a further aspect, the alkaloid content is measured from conditioned leaves, such as air-cured, flue-cured, fire-cured, or sun-cured leaves. In a further aspect, the alkaloid content is measured from flue-cured leaves. In a further aspect, the alkaloid content is measured from air-cured leaves.

[0366] The term "alkaloid content" is used herein to mean the concentration and / or total amount of the whole group of compounds classified as alkaloids, or the concentration and / or total amount of one or more compounds classified as alkaloids. The alkaloids generally present in tobacco include nornicotine, PON, anatabine, anabasine, nicotine and mithosamine. In some embodiments, one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as the content of all six alkaloids is regulated. In some embodiments, one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as the content of all six alkaloids is increased. In some embodiments, one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithofamine, for example, two or more alkaloids, for example, three or more alkaloids, for example, four or more alkaloids, for example, five or more alkaloids, for example, the content of all six alkaloids is reduced. In some embodiments, the total alkaloid content of the plant or cell is adjusted. In some embodiments, the total alkaloid content is increased. In some embodiments, the total alkaloid content is increased.

[0367] Any method known in the art for determining the concentration and / or total content of alkaloids may be used. A preferred method for analyzing alkaloid content involves analysis by gas chromatography-flame ionization detection (GC-FID) or by reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS).

[0368] In one embodiment, a method is provided for producing a plant (e.g., a tobacco plant) or part thereof obtainable or obtained by a plant of the present invention, a plant propagation material (e.g., a tobacco plant propagation material), a cell (e.g., a tobacco cell), a leaf (e.g., a tobacco leaf), a harvested leaf (e.g., a harvested tobacco leaf), a cut harvested leaf (e.g., a cut harvested tobacco leaf), a processed leaf (e.g., a processed tobacco leaf), a cut and processed leaf (e.g., a cut and processed tobacco leaf), a product (e.g., a tobacco industry product) comprising the plant or part thereof, or a combination thereof, wherein the plant of the present invention has a modulated alkaloid content, the method comprising: modifying the plant to modulate the activity or expression of a gene encoding an RNA binding protein. The modulated alkaloid content can be determined by comparing the alkaloid content in the following: plants (e.g., tobacco plants) or parts thereof, plant propagation material (e.g., tobacco plant propagation material), cells (e.g., tobacco cells), leaves (e.g., tobacco leaves), harvested leaves (e.g., harvested tobacco leaves), cut harvested leaves (e.g., cut harvested tobacco leaves), processed leaves (e.g., processed tobacco leaves), cut and processed leaves (e.g., cut and processed tobacco leaves), products comprising plants or parts thereof of the present invention, such as tobacco industry products, or combinations thereof, to comparable products.

[0369] Suitably, alkaloid content can be regulated in plant, for example tobacco plant, for example tobacco plant of modification. Suitably, alkaloid content can be regulated in blade (for example tobacco blade, for example tobacco blade from the tobacco plant of modification). Suitably, alkaloid content can be regulated in blade of results (for example tobacco blade from the tobacco plant of modification). Suitably, alkaloid content can be regulated in blade of cutting results (for example tobacco blade from the tobacco plant of cutting results). Suitably, alkaloid content can be regulated in processing blade (for example processing tobacco blade, for example processing tobacco blade from the tobacco plant of modification). Suitably, alkaloid content can be regulated in blade of cutting and processing (for example cutting and processing tobacco blade, for example cutting and processing tobacco blade from the tobacco plant of modification). Suitably, alkaloid content can be regulated in blade after modulation (for example tobacco blade after modulation from the tobacco plant of modification). Suitably, alkaloid content can be regulated in extract of green blade (for example green tobacco blade from the tobacco plant of modification). Suitably, alkaloid content can be regulated in the product (for example tobacco industry product, for example tobacco industry product produced by the tobacco plant of modification or its part) comprising plant of the present invention or its part. Suitably, alkaloid content can be regulated in any one of the above-mentioned products or its combination. Suitably, the regulation of above-mentioned alkaloid content can be the increase in the alkaloid content. Suitably, the regulation of above-mentioned alkaloid content can be the reduction in the alkaloid content (for example the reduction in nornicotine and / or PON content).

[0370] In one embodiment, the content of one or more alkaloids selected from nornicotine, PON, anatabine and anabasine is reduced. In one embodiment, the content of nornicotine is reduced. In one embodiment, the content of PON is reduced. In one embodiment, the content of anatabine is reduced. In one embodiment, the content of anabasine is reduced.

[0371] In one embodiment, the nicotine content of the modified plant (e.g., tobacco plant), plant propagation material (e.g., tobacco plant propagation material), leaf (e.g., tobacco leaf), harvested leaf (e.g., harvested tobacco leaf), cut harvested leaf (e.g., cut harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf), cut and processed leaf (e.g., cut and processed tobacco leaf), or tobacco industry product from the modified tobacco plant is not significantly reduced. Suitably, the nicotine content is at least 85% (e.g., at least 90%, e.g., at least 95%, e.g., at least 98%, e.g., at least 99%) of the nicotine content of comparable products.

[0372] In one embodiment, when compared with the alkaloid content of a plant (e.g., tobacco plant) or its part that has not been modified to regulate the activity or expression of at least one gene encoding RNA binding protein and has been grown under similar growth conditions, the alkaloid content of a plant (e.g., tobacco plant) or its part can be adjusted to at least 0.5, 1.5, 2, 3 or 4 times. Suitably, the alkaloid content can be adjusted to about 0.5 times to about 4 times. Suitably, the alkaloid content can be adjusted to about 4 times. Suitably, the modification can be an increase or reduction in alkaloid content. Suitably, the adjustment can be the adjustment of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithofamine. Suitably, the adjustment can be the adjustment of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine. Suitably, the nornicotine content can be reduced. Suitably, the PON content can be reduced. Suitably, the anabasine content can be reduced. Suitably, the anabasine content can be reduced. Suitably, the anabasine content can be reduced.

[0373] In one embodiment of the invention, compared with plant (for example tobacco plant) or its part that has not been modified according to the present invention, the alkaloid content of plant (for example tobacco plant) or its part can be regulated at least 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 30%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 40%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 50%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 60%.When compared with unmodified plant (for example tobacco plant) or its part, regulation can be an increase or reduction in the alkaloid content.

[0374] Suitably, regulation can be regulation of total alkaloid content. Suitably, regulation can be regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithoxamine. Suitably, regulation can be regulation of one or more alkaloids selected from nornicotine, nicotine, PON, anatabine and anabasine. Suitably, regulation can be regulation of nornicotine content, such as a reduction in nornicotine content. Suitably, regulation can be regulation of anabasine content, such as a reduction in anabasine content. Suitably, regulation is regulation of PON content, such as a reduction in PON content. Suitably, regulation can be regulation of anabasine content, such as a reduction in anabasine content.

[0375] Suitably, the modulation may be of more than one alkaloid selected from nicotine, nornicotine, PON, anatabine, anabasine and mithofamine, such as modulation of two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids.

[0376] In some embodiments, the alkaloid content of the plant can be adjusted from about 5% to about 100%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to 60%, from about 40% to 50%, or from about 50% to 60%.

[0377] Tobacco-specific nitrosamines (TSNA) content

[0378] In one embodiment, the invention provides a method of reducing the content of at least one tobacco-specific nitrosamine (TSNA) precursor in a plant (e.g., a tobacco plant) or a part thereof or a tobacco cell. Suitably, the method may comprise modifying the plant by modulating the activity or expression of at least one gene encoding an RNA binding protein. In one embodiment, the invention provides a method of producing a processed leaf having a reduced TSNA content (e.g., relative to a comparable product). The method of producing a processed leaf having a reduced TSNA content may comprise:

[0379] Modifying a plant by reducing or inhibiting the activity or expression of at least one gene encoding an RNA binding protein;

[0380] harvesting leaves from the plant;

[0381] and processing, e.g., conditioning, said harvested leaves.

[0382] TSNA can be measured in processed tobacco, such as cured tobacco or reconstituted tobacco. In one embodiment, TSNA content is measured and / or modified (eg, reduced) in cured tobacco plants or parts thereof (eg, cured tobacco leaves).

[0383] As used herein, the term "tobacco-specific nitrosamines" or "TSNAs" has its usual meaning in the art, i.e., nitrosamines found only in tobacco industry products or other nicotine-containing products. Suitably, the at least one tobacco-specific nitrosamine may be N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) or N'-nitrosoanatabine (NAB).

[0384] The term "precursor thereof" when used with respect to at least one tobacco-specific nitrosamine refers to one or more chemicals or compounds of the tobacco plant that cause the formation of the tobacco-specific nitrosamine or are involved in a nitrosative reaction leading to the production of tobacco-specific nitrosamines.

[0385] In one embodiment, the TSNA may be one or more selected from the group consisting of N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonicotinoline (NAT) and N'-nitrosoanatabine (NAB). Suitably, at least one tobacco-specific nitrosamine may be NNK or NNN. In one embodiment, the tobacco-specific nitrosamine is NNN. In another embodiment, the tobacco-specific nitrosamine is NNK.

[0386] In one embodiment, the precursor of TSNA is one or more selected from the group of nornicotine, anabasine, anatabine and oxidized derivatives of nicotine such as pseudooxynicotine (PON).

[0387] In one embodiment, the TSNA is N'-nitrosonornicotine (NNN) and / or the precursor is nornicotine. In one embodiment, the content of NNN is reduced. In one embodiment, the content of nornicotine is reduced. In one embodiment, the content of NNN and nornicotine are reduced.

[0388] In one embodiment, the TSNA is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and / or the precursor is PON. In one embodiment, the content of NNK is reduced. In one embodiment, the content of PON is reduced. In one embodiment, the content of NNK and PON is reduced.

[0389] In one embodiment, the TSNA is N'nitrosoanatabine (NAT) and / or the precursor is anatabine. In one embodiment, the content of NAT is reduced. In one embodiment, the content of anatabine is reduced. In one embodiment, the content of NAT and anatabine are reduced.

[0390] In one embodiment, the TSNA is an N'-nitrosoneonicotinoid (NAB) and / or the precursor is a neonicotinoid. In one embodiment, the content of NAB is reduced. In one embodiment, the content of neonicotinoid is reduced. In one embodiment, the content of NAB and neonicotinoid is reduced.

[0391] Precursors of TSNAs (e.g., NNK, NNN, NAB and / or NAT) can be measured in green tobacco leaves, e.g., prior to processing, e.g., prior to curing. In one embodiment, precursors of TSNAs (e.g., NNK, NNN, NAB and / or NAT) are measured and / or modified (e.g., reduced) in green tobacco leaves, e.g., prior to processing, e.g., prior to curing.

[0392] In one embodiment, performance of the methods and or uses of the invention results in a reduction in at least one TSNA or precursor thereof in the modified tobacco plant (or part thereof) when compared to a tobacco plant (or part thereof) which has not been modified according to the invention.

[0393] The term "reduction of at least one TSNA or a precursor thereof" or "reduction of at least one TSNA or a precursor thereof" is used herein to mean that the concentration and / or total content of at least one TSNA or a precursor thereof is lower in the product, method or use of the invention relative to a comparable product, method or use. For example, a comparable tobacco industry product would be derived from a tobacco plant that has not been modified according to the invention, but in which all other relevant characteristics are the same (e.g., plant species, growing conditions, methods of processing the tobacco, etc.).

[0394] Any method known in the art for determining the concentration and / or level of at least one TSNA or its precursor may be used. In particular, such a method may be used which may include the addition of a deuterium labeled internal standard, water extraction and filtration, followed by analysis using reverse phase high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). Other examples of methods for determining the concentration and / or level of tobacco specific nitrosamine precursors include methods such as CORESTA recommended method CRM-72: Determination of Tobacco Specific Nitrosamines in Tobacco and Tobacco Products by LC-MS / MS; the CRM being developed as ISO / DIS 21766 or the method detailed in Wagner et al. (2005) Analytical Chemistry, 77(4), 1001-1006, which are incorporated herein by reference in their entirety.

[0395] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can be reduced by implementing the method and / or use of the present invention. Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor can be reduced in the tobacco plant of the present invention (e.g., obtainable or obtained by the method and / or use of the present invention) when compared with the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor in the tobacco plant not yet modified according to the present invention.

[0396] The concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor may be reduced in tobacco leaves, harvested leaves, processed tobacco leaves, tobacco industry products, or a combination thereof, obtainable or obtained from a tobacco plant (or part of a tobacco plant or tobacco cell culture) of the present invention (or part of a tobacco plant or tobacco cell culture), when compared to tobacco leaves, harvested leaves, processed tobacco leaves, tobacco industry products, or a combination thereof, obtainable or obtained from a tobacco plant (or part of a tobacco plant or tobacco cell culture) that has not been modified according to the present invention.

[0397] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the processed tobacco leaves.

[0398] Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the tobacco industry product.

[0399] In one embodiment, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, or about 40% to 50%.

[0400] With respect to processed (e.g., cured) tobacco leaves (e.g., cured or reconstituted), at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5000 ng / g to about 50 ng / g, about 4000 ng / g to about 100 ng / g, about 3000 ng / g to 500 ng / g, or 2000 ng / g to 1000 ng / g. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 5000 ng / g, at least about 4000 ng / g, at least about 3000 ng / g, at least about 2000 ng / g, at least about 1000 ng / g, at least about 500 ng / g, at least about 100 ng / g, or at least about 50 ng / g.

[0401] Biomass production

[0402] In some cases, it may be desirable to produce plants or biomass with high alkaloid levels, such as high levels of nicotine content, so that the nicotine can be purified to produce a pure nicotine product, such as for use in devices that utilize liquids containing nicotine, such as e-cigarettes or tobacco heating devices. For example, producing nicotine in the manner described can reduce the cost of nicotine extraction for producing e-liquids for e-cigarettes.

[0403] In one aspect, the invention provides a method for producing biomass, comprising:

[0404] Cells are grown under conditions that produce biomass, the cells having been artificially modified to modulate (e.g., increase) the activity or expression of a gene encoding an RNA binding protein. Suitably, the activity or expression of the RNA binding protein may be increased to increase the concentration and / or total nicotine content.

[0405] In one embodiment, the present invention provides a method for producing biomass with a modified (e.g., increased) nicotine concentration and / or total content, comprising growing a cell that has been artificially modified to increase the activity or expression of at least one gene encoding an RNA binding protein, the RNA binding protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0406] The cell may be artificially modified by any method known in the art to modify the activity or expression of at least one gene encoding an RNA binding protein. Suitably, the cell may be artificially modified to express an exogenous gene encoding an RNA binding protein. Suitably, the cell may be artificially modified to overexpress a gene encoding an RNA binding protein.

[0407] Suitably, the biomass may contain a higher concentration and / or total content of nicotine than biomass produced from comparable cells which have not been modified according to the invention.

[0408] Suitably, the cells for biomass production may be plant cells, such as tobacco cells.

[0409] Suitably, the cells for biomass production may be yeast cells.

[0410] In one embodiment, the cell (e.g., yeast cell) may be further modified to include one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, these one or more sequences may be incorporated into a nucleic acid construct suitable for cell (e.g., yeast cell) transformation. One or more sequences may be overexpressed in a cell (e.g., yeast cell). The sequence may be selected from one or more of the following genes: MPO (or methylputrescine oxidase or MPO1 or MPO2); A622 (or isoflavone reductase-like protein or isoflavone reductase homolog (homolog) or isoflavone reductase-like protein); BBL (or berberine bridge enzyme or berberine bridge enzyme-like or BBE or NBB1); PMT (or putrescine N-methyltransferase or putrescine methyltransferase or S-adenosyl-L-methionine: putrescine N-methyltransferase or PMT or PMT1 or PMT2 or PMT3 or PMT4) and QPT (or quinolinate phosphoribosyltransferase). In one embodiment, the sequence may be selected from one or more of the following genes: BBL, A622, PMT, and MPO (MPO1 or MPO2). Genes suitable for modification in this manner may be taught, for example, in US2016032299, which is incorporated herein by reference.

[0411] Commercially desirable traits

[0412] In one embodiment, the plant of the invention has a modified (i.e. increased or decreased) total alkaloid content, and / or a modified (i.e. increased or decreased) content of one or more alkaloids, while the aroma characteristics and / or other commercially desirable traits are at least maintained. Suitably, the plant of the invention may have a reduced total alkaloid content, and / or a reduced content of one or more alkaloids, while the aroma characteristics and / or other commercially desirable traits are at least maintained.

[0413] In one embodiment, the plants of the invention produce leaves of similar grade and / or quality as plants which have not been modified according to the invention.

[0414] In one embodiment, a plant of the invention has a reduced nornicotine and / or PON and / or neonicotinoid and / or anatabine content without a significant change in the flavor profile of the plant (e.g. compared to the same plant which has not been modified according to the invention).

[0415] In one embodiment, the plants of the invention have a reduced TSNA precursor content (e.g., compared to the same plant that has not been modified according to the invention) without a significant change (e.g., a decrease) in other commercially desirable traits of the plant. In particular, the yield of the modified plant is preferably not reduced compared to the same plant that has not been modified according to the invention.

[0416] Thus, in one embodiment, the methods and uses of the present invention relate to reducing TSNA precursor content while maintaining flavor characteristics and / or other commercially desirable traits (eg, yield).

[0417] As used herein, the term "commercially desirable traits" will include traits such as yield, mature plant height, number of harvestable leaves, average node length, cutter leaf length, cutter leaf width, quality (e.g., leaf mass, appropriately modulated leaf mass), abiotic (e.g., drought) stress tolerance, herbicide tolerance, and / or biotic (e.g., insect, bacterial or fungal) stress tolerance.

[0418] For example, according to the United States Department of Agriculture (USDA) grades and standards, leaf quality can be measured based on the color, texture, and aroma of the cured leaves.

[0419] Tobacco grades are evaluated based on factors including, but not limited to, petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to the intensity and depth of color and brightness of the leaf), hygroscopicity (the ability of the tobacco leaf to absorb and retain moisture from the environment) and green nuance or cast.

[0420] Leaf grade can be determined using standard methods known in the art, for example, using the Official Standard Grades published by the Agricultural Marketing Service of the US Department of Agriculture (7 USC §511). See, e.g., the official standard grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55 FR 40645); the official standard grades for Flue-cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54 FR 7925); the official standard grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 FR 16854); the official standard grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44), effective December 8, 1963 (28 F.R. 11719 and 28 FR 7925). 11926); the official standard grades for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 FR 17061); the official standard grades for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grades for Georgia and Florida ShadeGrown Cigar-Wrapper Tobacco (U.S. 62), effective April 1971. USDA grade index values ​​may be determined based on industry recognized grade indices. See, e.g., Bowman et al., (1988) Tobacco Science, 32:39-40; Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al. (1990) Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated herein in their entirety).

[0421] In one aspect, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all shank positions. A higher grade index indicates higher quality. In another aspect, the blade grade can be determined via hyper-spectral imaging. See, for example, WO 2011 / 027315 (which is incorporated herein by reference).

[0422] In one embodiment, the tobacco plants of the present invention provide commercially acceptable grades of tobacco.

[0423] Suitably, the tobacco plants of the present invention provide commercially acceptable grades of cured tobacco.

[0424] In one embodiment, when growing under similar growth conditions, tobacco plant of the present invention can produce the leaf of at least about 70% USDA grade index value with the USDA grade index value of comparable plant leaves.Suitably, when growing under similar growth conditions, tobacco plant disclosed herein may be able to produce the leaf of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% USDA grade index value with the USDA grade index value of control plant. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA Grade Index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130% or 120% to 130% of the USDA Grade Index value of a comparable plant.

[0425] In one aspect, the tobacco plants of the present invention are capable of producing leaves having a USDA Grade Index value of at least 50. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA Grade Index value of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more.

[0426] Unless otherwise indicated, tobacco yield as used herein refers to cured leaf yield, which is calculated based on cured tobacco leaf weight per acre under standard field conditions following standard agronomic and curing practices.

[0427] In one aspect, plants of the invention (e.g., tobacco plants) have a yield that is 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%, 50% to 100%, 55% to 100%, 60% to 100% of the yield of comparable plants when grown under similar field conditions. %, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 100% to 150%, 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%.

[0428] In another aspect, the yield of plants (e.g., tobacco plants) of the invention is about 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.

[0429] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable plants that are fire-tube cured when grown under similar field conditions.

[0430] In one aspect, the tobacco plant of the invention provides 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.

[0431] 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.

[0432] 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.

[0433] Plant breeding

[0434] In one embodiment, the present invention provides a method for producing a plant having a modified alkaloid content and / or a modified tobacco-specific nitrosamine (TSNA) precursor content, comprising:

[0435] a. crossing a donor plant having a modified nicotine content and / or a modified tobacco-specific nitrosamine (TSNA) precursor content and wherein the activity or expression of at least one gene encoding an RNA-binding protein according to the invention has been modulated in the donor plant according to the invention with a recipient tobacco plant that does not have a modified nicotine content or a modified tobacco-specific nitrosamine (TSNA) precursor content and has a commercially desirable trait;

[0436] b. isolating genetic material from offspring of the donor plant hybridized with the recipient plant; and

[0437] c. performing molecular marker-assisted selection using molecular markers, comprising:

[0438] i. Identifying an introgressed region comprising a mutation in a polynucleotide sequence encoding a protein defined in a.

[0439] Suitably, the protein comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or orthologue thereof, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, when compared to a comparable plant; or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or orthologue of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or the activity or expression of a protein encoded by a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 is modulated in the donor plant. Suitably, the alkaloid content and / or TSNA precursor content is reduced by the method. Suitably, the alkaloid content and / or TSNA precursor content is reduced and the activity or expression of the RNA binding protein is reduced or inhibited.

[0440] Molecular marker-assisted selection can include performing PCR to identify introgressed nucleic acid sequences comprising a mutation that modulates the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0441] plant

[0442] In one embodiment, the invention relates to a method for modulating (eg, reducing) the alkaloid content in a plant or a part thereof or a plant cell.

[0443] Suitable plants according to the present invention include the Solanaceae ( Solanaceae ) plants, including, for example, datura, eggplant, mandrake, deadly nightshade (belladonna), capsicum (chili pepper, capsicum), potato and tobacco.

[0444] In one embodiment, a suitable genus of the Solanaceae family is Nicotiana ( Nicotiana ), such as Nicotiana tabacum or Nicotiana rustica ( Nicotiana rustica ).

[0445] A suitable species of the genus Nicotiana may be Nicotiana tabacum. Species of the genus Nicotiana may be referred to herein as tobacco plants, or simply tobacco.

[0446] Tobacco Plant

[0447] The invention provides methods, uses, and cells (eg, tobacco cells), plants (eg, tobacco plants), and plant propagation materials for plants (eg, tobacco plants).

[0448] As used herein, the term "tobacco plant" refers to plants in the genus Nicotiana used in the production of tobacco industry products. Non-limiting examples of suitable "tobacco" plants include Nicotiana tabacum and Nicotiana rustica (e.g., Nicotiana tabacum ( N. tabacum L. ), LA B21, LN KY171, Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico).

[0449] In one aspect, tobacco plant according to the present invention is a non-natural tobacco plant. Suitably, the tobacco plant can be a mutant, non-natural tobacco plant. Suitably, the tobacco plant can be a transgenic tobacco plant.

[0450] In one aspect, the tobacco plant according to the invention comprises a non-naturally occurring mutation that modulates (eg, reduces) the activity or expression of at least one RNA binding protein as defined herein. Suitably, the tobacco plant may comprise an introduced mutation.

[0451] Tobacco material can be derived from or derived from the variant of tobacco type, commonly referred to as burley tobacco variant, flue or bright and dark variants. In some embodiments, tobacco material is derived from burley, Virginia or dark tobacco plants. Tobacco plants can be selected from burley tobacco, rare tobacco, specialty tobacco, expanded tobacco, etc.

[0452] The present invention also contemplates the use of tobacco cultivars and elite tobacco cultivars. Thus, the tobacco plants used herein can be tobacco varieties or elite tobacco cultivars. Particularly useful tobacco varieties include flue-cured Virginia type, burley type and Oriental type.

[0453] In some embodiments, the tobacco plant can be, for example, selected from one or more of the following varieties: L. cultivar TI 1068, AA 37-1, B 13P, Xanthi (Mitchell-Mor), KT D#3 Hybrid 107, Bel-W3, 79-615, Samsun Holmes NN, F4 from the cross BU21 x Hoja Parado, line 97, KTRDC#2 Hybrid 49, KTRDC#4 Hybrid 1 10, Burley 21, PM016, KTRDC#5 KY 160 SI, KTRDC#7 FCA, KTRDC#6 TN 86SI, PM021, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KY 10, KY 14, KY 160, KY 17, KY 8959, KY 9, KY 907, MD 609, McNair 373, NC 2000, PG 01, PG 04, P01, P02, P03, RG 11, RG 17, RG 8, Speight G-28, TN 86, TN 90, VA 509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 319, Coker 347, Criollo Misionero, PM092, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona、PM102、Kutsage E1 , KY 14 x L8, KY 171, LA BU 21, McNair 944, 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, TRMadole, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, YakaJB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, PM204, PM205, Basma, TKF4028, L8, TKF2002, TN 90, GR141, Basma xanthi, GR149, GR153 and Petit Havana.

[0454] 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 501 LC, K 149, K326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY17, KY 171, KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371 LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, 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 No. 2 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 I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81. DVH 405. Galpao Comum, HB04P, Hicks Broadleaf, KabakulakElassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB156 / 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 Havana. Such low converter subvarieties are contemplated even though not specifically identified herein.

[0455] The tobacco plant may be Burley, Flue-cured Virginia, or Oriental.

[0456] In one embodiment, the plant propagation material may be obtainable from a plant of the invention, such as a tobacco plant.

[0457] "Plant propagation material" as used herein refers to any plant matter taken from a plant from which further plants can be produced. Suitably, the plant propagation material may be selected from seeds, plant callus and plant pieces. Suitably, the plant propagation material may be seeds. Suitably, the plant propagation material may be plant callus. Suitably, the plant propagation material may be plant pieces.

[0458] In one embodiment, the cell (eg tobacco cell), tobacco plant and / or plant propagation material may be obtainable (eg obtained) by a method according to the invention.

[0459] Suitably, when compared to an unmodified tobacco plant, a tobacco plant according to the present invention may have a regulated (e.g., reduced) nicotine content, wherein the tobacco plant has been modified to regulate (e.g., reduce) the activity or expression of at least one gene encoding an RNA binding protein. Suitably, when compared to an unmodified tobacco plant, a tobacco plant according to the present invention may have a reduced nicotine content, wherein the tobacco plant has been modified to reduce or inhibit the activity or expression of at least one gene encoding an RNA binding protein.

[0460] Suitably, the tobacco plant according to the present invention may have a modulated (e.g. reduced) tobacco-specific nitrosamine (TSNA) precursor content when compared to an unmodified tobacco plant, wherein the tobacco plant has been modified to modulate (e.g. increase) the activity or expression of at least one gene encoding an RNA binding protein.

[0461] Suitably, tobacco plants according to the invention may have reduced tobacco-specific nitrosamine (TSNA) precursor content when compared to unmodified tobacco plants, wherein the tobacco plants have been modified to reduce or inhibit the activity or expression of at least one gene encoding an RNA binding protein.

[0462] In one embodiment, a tobacco plant according to the invention comprises a tobacco cell of the invention.

[0463] In another embodiment, the plant propagation material may be obtainable (eg, obtained) from a tobacco plant of the present invention.

[0464] In one embodiment, there is provided use of a tobacco plant as described herein for breeding tobacco plants.

[0465] In another embodiment, the present invention also provides use of the tobacco plant of the preceding embodiment for producing tobacco industry products.

[0466] In another embodiment, there is provided use of the tobacco plants of the present invention to grow crops.

[0467] In one embodiment, provide cell, for example plant cell, for example tobacco plant cell, it has the activity or expression of at least one gene of the coding RNA binding protein as defined herein of regulating.Suitably, cell can be the cell of non-natural existence.Suitably, cell can be mutant cell.Suitably, cell can be the mutant cell of non-natural existence.For example, cell can comprise the sudden change that non-natural existence exists, and described sudden change regulates (for example reduces) the activity or expression of at least one gene of the coding RNA binding protein as defined herein.

[0468] In one embodiment, there is provided use of a cell as provided in the preceding embodiments for producing a tobacco industry product.

[0469] In one embodiment, the invention provides cell cultures (eg, in vitro cultures).

[0470] Tobacco cell culture can be a cell suspension culture. These in vitro cultured cells can be incorporated into tobacco industry products, for example as a substitute for conventional tobacco particles, shredded tobacco (shreds), finely cut or long cut tobacco flakes, as an additive component, or both as a substitute and as an additive. Suitably, cell culture can produce nicotine.

[0471] In one embodiment, there is provided the use of a cell culture, such as a harvested and / or processed cell culture according to the invention, for producing a tobacco industry product.

[0472] Tobacco cells harvested from in vitro culture may be dried, such as freeze-dried, for example to produce a powder.

[0473] In one embodiment, cell culture is tobacco cell culture.Technician will know the known methods for establishing the in vitro culture of tobacco cell.Only for example, can use following method: collect the seed from interested tobacco plant, and sterilize its outside to eliminate undesirable organism, plant described seed with growth interested tobacco plant, from tobacco plant (for example, from tobacco stem) take out tissue for use as explant, establish callus culture from tobacco explant, establish cell suspension culture from callus culture, and gather in the crops culture material (for example comprising tobacco cell) to produce tobacco cell culture.

[0474] 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 the remaining liquid is removed by filtration, such as vacuum filtration.

[0475] The harvested tobacco cell culture may be further processed, such as dried, such as air-dried and / or freeze-dried.The harvested tobacco cell culture or the dried harvested tobacco cell culture or an extract thereof may be incorporated into a tobacco industry product according to the invention.

[0476] In one embodiment, the invention provides plants (e.g., tobacco plants) or parts thereof for use in molecular agriculture. Suitably, plants or parts thereof modified according to the invention can be used to make proteins, e.g., therapeutic agents, such as antibiotics, virus-like particles, nutritional medicines or small molecules.

[0477] In one embodiment, the present invention provides a method for producing a protein (e.g., a therapeutic protein), the method comprising modifying a plant or part thereof capable of producing the protein (e.g., a therapeutic protein) by modulating the activity or expression of at least one gene encoding an RNA binding protein having an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein at least one gene encoding an RNA binding protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; and cultivating the plant under conditions sufficient to allow production of the protein (e.g., a therapeutic protein).

[0478] product

[0479] The invention also provides products obtainable or obtained from a plant according to the invention.Products obtainable or obtained from a plant in which the activity or expression of a gene encoding an RNA binding protein has been modulated are provided.

[0480] In one embodiment, the product may comprise a construct of the invention that modulates the activity or expression of at least one gene encoding an RNA-binding protein as defined herein. In one embodiment, the product may comprise a construct of the invention that modifies the nucleic acid sequence of at least one gene encoding an RNA-binding protein as defined herein.

[0481] The invention also provides products obtainable or obtained from tobacco according to the invention.

[0482] In one embodiment, there is provided use of a tobacco plant of the present invention for producing tobacco leaves.

[0483] Suitably, the tobacco leaves may be subjected to downstream applications, such as processing.

[0484] Therefore, in one embodiment, the use of the aforementioned embodiments can provide processed tobacco leaves. Suitably, the tobacco leaves can be subjected to modulation, fermentation, pasteurization or a combination thereof. In another embodiment, the tobacco leaves can be cut. In some embodiments, the tobacco leaves can be cut before or after being subjected to modulation, fermentation, pasteurization or a combination thereof.

[0485] In one embodiment, the invention provides harvested leaves of the tobacco plant of the invention.

[0486] In a further embodiment, the harvested leaves may be obtainable (eg, obtained) from a tobacco plant propagated from the propagation material of the invention.

[0487] In another embodiment, there is provided harvested leaves obtainable from the methods or uses of the invention.

[0488] Suitably, the harvested leaves may be cut harvested leaves.

[0489] In some embodiments, the harvested leaves may contain living tobacco cells.In other embodiments, the harvested leaves may be subjected to further processing.

[0490] Processed tobacco leaves are also provided.

[0491] The processed tobacco leaf blade may be obtainable from a tobacco plant of the present invention. Suitably, the processed tobacco leaf blade may be obtainable from a tobacco plant obtained according to any of the methods and / or uses of the present invention.

[0492] Suitably, the processed leaf may comprise a reduced content of one or more TSNAs selected from NNN, NNK, NAT and NAB. Suitably, the content of NNN may be reduced. Preferably, the content of NNK may be reduced. Suitably, the content of NAT may be reduced. Suitably, the content of NAB may be reduced. Suitably, the reduction in TSNA content is relative to a comparable product which has not been modified according to the invention.

[0493] In another embodiment, the processed tobacco leaf blade may be obtainable (eg, obtained) from a tobacco plant propagated from tobacco plant propagation material according to the invention.

[0494] The processed tobacco leaves of the present invention may be obtainable (eg, obtained) by processing the harvested leaves of the present invention.

[0495] 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.

[0496] The term "living cell" refers to a cell that is capable of growth and / or metabolic activity. Thus, if a cell is said to be not alive, also known as "non-viable", the cell does not exhibit the characteristics of a living cell.

[0497] The term "substantially free of living cells" means that less than about 5% of all cells are alive. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of all cells are alive.

[0498] In one embodiment, the processed tobacco leaves may be processed by one or more of: curing, fermenting, and / or pasteurizing.

[0499] Suitably, the processed tobacco leaves may be processed by curing.

[0500] 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 curing methods selected from the group consisting of air-curing, open flame curing, fire tube curing, and sun curing.

[0501] Suitably, the tobacco leaves may be air-cured.

[0502] Typically, air-curing is achieved by hanging the tobacco leaves in a well-ventilated curing 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.

[0503] Suitably, the tobacco leaves may be open fire cured. Open fire curing is generally achieved by hanging the tobacco leaves in large curing barns where a hardwood fire is kept smoldering low, either continuously or intermittently, and typically takes between three days and ten weeks, depending on the process and the tobacco.

[0504] In another embodiment, the tobacco leaves can be fire-tube cured. Fire-tube curing can include stringing the tobacco leaves onto tobacco rods and hanging them on tier-poles in a curing barn. The curing barn usually has a flame path that runs with an externally supplied firebox. Generally, this results in 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.

[0505] Suitably, the tobacco leaves may be cured. This process generally involves exposing uncovered tobacco to the sun.

[0506] Suitably, the processed tobacco leaves may be processed by fermentation.

[0507] Fermentation can be carried out in any manner known in the art. Generally, during fermentation, tobacco leaves are piled into a tobacco pile (pile) covered with a modulation in a sack, for example, to retain moisture. The combination of the residual water inside the leaf and the tobacco weight generates the 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 take out the leaf to shake and moisten, and the pile is rotated, so that the inner leaf is placed on the top of the pile outside and the bottom leaf. This ensures the uniform fermentation of the whole pile. The extra moisture on the leaf adds the actual rotation of the leaf itself to generate heat, thereby releasing the natural ammonia of tobacco and reducing nicotine, while also deepening the color and improving the aroma of tobacco. Generally, the fermentation process continues up to 6 months, which depends on the variety of tobacco, the handle position on the leaf, the thickness of the leaf and the intended use.

[0508] Suitably, the processed tobacco leaves may be processed by pasteurisation.Pasteurisation may be particularly preferred when the tobacco leaves are to be used in the preparation of smokeless tobacco industrial products, most preferably snus.

[0509] The pasteurization of tobacco leaves can be carried out by any method known in the art. For example, the pasteurization method can be as described 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.

[0510] During snus production, pasteurization is generally carried out by a process in which the tobacco is heat treated with steam for 24-36 hours, reaching a temperature of approximately 100° C. This results in a nearly sterile product, and without wishing to be bound by theory, one of the consequences of this is believed to be limiting further TSNA formation.

[0511] In one embodiment, the pasteurization process may be steam pasteurization.

[0512] In some embodiments, the processed tobacco leaves may be cut. The processed tobacco leaves may be cut before or after processing. Suitably, the processed tobacco leaves may be cut after processing.

[0513] In one embodiment, use of the foregoing embodiments can provide reconstituted tobacco.

[0514] In one embodiment, reconstituted tobacco is provided.

[0515] As used herein, "reconstituted" may also be referred to as reconstituted, recycled or homogenized sheet tobacco, and refers to tobacco material generated from tobacco leaf residues after processing. Reconstituted tobacco allows for the production of consistent, high quality blends, and allows for adjustment of the ratios of the various components.

[0516] Reconstituted tobacco can be nanofibre reconstituted (nanofibres can be extracted in solid or liquid form), paper reconstituted (which uses stems, scraps and midrib as raw materials), or slurry reconstituted (which uses a mixture of finely powdered material and tobacco stems that is ground into powder and mixed with water and a vegetable binder; the soluble residue is formed into sheets by extracting the water).

[0517] Any method known in the art can be used to prepare reconstituted tobacco, for example, see CORESTA Congress, Sapporo, 2012, Smoke Science / Product Technology Groups, SSPT 12 (incorporated herein by reference).

[0518] In some embodiments, the leaf of the results of tobacco plant, tobacco plant and / or processed tobacco leaf can be used to extract nicotine. The extraction of nicotine can be realized 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.

[0519] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0520] Suitably, the cured tobacco may contain a reduced content of one or more TSNAs selected from NNK, NNN, NAT and NAB. Suitably, the content of NNN may be reduced. Preferably, the content of NNK may be reduced. Suitably, the content of NAT may be reduced. Suitably, the content of NAB may be reduced. Suitably, the reduction in TSNA content is relative to a comparable product which has not been modified according to the invention.

[0521] 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 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.

[0522] Suitably, tobacco blends according to the present invention can include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 10% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 20% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 30% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 40% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 50% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can include about 60% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can comprise about 70% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can comprise about 80% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention. Suitably, tobacco blends can comprise about 90% tobacco from tobacco plant according to the present invention or its part or from tobacco cell culture according to the present invention.

[0523] In one aspect, the tobacco blend products of the present invention comprise at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 percent by dry weight of tobacco cured from a tobacco plant or part thereof according to the present invention or a tobacco cell culture according to the present invention.

[0524] Suitably, the tobacco material after curing can be air-cured. Suitably, the tobacco material after curing can be fire-tube cured. Suitably, the tobacco material after curing can be sun-cured. Suitably, the tobacco material after curing can be open flame cured.

[0525] Tobacco industry products or smoking articles according to the invention may comprise a tobacco material according to the invention (eg, a cured tobacco material or a reconstituted tobacco material).

[0526] In another aspect, the invention provides tobacco industry products.

[0527] In one embodiment, the tobacco industry product according to the invention may be a blended tobacco industry product. Suitably, a tobacco blend may comprise a cured tobacco material according to the invention.

[0528] In one embodiment, tobacco industry products can be prepared from the tobacco plants or parts thereof of the present invention.

[0529] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the invention.

[0530] The term " part " used in the context of tobacco plant as this paper refers to a part of tobacco plant. Suitably, " part " can be a leaf, root or stem or flower of tobacco plant. Suitably, " part " can be a leaf, root or stem of tobacco plant.

[0531] Delivery System

[0532] As used herein, the term "delivery system" is intended to include systems for delivering at least one substance to a user, and includes:

[0533] Combustible gas aerosol delivery systems, such as cigarettes, cigarillos, cigarillos, and tobacco (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokeable materials) for pipe smoking or for rolling or make-your-own cigarettes;

[0534] Non-combustible gas aerosol delivery systems that release compounds from an aerosol-generating material without burning the aerosol-generating material, such as electronic cigarettes, tobacco heating products, and hybrid systems that generate an aerosol using a combination of aerosol-generating materials; and

[0535] Non-aerosol delivery systems that deliver at least one substance to a user orally, nasally, transdermally or in another manner without forming an aerosol, including, but not limited to, lozenges, chewing gum, patches, preparations containing powders that can be inhaled, and oral products such as oral tobacco, including snus or moist snuff, wherein the at least one substance may or may not contain nicotine.

[0536] According to the present disclosure, a "combustible" aerosol supply system is a system in which an aerosol-generating material that is part of the aerosol supply system (or components thereof) burns or burns during use to facilitate delivery of at least one substance to a user.

[0537] In some embodiments, the delivery system is a combustible aerosol supply system, such as a system selected from the group consisting of a cigarette, a cigarillo, and a cigar.

[0538] In some embodiments, the present disclosure is directed to components for use in a flammable aerosol delivery system, such as a filter, a filter rod, a filter segment, a tobacco rod, a spill, an aerosol modifier release component such as a capsule, a string, or a bead, or a paper such as a plug wrap, a tipping paper, or a cigarette paper.

[0539] According to the present disclosure, a "non-combustible" aerosol supply system is a system in which the aerosol-generating materials that are part of the aerosol supply system (or components thereof) are not burned or combusted to facilitate delivery of at least one substance to a user.

[0540] In some embodiments, the delivery system is a non-combustible gas aerosol supply system, such as a powered non-combustible gas aerosol supply system.

[0541] In some embodiments, the non-combustible aerosol supply system is an electronic cigarette, also known as a vaping device or an electronic nicotine delivery system (END), although it is noted that the presence of nicotine in the aerosol-generating material is not a requirement.

[0542] In some embodiments, the non-combustible gas aerosol supply system is an aerosol-generating material heating system, also known as a heat-not-burn system. An example of such a system is a tobacco heating system.

[0543] In some embodiments, the non-combustible aerosol supply system is a hybrid system that uses a combination of aerosol-generating materials, and one or more of the aerosol-generating materials may be heated. Each type of aerosol-generating material may be, for example, in the form of a solid, liquid or gel and may or may not contain nicotine. In some embodiments, the hybrid system includes a liquid or gel aerosol-generating material and a solid aerosol-generating material. The solid aerosol-generating material may include, for example, tobacco or non-tobacco products.

[0544] Generally, a non-combustible gas aerosol supply system may include a non-combustible gas aerosol supply device and consumables for use with the non-combustible gas aerosol supply device.

[0545] In some embodiments, the present disclosure relates to consumables containing aerosol-generating materials and configured for use with non-combustible gas aerosol supply devices. These consumables are sometimes referred to as articles throughout the disclosure.

[0546] In some embodiments, a non-combustible gas aerosol supply system, such as a non-combustible gas aerosol supply device thereof, may include a power source and a controller. The power source may, for example, be an electrical power source or an exothermic power source. In some embodiments, the exothermic power source includes a carbon substrate that may be powered so as to distribute energy in the form of heat to a material that generates an aerosol or a heat transfer material adjacent to the exothermic power source.

[0547] In some embodiments, a non-combustible gas aerosol supply system may include an area for receiving a consumable product, an aerosol generator, an aerosol generating area, a housing, a mouthpiece, a filter, and / or an aerosol modifier.

[0548] In some embodiments, consumables for use with a non-flammable gas aerosol supply device may include an aerosol-generating material, an aerosol-generating material storage area, an aerosol-generating material transfer component, an aerosol generator, an aerosol generating area, a shell, a packaging material, a filter, a mouthpiece and / or an aerosol modifier.

[0549] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco plant or part thereof according to the invention.

[0550] Suitably, the delivery system may be prepared from a tobacco cell culture according to the invention.

[0551] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco plant or part thereof propagated from tobacco plant propagation material according to the invention.

[0552] Suitably, the delivery system may be prepared from (eg may comprise) harvested leaves of a tobacco plant according to the invention.

[0553] Suitably, the delivery system may be prepared from (eg may comprise) processed tobacco leaves according to the invention.

[0554] Suitably, the delivery system may be prepared from (eg may comprise) a cured tobacco material according to the invention.

[0555] Suitably, the delivery system may be prepared from (eg may include) a tobacco blend according to the invention.

[0556] In one embodiment, the delivery system is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0557] In one embodiment, the delivery system includes one or more components of a combustible smoking article, such as a filter, a filter rod, a filter rod segment, tobacco, a tobacco rod, a tobacco rod segment, a plug, an additive release member such as a capsule, a string, a bead, a paper such as a plug wrap, a tipping paper or a cigarette paper.

[0558] In one embodiment, the delivery system is a non-flammable gas aerosol supply system.

[0559] In one embodiment, the delivery system includes one or more components of a non-combustible gas aerosol supply system, such as a heater and an aerosolizable substrate.

[0560] In one embodiment, the aerosol supply system is an electronic cigarette, also known as a vaping device.

[0561] In one embodiment, the electronic cigarette includes 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.

[0562] In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is associated with or includes a heater.

[0563] In one embodiment, the delivery system is a heating product that releases one or more compounds by heating rather than burning a matrix material. The matrix material is an aerosolizable material, which may be, for example, tobacco or other non-delivery systems, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0564] In one embodiment, the heating product is an electronic device.

[0565] 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.

[0566] In one embodiment, the heating product is a non-electronic article.

[0567] In one embodiment, the heating product includes an aerosolizable substrate, such as a solid or gel material, and a heat source capable of supplying thermal energy to the aerosolizable substrate without any electrical means, such as by burning a combustion material such as charcoal.

[0568] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0569] In some embodiments, the aerosolizable base material may comprise a vapor or aerosol generating agent or a humectant, such as glycerol, propylene glycol, triacetin, or diethylene glycol.

[0570] In one embodiment, the delivery system is a hybrid system, which generates aerosols by heating without burning the combination of host materials. The host material can include, for example, solid, liquid or gel, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel matrix and a solid matrix. The solid matrix can be, for example, tobacco or other non-delivery systems, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel matrix and tobacco. In another embodiment, the product can include construct of the present invention, which regulates activity or expression of at least one protein containing the RNA binding domain, and reduces alkaloid content when expressed in a plant (for example, tobacco plant).

[0571] Tobacco industry products

[0572] As used herein, the term "tobacco industry product" is intended to include combustible smoking articles, such as cigarettes, cigarillos, cigarillos, tobacco for pipes or roll-your-own cigarettes (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokeable materials), non-combustible gas 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 generate aerosols from a combination of matrix materials, such as hybrid systems containing liquid or gel or solid matrices, and aerosolizable matrix materials used in these aerosol delivery systems; as well as non-aerosol delivery products, such as lozenges, chewing gum, patches, products containing inhalable powders, and smokeless tobacco industry products such as snus and snus, which may or may not deliver nicotine.

[0573] In one embodiment, a tobacco industry product can be prepared from (eg, can comprise) a tobacco plant or part thereof of the invention.

[0574] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the invention.

[0575] As used herein in the context of tobacco plants, the term "part thereof" refers to a part of the tobacco plant. Preferably, the "part thereof" is a leaf of the tobacco plant.

[0576] In another embodiment, tobacco industry products can be prepared from the harvested leaves of the present invention.

[0577] In further embodiments, tobacco industry products can be prepared from the processed tobacco leaves of the present invention.

[0578] Suitably, tobacco industry products may be prepared from tobacco leaves processed by one or more of: curing, fermenting and / or pasteurising.

[0579] Suitably, the tobacco industry product may comprise cut tobacco leaves, optionally processed in accordance with the preceding embodiments.

[0580] In another embodiment, tobacco industry products can be prepared from tobacco cell cultures according to the present invention.

[0581] In another embodiment, a tobacco industry product may be prepared from (eg may comprise) a cured tobacco material according to the invention.

[0582] In another embodiment, a tobacco industry product can be prepared from (eg, can include) a tobacco blend according to the invention.

[0583] In one embodiment, the tobacco industry product may be a smoking article.

[0584] As used herein, the term "smoking article" may include smokeable products such as cigarettes, cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes.

[0585] In another embodiment, the tobacco industry product can be a smokeless tobacco industry product.

[0586] As used herein, the term "smokeless tobacco industry product" refers to a tobacco industry product that is not intended to be smoked and / or subjected to combustion.

[0587] Smokeless tobacco industry products (including heat-not-burn materials) can contain tobacco in any form (including dry particles, shredded tobacco, granules, powders, or slurries) deposited on, mixed in, surrounded by, or combined with other ingredients, the other ingredients taking any form such as sheets, films, tabs, foams, or beads.

[0588] In one embodiment, smokeless tobacco industry products can include snus, snuff, chewing tobacco, and the like.

[0589] In one embodiment, the tobacco industry product is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0590] In one embodiment, the tobacco industry product comprises one or more components of a combustible smoking article, such as a filter, a filter rod, a filter rod segment, tobacco, a tobacco rod, a tobacco rod segment, a spill, an additive release component such as a capsule, a thread, a bead, a paper such as a plug wrap, a tipping paper or a cigarette paper.

[0591] In one embodiment, the tobacco industry product is a non-combustible gas aerosol delivery system.

[0592] In one embodiment, the tobacco industry product comprises one or more components of a non-combustible gas aerosol delivery system, such as a heater and an aerosolizable substrate.

[0593] In one embodiment, the aerosol supply system is an electronic cigarette, also known as a vaping device.

[0594] In one embodiment, the electronic cigarette includes 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.

[0595] In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is associated with or includes a heater.

[0596] In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating rather than 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.

[0597] In one embodiment, the heating product is an electronic device.

[0598] 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.

[0599] In one embodiment, the heating product is a non-electronic article.

[0600] In one embodiment, the heating product includes an aerosolizable substrate, such as a solid or gel material, and a heat source capable of supplying thermal energy to the aerosolizable substrate without any electrical means, such as by burning a combustion material such as charcoal.

[0601] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0602] In some embodiments, the aerosolizable base material may comprise a vapor or aerosol generating agent or a humectant, such as glycerol, propylene glycol, triacetin, or diethylene glycol.

[0603] In one embodiment, the tobacco industry product is a hybrid system, generates aerosol with the combination of the non-combustion host material by heating.The host material can include, for example, 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, for example, 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.

[0604] In further embodiments, the tobacco industry product may be a tobacco heating device or a hybrid device or an electronic cigarette, and the like.

[0605] Typically in tobacco heating devices or hybrid devices, an aerosol is generated by transferring heat from a heat source to a physically separate aerosol-forming substrate or material, which may be located within, 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 drawn in through the smoking article. As the released compounds cool, they condense to form an aerosol that is inhaled by the user.

[0606] 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.

[0607] Suitably, the tobacco heating device may be an aerosol generating device.

[0608] 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.

[0609] Examples of suitable heat not burn devices may be those taught in WO2013 / 034459 or GB2515502, which are incorporated herein by reference.

[0610] In one embodiment, the aerosol-forming substrate of the tobacco heating device may be a tobacco industry product according to the present invention.

[0611] In one embodiment, the tobacco heating device may be a hybrid device.

[0612] Polynucleotide / polypeptide / construct

[0613] In certain embodiments of the invention, constructs that modulate the activity or expression of at least one gene encoding an RNA binding protein as described herein may be transformed into plant cells, suitably under the direction of a promoter.

[0614] In certain embodiments of the present invention, under the guidance of a promoter, a construct that reduces (i.e., inhibits) the activity or expression of at least one gene encoding an RNA binding protein as described herein can be transformed into a plant cell. For example, the genetic construct can be a gene editing construct or can include an RNAi molecule, which can include a small interfering RNA (siRNA) molecule or a short hairpin (shRNA) molecule.

[0615] In certain embodiments of the invention, a construct that increases the activity or expression of a gene encoding an RNA binding protein as described herein, such as a construct encoding a gene encoding an RNA binding protein such as an endogenous RNA binding protein, can be transformed into a plant cell, suitably under the direction of a promoter.

[0616] The construct can be introduced into the plant according to the present invention with the aid of a suitable vector, such as a plant transformation vector. The plant transformation vector may comprise an expression cassette comprising a promoter sequence in the transcriptional direction 5'-3', a construct sequence targeting a gene encoding an RNA binding protein as described herein, and optionally a 3' untranslated terminator sequence, including a termination signal for RNA polymerase and a polyadenylation signal for polyadenylase. The promoter sequence may be present in one or more copies, and such copies may be identical or variants of the promoter sequence as described above. The terminator sequence may be derived from a plant, bacterial or viral gene. Suitable terminator sequences are, for example, pea E9 Terminator sequence, derived from Agrobacterium tumefaciens ( Agrobacterium tumefaciens )of Nopaline synthase Genetic nos terminator sequence, and from cauliflower mosaic virus 35S terminator Those skilled in the art will readily recognize other suitable terminator sequences.

[0617] The constructs of the present invention may also contain gene expression enhancing mechanisms to increase the strength of the promoter. Examples of such enhancer elements are derived from pea Plastocyanin The enhancer element is part of the promoter of the gene and is the subject of International Patent Application No. WO 97 / 20056, which is incorporated herein by reference. Suitable enhancer elements can be, for example, derived from Agrobacterium tumefaciens. Nopaline synthase Genetic nos Enhancer element, and from cauliflower mosaic virus 35S Enhancer elements.

[0618] These regulatory regions may be derived from the same gene as the promoter DNA sequence, or may be derived from tobacco or other organisms, such as from the Solanaceae family or from the subfamily Lycopodiellae ( Cestroideae All regulatory regions should be able to function in cells of the tissue to be transformed.

[0619] The promoter DNA sequence may be derived from the same gene as the gene of interest, e.g., the gene that the promoter is to direct, e.g., a gene encoding an RNA binding protein according to the invention, the coding sequence used in the invention, or may be derived from a different gene from tobacco or another organism, e.g., from the Solanaceae family or from the subfamily Lycopodiellae.

[0620] 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 known in the art. In addition to the expression cassette, the plant transformation vector will contain such sequences as are necessary for the transformation process. These may include Agrobacterium ( Agrobacterium ) vir gene, one or more T-DNA border sequences, and a selectable marker or other means of identifying transgenic plant cells.

[0621] The term "expression vector or plant transformation vector" means a construct capable of being expressed in vivo or in vitro. Preferably, the expression vector is incorporated into the genome of the organism. In one embodiment, the vector of the invention expresses a protein, such as an RNA binding protein as described herein. The term "incorporated" preferably includes stable incorporation into the genome.

[0622] Techniques for transforming plants are well known in the art and include, for example, Agrobacterium-mediated transformation. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant genome in order to obtain stable maintenance of the inserted genetic material. An overview of general techniques can be found in the literature by Potrykus ( Annu Rev Plant Physiol Plant Mol Biol

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

[0623] Generally, in Agrobacterium-mediated transformation, a binary vector carrying an exogenous DNA of interest, i.e., a construct according to the present invention, is transferred from an appropriate Agrobacterium strain to a target plant by co-cultivation of Agrobacterium with an explant from the target plant. The transformed plant tissue is then regenerated on a selective medium containing a selective marker and a plant growth hormone. An alternative method is the floral dip method (Clough & Bent, 1998 Plant J. 1998 December; 16 (6): 735-43, which is incorporated herein by reference), whereby the flower buds of a complete plant are contacted with a suspension of an Agrobacterium strain containing a chimeric gene, and after fruiting, the transformed individuals are germinated and identified by growth on a selective medium. Direct infection of plant tissue by Agrobacterium is a simple technique that has been widely used and described in Butcher et al. (1980), Tissue Culture Methods for Plant Pathologists , eds.: DS Ingrams and JP Helgeson, 203-208, which reference is incorporated herein by reference.

[0624] Further suitable transformation methods include, for example, the use of polyethylene glycol or electroporation technology, particle bombardment, microinjection and silicon carbide fibers to transfer genes directly into protoplasts. Use impact transformation (ballistic transformation) to transform plants and produce fertile transgenic maize plants mediated by silicon carbide whiskers in Frame et al. (1994) The Plant Journal 6 (6): 941-948 to obtain instruction, and the reference is incorporated herein by reference, and virus transformation technology is, for example, Meyer et al. (1992) Mol. Gen. Genet. 231 (3): 345-352 to obtain instruction, and the reference is incorporated herein by reference. Cassava mosaic virus is used as a vector system for plants in Meyer et al. (1992) Gene 110: 213-217 to obtain instruction, and the reference is incorporated herein by reference. Further instructions about plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

[0625] In a further aspect, the present invention relates to a vector system, which carries a construct and is introduced into a plant, suitably a tobacco plant, in the genome. The vector system can comprise a vector, but it can comprise 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 further detail in Gynheung et al. (1980) Binary Vectors, Plant Molecular Biology Manual A3, 1-19, which is incorporated herein by reference.

[0626] A widely adopted system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens or from Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) of the Ri plasmid, which was described by An et al. (1986) Plant Physiol. 81, 301-305 and Butcher et al. (1980) Tissue Culture Methods for Plant Pathologists,Editors: 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 plant cell transformation has been well studied and is described in EP-A-120516; Hoekema (1985) The Binary Plant Vector System, Offset-drukkerij Kanters B. B., Amsterdam, Chapter V; Fraley et al., Crit. Rev. Plant Sci., 4: 1-46; and An et al. (1985) EMBO J 4: 277-284, which are all incorporated herein by reference.

[0627] Plant cells transformed with one or more constructs that modulate the activity or expression of at least one gene encoding an RNA binding protein can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0628] The term "transgenic plant" in connection with the present invention includes any plant comprising a construct which modulates the activity or expression of at least one gene encoding an RNA binding protein according to the present invention. Accordingly, a transgenic plant is a plant which has been transformed with a construct according to the present invention. Preferably, according to the present invention, the transgenic plant shows a modulated (e.g. reduced) alkaloid content and / or a modulated (e.g. reduced) TSNA precursor content. The term "transgenic plant" does not include a native nucleotide coding sequence in its natural environment when under the control of its native promoter (which is also in its natural environment).

[0629] In one aspect, the gene, construct, plant transformation vector or plant cell encoding RNA binding protein according to the present 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.

[0630] In one aspect, the gene, construct, plant transformation vector or plant cell encoding RNA binding protein according to the present invention is in a purified form. The term "purified" means being in a relatively pure state, such as at least about 90% pure, or at least about 95% pure, or at least about 98% pure.

[0631] As used herein, the term "nucleotide sequence" refers to an oligonucleotide sequence or a polynucleotide sequence, as well as variants, homologues, fragments and derivatives (e.g., portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, it may be double-stranded or single-stranded, whether representing the sense strand or the antisense strand.

[0632] The term "nucleotide sequence" related to the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it means a DNA encoding the present invention, more preferably a cDNA sequence.

[0633] In a preferred embodiment, when relevant to and when encompassed by the scope of the invention itself, the nucleotide sequence, i.e., the gene encoding the RNA binding protein according to the invention, comprises the native nucleotide sequence when in its natural environment and when it is linked to one or more of its naturally associated sequences that are also in its natural environment. For ease of reference, we shall refer to this preferred embodiment as the "native nucleotide sequence". In this regard, the term "native nucleotide sequence" means the entire nucleotide sequence, which is in its natural environment, and when operably linked to the entire promoter to which it is naturally associated, the promoter is also in its natural environment.

[0634] The nucleotide sequence for use of the present invention can be present in a vector, wherein the nucleotide sequence is operably linked to a regulatory sequence that can ensure expression of the nucleotide sequence by a suitable host organism. The construct for use of the present invention can be transformed into a suitable host cell as described herein to ensure expression of the polypeptide of the present invention. The selection of a vector such as a plasmid, cosmid or phage vector will often depend on the host cell to be introduced therein. The vector can be used, for example, for the production of RNA in vitro, or for transfection, transformation, transduction or infection of a host cell.

[0635] In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence that is capable of ensuring expression of the nucleotide sequence, for example, by a selected host cell. For example, the present invention includes a vector comprising a nucleotide sequence of a gene encoding an RNA binding protein as described herein operably linked to such a regulatory sequence, i.e., the vector is an expression vector.

[0636] The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0637] 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, such as an RNA polymerase binding site. The nucleotide sequence within the construct encoding a gene can be operably linked to at least one promoter, the gene encoding an RNA binding protein.

[0638] 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 attached directly or indirectly to a promoter.

[0639] An example of indirect attachment is between the promoter and the nucleotide sequence of the present invention, providing a suitable spacer group, such as an intron sequence, such as the Sh1-intron or the ADH intron. The same is true for the term "fused" related to the present invention, which includes direct or indirect attachment. In some cases, the term does not include the natural combination of nucleotide sequences of protein encoding that are usually combined with wild-type gene promoters and when they are all in their natural environment. Constructs can even contain or express markers that allow the selection of genetic constructs.

[0640] In some embodiments, the promoter can be operably linked to a nucleotide sequence in a construct or vector for regulating nicotine concentration and / or total content in a cell or cell culture or a tobacco plant or part thereof.

[0641] In some embodiments, the promoter can be selected from the group consisting of: a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, and an inducible promoter.

[0642] In one embodiment, the promoter may be a constitutive promoter.

[0643] 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), the rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991). Analysis of rice Act1 5' region activity in transgenic rice plants. Plant Cell 3 1155-65) 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). A constitutive promoter, for example, the carnation etched ring virus (CERV) promoter (Hull R, Sadler J, Longstaff M (1986) (CaMV / 35S), figwort mosaic virus 35S promoter. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses. EMBO Journal, 5 (2): 3083-3090).

[0644] 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.

[0645] The promoter may 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, usually throughout the lifetime of those plant parts. The category of tissue-specific promoters also generally includes promoters whose specificity is not absolute, i.e., they may also direct expression at lower levels in tissues other than preferred tissues. Tissue-specific promoters include the phaseolin promoter, the legumin b4-promoter, the usp-promoter, the sbp-promoter, the ST-LS1 promoter, and the B33 (patatin class I promoter).

[0646] In another embodiment, the promoter can be a developmentally regulated promoter.

[0647] Developmentally regulated promoters direct changes in the expression of a gene in one or more parts of a plant at specific times during plant development. A gene may be expressed at different (usually lower) levels in said plant part at other times, and may also be expressed in other plant parts.

[0648] In one embodiment, the promoter can be an inducible promoter.

[0649] Inducible promoter can respond to inducer to guide the expression of gene. In the absence of inducer, gene will not be expressed. Inducer can act directly on promoter sequence, or can work by offsetting the effect of repressor molecule. Inducer can be chemical reagent such as metabolite, albumen, growth regulator (for example auxin and salicylic acid that activate OCS promoter) or toxic element, physiological stress such as heat, light (for example soybean SSU promoter), damage (for example nos, nopaline synthase promoter), or osmotic pressure, or the indirect consequence of pathogen or pest action. The promoter of development regulation can be described as a specific type of inducible promoter, which responds to the environmental stimulation of the specific moment of endogenous inducer produced by plant or in the life history of plant. Examples of known inducible promoters include those associated with wound response as described, for example, by Warner SA, Scott R, Draper J. ((1993) Plant J. 3 191-201), temperature response as disclosed by Benfey & Chua (1989) (Benfey, PN, and Chua, NH. ((1989) Science 244 174-181), and chemical induction as described by Gatz ((1995) Methods in Cell Biol. 50 411-424).

[0650] Can from produce described albumen any cell or biology, identify and / or separate and / or purify nucleotide sequence, described nucleotide sequence coding has the albumen of the specific property of the gene of coding RNA binding protein as defined herein or is suitable for the albumen of modification.The various methods for identification and / or separation and / or purification of nucleotide sequence are well-known in the art.For example, once suitable sequence has been identified and / or separated and / or purified, just can use PCR amplification technique to prepare more sequence.

[0651] In a further alternative, the nucleotide sequence encoding the RNA binding protein can be prepared synthetically by established standard methods, such as the phosphoramidite method described by Beucage et al. (1981) Tetrahedron Letters 22, pp. 1859-1869, or by the method described by Matthes et al. (1984) EMBO J. 3, pp. 801-805, which is incorporated herein by reference. In the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned in appropriate vectors, for example, in an automatic DNA synthesizer.

[0652] 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".

[0653] The present invention also includes the use of sequences that have a certain degree of sequence identity or sequence homology (hereinafter referred to as "one or more homologous sequences") with one or more amino acid sequences of a polypeptide having the specific properties defined herein, or any nucleotide sequence, i.e., a gene encoding an RNA binding protein. Here, the term "homolog" means an entity that has a certain homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" can be equivalent to "identity".

[0654] Homologous amino acid sequences and / or nucleotide sequences and / or fragments should provide and / or encode polypeptides that retain the functional activity of RNA-binding proteins and / or enhance the activity of RNA-binding proteins. Generally, homologous sequences will contain, for example, the same active site as 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, it is preferably represented in terms of homology of sequence identity. Homologous sequences generally retain functional domains or motifs.

[0655] In one embodiment, homologous sequences are employed to include amino acid sequences or nucleotide sequences that have one, two or several additions, deletions and / or substitutions compared to the subject sequence.

[0656] Sequence identity

[0657] Sequence identity comparisons can be performed by eye, or more generally, by means 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 adjacent sequences, i.e., one sequence is compared 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 called a "no gap" comparison. Typically, this no gap comparison is performed only on a relatively short number of residues.

[0658] Although this is a very simple and consistent method, it fails to take into account that, for example, in otherwise identical sequence pairs, an insertion or deletion will cause the amino acid residues in the back to be unable to be compared, thus potentially leading to a significant reduction in % homology when performing an overall comparison. Thus, most sequence comparison methods are designed to produce an optimal comparison that takes into account possible insertions and deletions without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to attempt to maximize local homology.

[0659] However, these more sophisticated methods assign "gap penalties" 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 relatedness between the two compared sequences - will receive a higher score than an alignment with many gaps. An "affine gap cost" is generally used, which imposes a relatively high cost for the presence of a gap, and a smaller penalty for each residue after 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 such software for sequence comparison, it is preferably to use the default value.

[0660] 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, pp. 7-58 to 7-60).

[0661] Although final % homology can be measured according to identity, the comparison process itself is generally not based on all-or-no paired comparisons. On the contrary, a similarity score matrix with a change in ratio is usually used, which is based on chemical similarity or evolutionary distance to each paired comparison assignment score. 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 a disclosed default value or a conventional symbol comparison table (if provided) (for further details, referring to the user manual). For some applications, the default values ​​of the Vector NTI program package are preferably used.

[0662] 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). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software generally performs this as part of a sequence comparison and generates a numerical result.

[0663] If a gap penalty should be used when determining sequence identity, then preferably the following parameters are used for pairwise alignments:

[0664] For BLAST GAP OPEN 0 GAP EXTENSION 0

[0665] For CLUSTAL DNA protein Word length 2 1 K triplet Gap Penalty 15 10 Gap Extension 6.66 0.1

[0666] In one embodiment, CLUSTAL can be used together with gap penalty and gap extension set as defined above.In some embodiments, the gap penalty for BLAST or CLUSTAL comparison can be different from those described in detail above.Technical staff will understand that the standard parameters for performing BLAST and CLUSTAL comparison may change regularly, and will be able to select appropriate parameters based on the standard parameters detailed for BLAST or CLUSTAL comparison algorithm at that time.

[0667] Suitably, the degree of identity with respect to a nucleotide sequence is determined over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 70 contiguous nucleotides, preferably over at least 80 contiguous nucleotides, preferably over at least 90 contiguous nucleotides, preferably over at least 100 contiguous nucleotides, preferably over at least 150 contiguous nucleotides, preferably over at least 200 contiguous nucleotides, preferably over at least 250 contiguous nucleotides, preferably over at least 300 contiguous nucleotides, preferably over at least 350 contiguous nucleotides, preferably over at least 400 contiguous nucleotides, preferably over at least 450 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 550 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 650 contiguous nucleotides, or preferably over at least 700 contiguous nucleotides.

[0668] Suitably, the degree of identity with respect to nucleotide, cDNA, cds or amino acid sequences may be determined over the entire sequence.

[0669] The sequences may also have deletions, insertions or substitutions of amino acid residues which produce silent changes and result in functionally equivalent substances. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties 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 having similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0670] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same group in the second column, and preferably in the same row in the third column, can be substituted for each other:

[0671]

[0672] The present invention also includes homologous substitutions (substitution and replacement are both used herein to mean the exchange of an existing amino acid residue with an alternative residue), i.e. like-for-like substitutions, such as basic substitutions for basic, acidic substitutions for acidic, polar substitutions for polar, etc. Non-homologous substitutions may also occur, i.e. from one class of residues to another, or on the other hand involving 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), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0673] 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-allyl-glycine*, 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-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline (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 notation * has been used for the purposes discussed above (in relation to homologous or nonhomologous substitution) to indicate the hydrophobic character of the derivative, while # has been used to indicate the hydrophilic character of the derivative, with #* indicating amphiphilic character.

[0674] In addition to amino acid spacers such as glycine or β-alanine residues, the variant amino acid sequence may include a suitable spacer, which may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl. Further variant forms relate to the presence of one or more amino acid residues in peptidomimetic form, which will be fully understood by those skilled in the art. For the avoidance of doubt, "peptidomimetic 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 peptidomimetic form are known in the art, such as Simon et al. (1992) PNAS 89 (20), 9367-9371 and Horwell (1995) Trends Biotechnol. 13 (4), 132-134.

[0675] Nucleotide sequences for use 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 methylphosphonate and phosphorothioate backbones and / or acridine or polylysine chain additions at the 3' and / or 5' ends of the molecule. For 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 performed to enhance the in vivo activity or lifespan of the nucleotide sequences of the present invention.

[0676] The present invention also includes sequences complementary to the nucleic acid sequences of the present invention, or sequences that can hybridize with the sequences of the present invention or with sequences complementary thereto. The term "hybridization" as used herein should include "a process by which a nucleic acid strand binds to a complementary strand via base pairing", as well as amplification processes such as those performed in polymerase chain reaction (PCR) technology.

[0677] The present invention also relates to nucleotide sequences that can hybridize with the nucleotide sequences of the present invention (including the complementary sequences of those presented herein). Preferably, hybridization is determined under stringent conditions (e.g., 50° C. and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3 citric acid, pH 7.0}). More preferably, hybridization is determined under high stringency conditions (e.g., 65° C. and 0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3 citric acid, pH 7.0}).

[0678] A review of general techniques for transforming plants can be found in papers such as Potrykus et al. (1991) Annu Rev Plant Physiol Plant Mol Biol 42:205-225 and Christou et al. (1994) Agro-Food-Industry Hi-Tech Mar / Apr 17-27, which are incorporated herein by reference. Further teachings on plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

[0679] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present 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 a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

[0680] 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 numerical values ​​of the limited ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

[0681] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0682] 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".

[0683] 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.

[0684] 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, as these may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0685] Where a range of values ​​is provided, it is understood that, unless the context clearly indicates otherwise, each intermediate value based on one-tenth of the unit of the lower limit between the upper and lower limits of the range 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. Depending on any specifically excluded limits in the specified range, the upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which any limit, no limit, or both limits are included in the smaller range is also included in the present disclosure. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0686] 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 plurality of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

[0687] advantage

[0688] It has surprisingly been found that the alkaloid and / or TSNA precursor content of a plant can be modulated by modulating the activity or expression of at least one gene encoding an RNA binding protein as taught herein, which acts as a positive regulator of alkaloid content in tobacco. Thus, tobacco industry products can be produced having a modulated (e.g., reduced) alkaloid content) and / or reduced TSNA precursor content and commercially desirable traits sought by consumers of tobacco industry products.

[0689] The present inventors have surprisingly identified a method for regulating the alkaloid content and / or TSNA precursor content of a plant, such as a tobacco plant, by modulating the activity or expression of a gene encoding an RNA binding protein. The alkaloid or TSNA precursor content of a plant, such as a tobacco plant, can be reduced by reducing or inhibiting the activity or expression of a gene encoding an RNA binding protein as described herein. Prior to the present invention, it was not known that modulation of the activity or expression of a gene encoding an RNA binding protein as described herein could be used to modulate the alkaloid (and / or TSNA precursor) content of a plant, such as a tobacco plant.

[0690] 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 publication constitutes prior art for the claims appended hereto. Example

[0691] Example 1 - Transient overexpression of a gene encoding an RNA binding protein increases alkaloid content in leaves

[0692] Methods and Materials

[0693] Cloning RNA Binding Protein Expression Vector

[0694] The Nitab4.5_0013685g0010.2 gene sequence (SEQ ID NO. 3) encoding the RNA binding protein according to the present invention was amplified from a Gateway™ compatible cDNA library using primers located outside of the restriction sites flanking the gene sequence.

[0695] The resulting plasmid was sequenced and transformed into Agrobacterium tumefaciens GV3101pMP90 by heat shock and transiently expressed in tobacco leaves.

[0696] Transient gene expression

[0697] Agrobacterium tumefaciens GV3101 strains carrying the construct of interest were grown overnight in Luria-Bertani (LB) medium supplemented with appropriate antibiotics. The culture was quickly spun down and resuspended to OD600=0.6 in a buffer containing 10 mM MgCl2, 10 mM MES pH 5.6 and 100 μM acetosyringone, and incubated at room temperature for 1 hour. Infiltration into tobacco leaves was performed with a needleless syringe. Samples were obtained 5 days after infiltration.

[0698] Tests were performed in two biological replicates.

[0699] Alkaloid measurement

[0700] The relative contents of pyridine alkaloids were determined by reversed-phase high-performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). Chromatographic separation was achieved using a Gemini-NX column (100 mm × 3.0 mm, 3 μm particle size, Phenomenex) and a gradient chromatographic separation using 6.5 mM ammonium acetate buffer (aq) (pH 10) and methanol.

[0701] The mass spectrometer was operated in electrospray (ESI) positive mode using scheduled MRM data acquisition. Two MRM transitions were monitored for each analyte and one for an isotopically labeled internal standard.

[0702]

[0703]

[0704] result

[0705] The alkaloid content of 5-week-old tobacco leaves expressing the Nitab4.5_0013685g0010.2 construct is shown in Figure 1 In. Alkaloid content is expressed relative to the control and contains two biological replicates analyzed by a one-way t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P values ​​≤ 0.01.

[0706] Overexpression of Nitab4.5_0013685g0010.2 resulted in a significant increase in alkaloid content in leaves.

[0707] in conclusion

[0708] Nitab4.5_0013685g0010.2 is a positive regulator of alkaloid content and is a regulator of pyridine alkaloids in tobacco.

[0709] Example 2 - Transient expression of antisense RNA targeting Nitab4.5_0013685g0010.2 reduces Alkaloid content

[0710] Materials and methods

[0711] The Nitab4.5_0013685g0010.2 coding sequence was cloned in reverse direction into a plant expression vector driven by the CERV promoter.

[0712] result

[0713] The alkaloid content of 5-week-old tobacco leaves expressing Nitab4.5_0013685g0010.2 antisense RNA is shown in Figure 2In. Alkaloid content is expressed relative to the control and contains two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P value ≤ 0.01.

[0714] Inhibition of Nitab4.5_0013685g0010.2 expression using antisense RNA resulted in a decrease in alkaloid content in leaves, particularly nornicotine, PON, nicotine, anabasine, and anatabine.

[0715] in conclusion

[0716] Nitab4.5_0013685g0010.2 is a positive regulator of alkaloid content, especially in leaves, and is a regulator of pyridine alkaloids in tobacco.

[0717] Example 3 - Transient expression of Nitab4.5_0013685g0010.2_deltaRBD-RGG results in the production of alkaloids Significantly reduced.

[0718] Materials and methods

[0719] The Nitab4.5_0013685g0010.2_deltaRBD-RGG coding sequence (SEQ ID NO: 34) was cloned into the expression vector as described in Example 1.

[0720] result

[0721] The alkaloid content of 5-week-old tobacco leaves expressing Nitab4.5_0013685g0010.2_deltaRBD-RGG is shown in Figure 3 In. Alkaloid content is expressed relative to the control and contains two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P value ≤ 0.05.

[0722] in conclusion

[0723] Disruption of the RBD-RGG domain in RNA-binding proteins regulates alkaloid content in tobacco leaves.

[0724] Example 4 - Homolog Testing

[0725] Homologues of SEQ ID NO. 3, namely SEQ ID NOs 6, 9, 12, 15, 18, 21, 24, 27 and 30, were tested for their effects in the assays described in the above examples.

[0726] Example 5 - Interactome study of Nitab4.5_0013685g0010.2

[0727] method:

[0728] The triplicate co-immunoprecipitation samples of Nitab4.5_0013685g0010.2 were submitted to mass spectrometry analysis. The samples were digested with trypsin and analyzed on a Thermo Scientific™ Orbitrap Fusion™ mass spectrometer. Using a 5% false discovery rate, data were analyzed using Proteome Discoverer 2.1 for the BAT tobacco proteome database and the common pollutant database. The sorting proteins were based on the following: identification in all the same samples of the candidate samples; the number of identified peptides, in the candidate samples; and not present in the control (GFP) or other candidate samples. A list of candidate interactors for Nitab4.5_0013685g0010.2 was generated using these criteria.

[0729] Results and Conclusions:

[0730] Mass spectrometry analysis of triplicate samples correctly identified the bait protein in each sample. In addition, other proteins were identified in each sample that enabled characterization of the Nitab4.5_0013685g0010.2 proteome (data not shown). Several interactors are RNA binding proteins. Such glycine-rich RNA binding proteins are known to bind RNA and interact with other proteins associated with RNA processing.

Claims

1. A method for regulating the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content of a tobacco plant or part thereof, or a tobacco cell or a tobacco cell culture, the method comprising modifying the tobacco plant or part thereof, or a tobacco cell or a tobacco cell culture by regulating the activity or expression of at least one gene encoding an RNA binding protein, wherein the RNA binding protein consists of the amino acid sequence shown in SEQ ID NO.

1.

2. Use of at least one gene encoding an RNA binding protein consisting of the amino acid sequence shown in SEQ ID NO.1 for regulating the content of nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine in tobacco cells, or tobacco plants or parts thereof, or tobacco cell cultures.

3. A method for producing a tobacco plant or part thereof, a tobacco cell or a tobacco cell culture having a modulated nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content, the method comprising modifying the tobacco plant or part thereof, or the tobacco cell or tobacco cell culture to modulate the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence shown in SEQ ID NO.

1.

4. The method of claim 3, comprising the step of obtaining plant propagation material from the tobacco plant or part thereof, or tobacco cells or tobacco cell cultures.

5. The method of claim 3, wherein the portion thereof is a leaf.

6. The method of claim 3, comprising the step of harvesting leaves from the plant.

7. A method as claimed in claim 5 or 6, comprising the step of machining the blade.

8. The method of claim 5 or 6, comprising the step of cutting the leaves.

9. The method according to claim 1 or 3 or the use according to claim 2, wherein the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content of the modified tobacco plant or part thereof, or tobacco cell or tobacco cell culture is reduced compared to a tobacco plant or part thereof, or tobacco cell or tobacco cell culture that has not been modified to reduce the activity or expression of at least one gene encoding the RNA binding protein, and the RNA binding protein consists of the amino acid sequence shown in SEQ ID NO.

1.

10. The method or use according to claim 9, wherein the part thereof is plant propagation material or leaves.

11. The method according to claim 1 or 3 or the use according to claim 2, wherein the nornicotine content is adjusted.

12. The following uses for plant cultivation: (1) A tobacco plant or part thereof, or a tobacco cell culture, which has been modified to achieve regulation of the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof, or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence as shown in SEQ ID NO.1; or (2) a tobacco plant produced by the method of claim 1 or 3.

13. The following are used to produce products: (1) A tobacco plant or part thereof, or a tobacco cell culture, which has been modified to achieve regulation of the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof, or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence as shown in SEQ ID NO.1; or (2) a tobacco plant produced by the method of claim 1 or 3.

14. The use according to claim 13, wherein the product is a tobacco industry product.

15. The following uses for growing crops: (1) A tobacco plant or part thereof, or a tobacco cell culture, which has been modified to achieve regulation of the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof, or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence as shown in SEQ ID NO.1; or (2) a tobacco plant produced by the method of claim 1 or 3.

16. The following are used for the production of blades: (1) A tobacco plant or part thereof, or a tobacco cell culture, which has been modified to achieve regulation of the nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof, or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence as shown in SEQ ID NO.1; or (2) a plant produced by the method of claim 1 or 3.

17. Non-viable processed tobacco leaves, which: (a) obtained by processing a tobacco plant which has been modified to achieve regulation in the content of nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine compared to an unmodified plant, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence shown in SEQ ID NO.1; or (b) obtained from a tobacco plant obtained by the use according to claim 2; or (c) a tobacco plant propagated from plant propagation material obtained from: (i) a tobacco plant as defined in (a); or (ii) a tobacco plant or tobacco cell culture produced by the method of claim 1 or 3; or (d) obtained by processing harvested leaves of: (i) a tobacco plant as defined in (a) or (c); (ii) a tobacco plant obtained by the use of claim 2; or (iii) a tobacco plant produced by the method of claim 1 or 3; or (e) obtained from a tobacco plant produced by the method of claim 1 or 3.

18. The non-viable processed tobacco leaf of claim 17, wherein the leaf is processed by curing, fermenting, pasteurizing, or a combination thereof.

19. The non-viable processed tobacco leaf blade of claim 17, wherein the content of one or more TSNAs selected from the group consisting of N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanatabine (NAB) is reduced.

20. The non-viable processed tobacco leaf blade according to claim 19, wherein the content of NNN and / or NNK is reduced.

21. The non-viable processed tobacco leaf blade of claim 19, wherein at least the NNN content is reduced.

22. The non-viable processed tobacco leaf blade of claim 17, wherein the processed tobacco leaf blade is a cut processed leaf blade.

23. A cured tobacco material or extract thereof, made from: (a) a tobacco plant or part thereof, or a tobacco cell culture, which has been modified to achieve regulation in the content of nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine compared to an unmodified tobacco plant or part thereof or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence as shown in SEQ ID NO.1; or (b) harvested leaves of: (i) a tobacco plant as defined in (a); (ii) a tobacco plant propagated from plant propagation material obtained from a tobacco plant as defined in (a) or a tobacco plant or tobacco cell culture produced by the method of claim 1 or 3; (iii) a tobacco plant obtained by the use of claim 2; or (iv) a tobacco plant produced by the method of claim 1 or 3.

24. A tobacco blend comprising the cured tobacco material of claim 23.

25. A tobacco delivery system product prepared by: (a) a tobacco plant or part thereof, or a tobacco cell or cell culture, which has been modified to achieve regulation of nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence shown in SEQ ID NO.1; or (b) a tobacco plant or part thereof propagated from tobacco plant propagation material obtained from: (i) a tobacco plant as defined in (a); or (ii) a tobacco plant or tobacco cell culture produced by the method of claim 1 or 3; or (c) harvested leaves of: (i) a tobacco plant as defined in (a) or (b); (ii) a tobacco plant obtained by the use of claim 2; or (iii) a tobacco plant produced by the method of claim 1 or 3; or (d) a tobacco plant produced by the method of claim 1 or 3; or (e) a cured tobacco material according to claim 23; or (f) The tobacco blend of claim 24.

26. The tobacco delivery system product of claim 25, wherein the tobacco delivery system product is a combustible smoking article.

27. The tobacco delivery system product of claim 25, wherein the tobacco delivery system product is a smokeless tobacco product.

28. The tobacco delivery system product of claim 25, wherein the tobacco delivery system product is a non-combustible gas aerosol supply system.

29. The tobacco delivery system product of claim 28, wherein the non-combustible gas aerosol supply system is a tobacco heating device.

30. The tobacco delivery system product of claim 28, wherein the non-combustible gas aerosol supply system is an aerosol generating device.

31. Combustible smoking articles, non-combustible gas aerosol delivery systems, or smokeless tobacco products, comprising: (a) a tobacco plant or part thereof, or a tobacco cell or cell culture, which has been modified to achieve regulation of nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content compared to an unmodified tobacco plant or part thereof or an unmodified tobacco cell culture, wherein the modification is regulation of the activity or expression of at least one gene encoding an RNA binding protein consisting of the amino acid sequence shown in SEQ ID NO.1; or (b) harvested leaves of: (i) a tobacco plant as defined in (a); (ii) a tobacco plant propagated from plant propagation material obtained from a tobacco plant as defined in (a) or a tobacco plant or tobacco cell culture produced by the method of claim 1 or 3; (iii) a tobacco plant obtained by the use of claim 2; or (iv) a tobacco plant produced by the method of claim 1 or 3; or (c) a non-viable processed tobacco leaf according to any one of claims 17 to 22; or (d) or a cured tobacco material according to claim 23; or (e) The tobacco blend of claim 24.

32. The combustible smoking article, non-combustible gas aerosol delivery system, or smokeless tobacco product of claim 31, wherein the non-combustible gas aerosol delivery system is a tobacco heating device.

33. Use of at least one gene encoding an RNA binding protein in selecting tobacco plants with modulated nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content, wherein the sequence of the RNA binding protein is SEQ ID NO.1, or wherein the at least one gene encoding the RNA binding protein consists of the nucleotide sequence as shown in SEQ ID NO.2 or 3.

34. The use according to claim 33, wherein the tobacco plant has a reduced nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content.

35. A method for producing a mutant of a tobacco plant carrying a heritable mutation in at least one gene encoding an RNA binding protein, the nucleotide sequence of the gene being selected from SEQ ID NO. 2 or 3; wherein the method comprises introducing a heritable mutation into the nucleotide sequence, the heritable mutation eliminating the activity of the RNA binding protein encoded by the at least one gene or eliminating the expression of the at least one gene encoding the RNA binding protein, and wherein the mutant of the tobacco plant has a reduced nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content relative to a comparable plant not carrying the heritable mutation.

36. The method of claim 35, wherein the heritable mutation that eliminates the activity of the RNA binding protein encoded by at least one gene is a deletion of the RNA binding domain and / or the RGG domain of the RNA binding protein.

37. The method according to claim 35, comprising the step of obtaining offspring from the mutant of the tobacco plant, wherein the offspring carries the heritable mutation according to claim 35 or 36.

38. The method according to claim 35, comprising the step of obtaining seeds from the mutant of the tobacco plant, wherein the seeds carry the heritable mutation according to claim 35 or 36.

39. Non-viable processed tobacco leaves produced by a tobacco plant, the tobacco plant comprising a modification in at least one gene encoding an RNA binding protein, the nucleotide sequence of the gene being selected from SEQ ID NO. 2 or 3, wherein the modification eliminates the activity of the RNA binding protein encoded by the at least one gene or eliminates the expression of the at least one gene encoding the RNA binding protein, and wherein the tobacco plant has a reduced nicotine, nornicotine, pseudooxynicotine, anabasine or anatabine content relative to a comparable plant that does not carry the modification in the at least one gene encoding the RNA binding protein.

40. The non-viable processed tobacco leaf blade of claim 39, wherein the non-viable processed tobacco leaf blade is a cured tobacco material.

41. The non-viable processed tobacco leaf blade of claim 39, wherein the modification that eliminates the activity of an RNA binding protein encoded by at least one gene is a deletion of an RNA binding domain and / or an RGG domain of the RNA binding protein.

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