Methods for reducing the alkaloid content of tobacco plants

Abstract

The present invention provides a method for regulating (e.g., reducing) the alkaloid content of a plant (e.g., a tobacco plant), the method comprising modifying the plant by regulating the activity or expression of at least one gene encoding a SOUL heme-binding protein. The present invention also provides the use of at least one gene encoding a SOUL heme-binding protein for regulating the alkaloid content of a plant, and tobacco cells, plants, plant propagation materials, harvested leaves, processed tobacco or tobacco products obtainable according to the present invention.

Description

Field of the Invention

[0001] The present invention relates to methods for regulating the alkaloid content, such as nicotine content, of plants or parts thereof. The present invention also extends to methods for regulating the expression and / or activity of polypeptides that regulate the alkaloid content in plants. Alternatively, 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 have been modified to achieve regulation of alkaloid content. The present invention also relates to processed and harvested leaves from such regulated plants and their use in tobacco industry products, including combustible smoking products.

[0002] background

[0003] 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.

[0004] Alkaloids can be classified based on similarities in their carbon skeletons, such as indole, isoquinoline, and pyridine. Pyridine derivatives are a class of monomeric alkaloids; this category includes simple pyridine derivatives, polycyclic condensed and non-condensed pyridine derivatives, and sesquiterpene pyridine derivatives. Examples include nicotine, nornicotine, anabasine, mithosamine, and anatabine.

[0005] 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 signal transduction cascades that monitor gene expression, enzyme activity, and alkaloid concentration. The fine regulation of alkaloid content in plants involves negative feedback loops and degradation pathways.

[0006] Nicotine occurs naturally in several plant species, but is found at its highest levels in tobacco plants. Cultivated tobacco produces 2-4% of the alkaloid by weight. Nicotine is present in both wild and cultivated Nicotiana species ( 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).

[0007] The regulation of alkaloid content in tobacco is complex. Several factors, including genotype, environment, fertilization, and agronomic practices (e.g., topping), 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 family of transcription factors 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.

[0008] The alkaloid content in the modification plant (for example tobacco) can have several commercial advantages.For example, reducing the total alkaloid content in the plant can increase the value of the plant as a biomass resource.For example, modifying the alkaloid content can comprise reducing the alkaloid content in the tobacco plant, for example nicotine content. In view of " nicotine ceiling (nicotineceiling) ", i.e. the potential regulation of the average nicotine upper limit in the tobacco product, the tobacco plant and the product with the nicotine reduced may be desired. Or, increase the alkaloid content in the plant for example tobacco plant, can help protect plants with anti-insect and herbivorous animals. Still need plant and preparation method thereof, the plant has the alkaloid content of regulation when there is the proterties expected commercially of improvement, for example, has the nicotine content of regulation.

[0009] During post-harvest leaf conditioning, reactions between pyridine alkaloids and nitrosating substances lead to the formation of tobacco-specific nitrosamines (TSNAs). Nornicotine and PON are precursors of the TSNAs NNN and NNK, respectively. Reducing the production and accumulation of nornicotine and PON is crucial. The CYP82E family of nicotine demethylase genes is one of the main regulators of the conversion of nicotine to nornicotine, and altering their activity or accumulation could lead to reduced NNN levels. However, to date, the enzyme or gene responsible for PON production has not been identified.

[0010] As described in the examples, the inventors sought to investigate the genes responsible for alkaloid synthesis with the aim of modulating the alkaloid content in plants, for example reducing the nicotine content in tobacco plants. SUMMARY OF THE INVENTION

[0012] Surprisingly, it has been discovered that by modulating the activity or expression of a gene encoding a SOUL heme-binding protein, the alkaloid content and / or TSNA content or TSNA precursor content of a plant can be modulated. A SOUL heme-binding protein, such as Nitab4.5_0013616g0010.2, as taught herein, is a regulator of nicotine, nornicotine, anabasine, PON, miosamine, and anatabine in cultivated tobacco. Nitab4.5_0013616g0010.2 encodes a SOUL heme-binding protein according to the present invention. Nitab4.5_0000652g0130.2, Nitab4.5_0001140g0220.2, Nitab4.5_0003235g0060.2, Nitab4.5_0004868g0020.2, Nitab4.5_0006614g0010.2, Nitab4.5_0006991g0050.2 and Nitab4.5_0009023g0010.2 encode homologues of Nitab4.5_0013616g0010.2 according to the present invention. The SOUL heme-binding protein according to the present invention contains a conserved three-dimensional structure called the SOUL motif. The SOUL heme-binding protein according to the present invention binds heme.

[0013] According to the present invention, tobacco products having regulated alkaloid content and commercially desirable traits sought after by consumers of tobacco products can be produced. In some cases, consumers may desire products with low levels of alkaloid content, such as low levels of TSNA or low nicotine content.

[0014] The present invention may be particularly useful in the field of plant molecular farming, where plants (such as tobacco and other Nicotiana species) are used to produce proteins, peptides and metabolites, for example for the production of therapeutics 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 participated in a tobacco-based platform for the production of virus-like particles for influenza vaccine manufacturing.

[0015] Thus, plants according to the present invention can be used in molecular farming to reduce or eliminate the presence of nicotine, nornicotine, PON and / or other nicotine alkaloids, such as anabasine or anatabine or mithosamine. The use of low-nicotine plants or roots is beneficial in molecular farming and will reduce downstream processing costs associated with purification.

[0016] In other cases, it may be desirable to produce plants with high alkaloid levels, such as high levels of nicotine content, so that nicotine can be purified from tobacco plants to produce pure nicotine products, such as for use in devices that utilize nicotine-containing liquids (e.g., e-cigarettes) or tobacco heating devices. For example, the production of plants with leaves containing high levels of nicotine can reduce the cost of nicotine extraction for the production of e-liquids for e-cigarettes.

[0017] The present inventors have surprisingly identified methods for modulating the alkaloid content (e.g., nicotine content) of a plant (e.g., a tobacco plant) by modulating the activity or expression of a gene encoding a SOUL heme-binding protein. The alkaloid content (e.g., the content of one or more of nicotine, nornicotine, anabasine, PON, miosamine, or anatabine) of a plant (e.g., a tobacco plant) can be reduced by reducing the activity or expression of a gene encoding a SOUL heme-binding protein or can be increased by increasing the activity or expression of a gene encoding a SOUL heme-binding protein (e.g., by increasing the heme-binding affinity of the SOUL heme-binding protein). Prior to the present invention, it was not known that modulation of the activity or expression of a gene encoding a SOUL heme-binding protein as described herein could be used to modulate alkaloid content.

[0018] In one aspect, a method of modulating (e.g., reducing) the alkaloid content of a plant or part thereof, or a cell or cell culture is provided, the method comprising modifying the plant or cell or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0019] In one aspect, a method of reducing the alkaloid content of a plant or part thereof, or a cell or cell culture is provided, the method comprising modifying the plant or cell or cell culture by reducing the activity or expression of at least one SOUL heme-binding protein.

[0020] In another aspect, a method is provided for modulating (e.g., reducing) the level of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant, or a plant part or cell thereof, the method comprising modifying the plant, cell, or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0021] In another aspect, a method is provided for reducing the level of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant, or a plant part or cell thereof, the method comprising modifying the plant or cell or cell culture by reducing the activity or expression of at least one SOUL heme-binding protein.

[0022] In a further aspect, there is provided the use of at least one gene encoding a SOUL heme-binding protein for modulating the alkaloid content of a cell or a plant or part thereof, or a cell or cell culture.

[0023] In yet another aspect, a method is provided for producing a plant or part thereof, cell or cell culture, plant propagation material, leaves, cut harvested leaves, processed leaves or cut and processed leaves having a modulated (e.g., reduced) alkaloid content, the method comprising modifying the plant or cell or cell culture to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0024] Suitably, the alkaloid content may be modulated (eg reduced) compared to a plant or cell or cell culture which has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0025] In a further aspect, there is provided a plant or part thereof, or a cell or cell culture, which has been modified to achieve modulation (e.g., reduction) of alkaloid content compared to an unmodified plant or unmodified cell or cell culture, wherein the modulation is a reduction in the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0026] In a further aspect, there is provided plant propagation material obtainable from a plant according to the invention, or a plant or a cell or cell culture produced by a method according to the invention.

[0027] In a further aspect, there is provided a method or use according to the invention, or a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention, wherein the alkaloid content of the plant is reduced compared to a plant or a cell or cell culture which has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0028] Suitably, the activity or expression of the at least one gene encoding a SOUL heme-binding protein may be reduced compared to a plant or cell or cell culture which has not been modified to modulate the activity or expression of the at least one gene encoding a SOUL heme-binding protein.

[0029] Suitably, the alkaloid content of the plant or cell or cell culture may be increased compared to a plant which has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0030] Suitably, the plant may be modified to increase the activity or expression of at least one gene encoding a SOUL heme-binding protein and the plant or cell or cell culture exhibits an increased alkaloid content compared to a plant or cell or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0031] Suitably, the total alkaloid content of the plant or cell or cell culture may be modulated (eg reduced).

[0032] Suitably, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anabasine, mithosamine and anatabine is regulated (e.g., reduced), preferably the content of nicotine, nornicotine and / or PON is regulated (e.g., reduced). In some embodiments, two or more (or three or more) alkaloids selected from nicotine, nornicotine, PON, anabasine, mithosamine and anatabine may be regulated (e.g., reduced). In some embodiments, the total alkaloid content of the plant or cell is regulated (e.g., reduced).

[0033] In one aspect, the plant or plant cell is from the Solanaceae family.

[0034] Suitably, the plant or plant cell may be from the genus Nicotiana.

[0035] Suitably, the nicotine content may be adjusted. Suitably, the nicotine content may be reduced.

[0036] In one aspect, a method or use according to the invention, a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention is provided, wherein the at least one gene encoding a SOUL heme-binding protein encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22 or a functional variant or functional fragment or orthologue thereof; or

[0037] wherein the at least one gene encoding a SOUL heme-binding protein encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19 or 22; or

[0038] wherein the at least one gene encoding the SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof; or

[0039] wherein the at least one gene encoding the SOUL hemoglobin-binding protein comprises a nucleotide 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 or 24.

[0040] In a further aspect, there is provided a method or use according to the invention, a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention, wherein an additional gene encoding a SOUL heme-binding protein is also regulated, wherein the additional gene encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22 or a functional variant or functional fragment or orthologue thereof; or

[0041] wherein the additional gene encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19 or 22; or

[0042] wherein the additional gene 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 or 24, or a functional variant or functional fragment or ortholog thereof; or

[0043] wherein the additional gene comprises a nucleotide 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 or 24.

[0044] In one aspect, there is provided use of a plant or part thereof, or a cell or cell culture according to the invention, or a plant produced by a method according to the invention, for growing a plant.

[0045] In another aspect, there is provided use of a plant or part thereof, or a cell or cell culture according to the invention, or a plant produced by a method according to the invention for producing a product.

[0046] In yet another aspect, there is provided 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.

[0047] In another aspect, there is provided use of a plant according to the invention or a part thereof, or a plant produced by a method according to the invention, to produce leaves.

[0048] In another aspect, there is provided harvested leaves of a plant according to the invention, or of a plant obtainable from a propagation material according to the invention, or of a plant obtainable from a use according to the invention, or of a plant produced by a method according to the invention.

[0049] Suitably, the harvested leaves may be cut harvested leaves.

[0050] In another aspect, there is provided a processed leaf, preferably a processed tobacco leaf, preferably a non-viable processed tobacco leaf, which:

[0051] obtainable from plants obtainable for use according to the invention;

[0052] Obtainable by processing the plant according to the invention;

[0053] obtainable from a plant propagated from the plant propagation material according to the invention; or

[0054] Obtainable by processing harvested leaves of a plant according to the invention; or

[0055] Obtainable from plants produced by the method according to the invention.

[0056] Suitably, the leaves may be processed by curing, fermenting, pasteurising or a combination thereof.

[0057] Suitably, the tooling blade may be a cut tooling blade.

[0058] In another aspect, there is provided a cured tobacco material made from a plant or part thereof or extract thereof according to the present invention.

[0059] In another aspect, there is provided a tobacco blend comprising the cured tobacco material according to the present invention.

[0060] In another aspect, a tobacco industry product is provided, which is prepared from:

[0061] a tobacco plant according to the invention or a part thereof, or a tobacco cell or cell culture according to the invention;

[0062] Tobacco plants or parts thereof propagated from tobacco plant propagation material according to the invention;

[0063] harvested leaves of a plant according to the present invention, wherein the plant is tobacco;

[0064] The processed leaf according to the present invention, wherein the plant is tobacco;

[0065] or

[0066] Plants produced by the method according to the present invention.

[0067] Suitably, the tobacco product may be a combustible smoking article.

[0068] Suitably, the tobacco product may be a smokeless tobacco product.

[0069] Suitably, the tobacco product may be a non-combustible gas aerosol delivery system, such as a tobacco heating device or an aerosol generating device.

[0070] In one aspect, there is provided use of tobacco cells according to the present invention for modulating alkaloid content in cell culture.

[0071] In another aspect, a combustible smoking article, a non-combustible gas aerosol delivery system, a smokeless tobacco product or a tobacco heating device is provided, which comprises a plant or part thereof or an extract thereof (e.g., a tobacco extract) according to the invention, or a tobacco cell or cell culture according to the invention; or a modulated tobacco material according to the invention; or a tobacco blend according to the invention.

[0072] In one aspect, there is provided the use of a nucleotide sequence of at least one gene encoding a SOUL heme-binding protein to select plants having a modulated (e.g., reduced) alkaloid content and / or a modulated (e.g., reduced) content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors, preferably, wherein the sequence of the SOUL heme-binding protein is selected from SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22 or a functional variant or functional fragment or ortholog thereof; or wherein the sequence of the SOUL heme-binding protein is a sequence having at least 80% identity to SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a sequence of SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene encoding the SOUL hemoglobin-binding protein comprises a nucleotide 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 or 24.

[0073] In another aspect, a mutant of a plant is provided that carries a heritable mutation in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, preferably wherein the 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 or 24 or a functional variant or functional fragment or ortholog thereof; or wherein the gene is selected from the group consisting of sequences having at least 80% identity to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; wherein the heritable mutation modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the mutant plant has a modulated (e.g., reduced) alkaloid content and / or a modulated content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs relative to a comparable plant not carrying the heritable mutation.

[0074] In another aspect, progeny or seeds of a mutant plant carrying a heritable mutation according to the present invention are provided.

[0075] In another aspect, there is provided a harvested leaf, processed leaf or cured tobacco material produced from a plant comprising a modification in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, wherein the at least one gene is selected from SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene is selected from the group consisting of SEQ ID No. No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 having a sequence with at least 80% identity; wherein the modification modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the plant has a modulated (e.g., reduced) alkaloid content and / or a modulated tobacco-specific nitrosamine (TSNA) or TSNA precursor content relative to a comparable plant not carrying the modification in the at least one gene encoding a SOUL heme-binding protein.

[0076] In another aspect, there is provided a method, leaf, plant, plant propagation material, harvested leaf, processed tobacco, tobacco product, use, or combination thereof as described herein with reference to the specification and figures. Sequence Listing <110> British American Tobacco (Investments) Limited <120> Method for reducing the alkaloid content of tobacco plants <130> P116498PCT <150> GB 1900940.6 <151> 2019-01-23 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 167 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 1 Met Leu Gly Lys Tyr Gly Phe Asp Phe Asn Gly Ala Ser Gln Ser Phe 1 5 10 15 Asn Thr Leu Ala Glu Tyr Leu Phe Gly Lys Asn Thr Lys Lys Glu Ser 20 25 30 Met Ala Met Thr Thr Pro Val Ile Thr Arg Arg Thr Gln Ser Asp Gly 35 40 45 Glu Lys Met Glu Met Thr Thr Ser Val Ile Thr Lys Arg Val Glu Asp 50 55 60 Gln Gly Lys Trp Arg Met Ser Phe Val Met Pro Ser Lys Tyr Gly Ser 65 70 75 80 Asp Leu Pro Leu Pro Lys Asp Ser Ser Val Thr Ile Lys Glu Val Pro 85 90 95 Arg Lys Thr Val Ala Val Val Ala Phe Ser Gly Phe Val Thr Asp Glu 100 105 110 Glu Val Lys Ala Arg Glu Ser Arg Leu Cys Ala Ala Leu Lys Gly Asp 115 120 125 Ala Glu Phe Arg Val Lys Asp Gly Ala Ser Ile Glu Val Ala Gln Tyr 130 135 140 Asn Pro Pro Phe Thr Leu Pro Phe Thr Arg Arg Asn Glu Ile Ser Leu 145 150 155 160 Glu Val Glu Arg Lys Gln Glu 165 <210> 2 <211> 2592 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 2 taatattgtg actttaaaca gccttacttt gcggcggagg ctacaatgct tgggaaatat 60 gggtttgatt ttaatggtgc atctcaatcc ttcaacacat tggcagagta cttgtttggt 120 aaggtagtta aaggtactta tagagagtca aacttagatc gcctccaacc acattgtaat 180 ggtgttcttc tttggttatg ttctctttct tgatgatttc cagaacacaa agaaggaaag 240 tatggcgatg acaacacccg taatcactcg tagaactcaa tctgatgggg agaagatgga 300 aatgactact tcagtgataa ctaaaagggt acagatcgtc ttctggtcat tcctttttcc 360 cttttagttc ttgtgctgtg tatattact acgtcattcg tgacatatgg ttggttact 420 aatgcgcatg cattttgta tgacatggtg ttactcaaaa tattaactgc taatctattg 480 aagttgagtc accacataac ttgggaatg gtaaatgaaa actactcta aaatgaccac 540 aggtcgtcaa atggaatac agactcact gattctattg aaagaaaca tcatctggtt 600 acctatcaca aggaaaacat ttaagctt gatctggaat tgataaaga ggacatgtgc 660 tttagattga gatttgagga tcttcatt gtatgctagg cctatacgcc gggggatggg 720 ggtacatata aaacgtaaac attctggatg cttatagttt aaacctgtaa tactctcaga 780 tagtattgca aatccatatc tgggagtg gaaactgtta gattccaggt ttgattgt 840 tgcatggatc cttcaacaa gtgtgacatg ggtgtgggta cttcttgcag gttcttcaa 900 gtatactaat attaggaaa aaatgcaca aactcctat cagatactta cacacacaca 960 aacacacaca catggtatat acctataggt ttgtccatat atgcgatat ctaatcttag 1020 tcgaaca gtgtcgaatg gaataacagt cacaaca gatcttctta atgataagg 1080 ttatggtacg aataaaatat acacccctat ataaacgata acacattttc ttgtaattct 1140 taagttaatt tctgagaata acattttgta acataaaaaa ccaattttat gtgaaaatgg 1200 gccctccata tagattggca tcctataaga attctttact cccagaaacc ttgaaaccgg 1260 atttagtcca ttctagaaaa gaaaaactca tttagcttg atgtatgatc ctatgttata 1320 gggcttaaac ccagaagaaa aaaagataaa cattgtgtat atagtttttg tatttcggaa 1380 tgtcgtgcgc acatattttc accaaaaaat atacccactg gaatttcatt aagaatttca 1440 ttaggtggaa gatcaaggaa agtggaggat gtcctttgtc atgccctcaa agtatggttc 1500 ggacttgcca ctaccaaagg attcctccgt aactatcaag gaggtgccta ggaaaactgt 1560 cgccgttgtt gctttttcag gtttgtgatt tcagatttta gcatgtcaat ataaagttct 1620 attttatatg atgatgatat gagttgagct tatcgaaaga agtgaggatt catagcgccg 1680 accccaactt tgtgggactg aggcatagtt gttgctgtta atacaaagtt ctagacagat 1740 ttgcattaaa gtttatttga aattcaatat cctagtaatt ggaagccgag ctctgtctca 1800 ttggtctata gaagtaatc ttgtttaagt ttacttagtg ctttattgat agcttctctt 1860 gttttgatat gagagaaact tgcatctagt tgtttttct gtgcctttcc tttgacgtgc 1920 tatatgcctg ttgtggtaga tagcaatttc cattcagagt tcaaaagcaa aaacatttag 1980 gaattctatt ttcaagttt atatcctaac ttagctagct ttctcccatt tgattcacca 2040 aaggttttgt gactgatgaa gaagttaaag cccgagaatc aagactatgt gctgcgttaa 2100 agggagatgc agagtttcgg gtaaaagatg gtgcctcgat agaagttgca caggtatatt 2160 cgtagaacca tttcaatagt acttgtaatt ctcctgggga acagattttg gcctagtgta 2220 acccccaagt gacatactag ttgtgttttt tttatacttc tgcttgcagt ataatccacc 2280 attcactctt ccattcacac gtcggaatga gattagcctg gaagttgaaa ggaaacagga 2340 atagctagtc agggtgcaac atcttgcaga taaatccgtg cactgatata tagaaccag 2400 tgaagcaaaa catatatggc gtaaatatat tgtgttgata tactccttgt atatatatat 2460 atgtatatat atatatttgg tgattgcttg acattttggt aacaaggtta tgtacatgca 2520 cagaaatgta aggtcactta tgcttcaact ctgaatataa taacactgtc tttaatactc 2580 ttctcgatcc tc 2592 <210> 3 <211> 504 <212> DNA <213> Nicotiana tabacum <400> 3 atgcttggga aatatgggtt tgattttaat ggtgcatctc aatccttcaa cacattggca 60 gagtacttgt ttggtaagaa cacaaagaag gaaagtatgg cgatgacaac acccgtaatc 120 actcgtagaa ctcaatctga tggggagaag atggaaatga ctacttcagt gataactaaa 180 agggtggaag atcaaggaaa gtggaggatg tcctttgtca tgccctcaaa gtatggttcg 240 gacttgccac taccaaagga ttcctccgta actatcaagg aggtgcctag gaaaactgtc 300 gccgttgttg ctttttcagg ttttgtgact gatgaagaag ttaaagcccg agaatcaaga 360 ctatgtgctg cgttaaaggg agatgcagag tttcgggtaa aagatggtgc ctcgatagaa 420 gttgcacagt ataatccacc attcactctt ccattcacac gtcggaatga gattagcctg 480 gaagttgaaa ggaaacagga atag 504 <210> 4 <211> 199 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 4 Met Gly Met Ile Leu Gly Lys Ile Cys Val Glu Thr Pro Lys Tyr Glu 1 5 10 15 Leu Ile Gln Ser Thr Ala Asp Tyr Glu Ile Arg Lys Tyr Pro Ala Ser 20 25 30 Val Ile Ala Gln Val Thr Tyr Asp Pro Thr Gln Phe Lys Gly Asn Lys 35 40 45 Asp Gly Gly Phe Met Leu Leu Ala Asn Tyr Ile Gly Ala Leu Gly Asn 50 55 60 Pro Gln Asn Ala Lys Pro Glu Thr Ile Ala Met Thr Ala Pro Val Ile 65 70 75 80 Thr Lys Ser Ser Glu Lys Ile Ala Met Thr Ala Pro Val Val Thr Lys 85 90 95 Asn Gly Asp Gly Glu Ser Asn Met Val Thr Met Gln Phe Ile Leu Pro 100 105 110 Ala Lys Tyr Thr Lys Ala Glu Glu Ala Pro Lys Pro Leu Asp Glu Arg 115 120 125 Val Met Ile Lys Glu Glu Gly Glu Arg Lys Phe Gly Val Val Gln Phe 130 135 140 Ser Gly Thr Ala Ser Asp Lys Ala Val Lys Glu Lys Val Glu Asn Leu 145 150 155 160 Arg Lys Cys Leu Glu Arg Asp Gly Tyr Lys Ile Ile Gly Asp Tyr Glu 165 170 175 Leu Ala Arg Tyr Asn Pro Pro Trp Thr Ile Pro Pro Phe Lys Thr Asn 180 185 190 Glu Val Met Ile Pro Val Glu 195 <210> 5 <211> 1204 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 5 ctgatttcgt aatgcagatt ggtttaacct tttacagagc aagtcaattt taacggcgat 60 actaaaatta ctgtgtgcac aaatagtgca caaactattc ctgtataggt ttatctccac 120 cagcttatgg tgtaaacgtg gcaaaacaaa ctccacagca tacttccctg atctgccgcc 180 actcattttg cagcaaacaa aacccctttc ccaagtcttc tgatcgtctc tttccgaatt 240 cccacatgaa acttcctctt ttcttccctt atatgtaaca attcacagcc cccatttact 300 atcaaaacat tctcaaattc cattttcaat aactaatttc ccattccatc acaaaaatgg 360 gcatgatttt gggtaagatc tgtgtggaaa caccaaaata cgagttgatt caatctacag 420 ctgactacga aatccgcaaa tacccagcat ctgttatagc aaagtcaca tatgatccaa 480 cccagttcaa aggaaaaa gacggtgggt ttatgctatt agccaattac atcggcgcac 540 tgggcaatcc tcaaaacgct aagcctgaaa caatcgccat gacagctcct gtaatcacca 600 aatcgtccga aaaaatcgcg atgactgcac cagatagtgac taagaatggt gacggagaga 660 720 ctaagccgtt ggatgagaga gtgatgatta aagaagaagg tgaaaggaag tttggtgtgg 780 tgcagtttag tgggactgca agtgataaag cggtaaaaga gaagtggag aatttaagga 840 aatgtttaga gagagatggg tataagaataa ttggtgatta tgagttggct aggtataatc 900 ctccttggac aattcctcca tttaagacca atgaagttat gattccagtt gagtgagctg 960 1020 actttatctg gtgtttatta ttgaattgtg tatttcttgt ttatatgttt acgctgttga 1080 caatgtcttt gcatacacta gtgtcaatta ttcatagtta taattagcac atattttgtc 1140 ttatttttat actttgccat agtaaagcag taaaactgat gcagttttgc aagcatctaa 1200 tgtg 1204 <210> 6 <211> 600 <212> DNA <213> Nicotiana tabacum <400> 6 atgggcatga ttttgggtaa gatctgtgtg gaaacaccaa aatacgagtt gattcaatct 60 acagctgact acgaaatccg caaataccca gcatctgtta tagcacaagt cacatatgat 120 ccaacccagt tcaaaggaaa caaagacggt gggtttatgc tattagccaa ttacatcggc 180 gcactgggca atcctcaaaa cgctaagcct gaaacaatcg ccatgacagc tcctgtaatc 240 accaaatcgt ccgaaaaaat cgcgatgact gcaccagtag tgactaagaa tggtgacgga 300 gagagcaata tggtgacgat gcagtttatt ttacctgcaa agtatacaaa agctgaagag 360 gcacctaagc cgttggatga gagagtgatg attaaagaag aaggtgaaag gaagtttggt 420 gtggtgcagt ttagtgggac tgcaagtgat aaagcggtaa aagagaaagt ggagaattta 480 aggaaatgtt tagagagaga tgggtataag ataattggtg attatgagtt ggctaggtat 540 aatcctcctt ggacaattcc tccatttaag accaatgaag ttatgattcc agttgagtga 600 <210> 7 <211> 230 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 7 Met Gln Gly Val Met Asn Phe Phe Thr Arg Leu Ser Phe Phe Leu Ile 1 5 10 15 Leu Val Ser Lys Leu Ser Asn Gly His Trp Thr Asn Asn Leu Lys Leu 20 25 30 Asp Phe Tyr Pro Pro Thr Cys Asn Arg Ile Glu Cys Pro Asn Tyr Asp 35 40 45 Leu Ile Gln Ser Gly Lys Asp Tyr Glu Ile Arg Arg Tyr Asn Ser Ser 50 55 60 Met Trp Met Ser Thr Ala Pro Ile Asp Asp Ile Asn Leu Tyr Ser Ala 65 70 75 80 Thr Arg Thr Gly Phe Leu Arg Leu Phe Asp Tyr Ile Gln Gly Lys Asn 85 90 95 Ser Tyr Gln Glu Lys Ile Glu Met Thr Ala Pro Val Ile Thr Gln Val 100 105 110 Lys Pro Ser Asp Gly Pro Phe Cys Ala Ser Ser Phe Val Val Ser Phe 115 120 125 Tyr Val Pro Lys Lys Asn Gln Pro Asn Pro Pro Pro Ala Lys Gly Leu 130 135 140 His Val Gln Lys Trp Ser Asn Thr Tyr Val Ala Val Arg Gln Phe Gly 145 150 155 160 Gly Phe Val Ala Asp Val Asp Val Ala Lys Glu Ala Ala Ala Leu Ser 165 170 175 Ala Ser Ile Ala Asp Thr Lys Trp Ala Ala Ala Val Glu Lys Ser His 180 185 190 Ala Ala Asp Asn Thr Thr Met Tyr Thr Val Ala Gly Tyr Asn Ser Pro 195 200 205 Phe Glu Phe Lys Asp Arg Val Asn Glu Ile Trp Phe Thr Phe Asp Leu 210 215 220 Asp Lys Ala Ser Ala Ile 225 230 <210> 8 <211> 1522 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 8 taccaattca ttctggtcag ttcatctctt tccctataat ttctgctgca attaaaccac 60 CAAAAAAAAA CAAAgatgc agggcttt gatttctc acgaggctat ctttctttct 120 catccttgtt tctaaattaa gcaatgggca ttggacaac aatttgaat tggatttcta 180 tcctccaacc tgtaccgaa tgagtgccc aaattatgac ttgattcaat ctggtaaaga 240 ctatgaaatt cgtcgttaca attcatccat gtggatgtct actgcaccca ttgatgatat 300 taatctttat tccgccacca gaactggttt cctcaggtat ctatggtttt ttttctctt 360 tttggtcatg aattccttc ggttgtgttg catatttgga tacgttagct actgtgcaat 420 accaattaaa gcatgtctgt tttctgtcca agattcctta agaaaacac taagggttac 480 ttacatttca attackgaa attackacgac tattaaaga agagaa aaaaaaaaga 540 tagttatctg windowtt gacttatata tgctgataat gtaagtatt cattcact 600 tttcaagca cgtaacctgc gtggttttc aagatagtaa tctcacttt tatggatgaa 660 tactaatctc acttttg gatatgatta ttgaagcgt aaagttctt ttgcactgtt 720 agtcagtaga atattttcgg gtgagcttgg ttttattact caaatgtta gtgtatctat 780 tcgcgaaaac ataaaagggg tcgttgtgaa ctttgccttg gcaggctatt cgattacatt 840 caagggaaga acagttacca ggagaaaata gagatgacag ctccagttat cactcaagta 900 aaaccaagtg atggaccatt ttgtgcatct tcatttgttg tgagcttcta tgtaccaaag 960 aagaaccaac caaatcctcc tccagctaaa ggccttcacg tccaaaaatg gagcaatact 1020 tatgtggccg tcaggcaatt cggcggattt gtagctgatg ttgatgttgc aaaagaagct 1080 gctgctttga gtgctagtat tgctgacact aaatgggcag ctgctgttga aaaaagccat 1140 gctgcagata acactaccat gtatacagtg gcgggataca actctccatt tgagttcaag 1200 gacagagtta atgagatttg gtttactttt gatttggaca aagcatctgc catttgagag 1260 gctcttattc tggactcttt aacatttcag gagacatgaa actatcttat tttcttgggt 1320 ttgtattatt aaacagcagt tttgtatgac cacaacgcta cattatatat agcaaataag 1380 ataggttgta taatacaatc tgattctgag gatttagtgg ttaagatacc tctttcaaga 1440 cctttcaaaa taatccgata tatatggaaa agggctagac catattgagt ttattgtatg 1500 tacgcagttt taccttccat tt 1522 <210> 9 <211> 693 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 9 atgcagggcg ttatgaattt cttcacgagg ctatctttct ttctcatcct tgtttctaaa 60 ttaagcaatg ggcattggac aaacaatttg aaattggatt tctatcctcc aacctgtaac 120 cgaattgagt gcccaaatta tgacttgatt caatctggta aagactatga aattcgtcgt 180 tacaattcat ccatgtggat gtctactgca cccattgatg atattaatct ttattccgcc 240 accagaactg gtttcctcag gctattcgat tacattcaag ggaagaacag ttaccaggag 300 aaaatagaga tgacagctcc agttatcact caagtaaaac caagtgatgg accattttgt 360 gcatcttcat ttgttgtgag cttctatgta ccaaagaaga accaaccaaa tcctcctcca 420 gctaaaggcc ttcacgtcca aaaatggagc aatacttatg tggccgtcag gcaattcggc 480 ggatttgtag ctgatgttga tgttgcaaaa gaagctgctg ctttgagtgc tagtattgct 540 gacactaaat gggcagctgc tgttgaaaaa agccatgctg cagataacac taccatgtat 600 acagtggcgg gatacaactc tccatttgag ttcaaggaca gagttaatga gatttggttt 660 acttttgatt tggacaaagc atctgccatt tga 693 <210> 10 <211> 386 <212> PRT <213> Nicotiana tabacum <400> 10 Met Ala Thr Ser Gln Leu Ser Asp Leu Ile Phe Arg Pro Ser Leu His 1 5 10 15 Arg Arg Thr Asn Phe Arg Gln Cys His Pro Thr Ser Val Phe Leu Thr 20 25 30 Pro Pro Lys Asn Ile Lys Thr Lys Thr Leu Lys Tyr Asp Arg Lys Ile 35 40 45 Lys Trp Leu Ile Lys Phe Ser Leu Val Asp Lys Gln Ser Pro Thr Lys 50 55 60 Lys Pro Thr Val Asp Met Asn Gln Leu Val Glu Phe Leu Tyr Glu Asp 65 70 75 80 Leu Pro His Leu Phe Asp Asp Gln Gly Ile Asp Arg Lys Ala Tyr Asp 85 9� 95 Asp Tyr Val Lys Phe Arg Asp Pro Ile Thr Lys His Asp Ser Ile Asp 100 105 110 Gly Tyr Leu Phe Asn Ile Ala Met Leu Lys Gln Leu Phe Lys Pro Asp 115 120 125 Phe Gln Leu His Trp Ala Lys Gln Thr Gly Pro Tyr Glu Ile Thr Thr 130 135 140 Arg Trp Thr Met Val Met Lys Phe Ile Leu Leu Pro Trp Lys Pro Glu 145 150 155 160 Leu Val Phe Thr Gly Thr Ser Val Met Gly Val Asn Pro Glu Thr Asn 165 170 175 Lys Phe Asn Ser His Val Asp Tyr Trp Asp Ser Ile Lys Asn Asn Asp 180 185 190 Tyr Phe Ser Val Glu Gly Leu Leu Glu Val Ile Lys Gln Leu Arg Ile 195 200 205 Tyr Lys Thr Pro Asp Leu Glu Thr Pro Ser Tyr Gln Ile Leu Arg Arg 210 215 220 Thr Ala Thr Tyr Glu Val Arg Lys Tyr Asp Pro Phe Ile Val Val Glu 225 230 235 240 Thr Glu Gly Asp Lys Leu Ala Gly Asn Arg Gly Phe Asn Asp Val Ala 245 250 255 Gly Tyr Ile Phe Gly Lys Asn Ser Ala Thr Glu Lys Ile Pro Met Thr 260 265 270 Thr Pro Val Phe Thr Gln Ala Phe Asp Ala Glu Lys Ser Lys Val Ser 第275位 第280位 第285位 Ile Gln Ile Val Leu Pro Ser Asp Lys Ser Leu Ser Ser Leu Pro Ala 第290位 第295位 第300位 Pro Asn Gln Glu Gly Ile Ser Leu Arg Lys Thr Glu Gly Gly Ile Ala 第305位 第310位 第315位 第320位 Ala Ala Leu Lys Phe Ser Gly Lys Pro Thr Asp Asp Val Val Arg Glu 第325位 第330位 第335位 Lys Glu Lys Gln Leu Arg Ser Ser Leu Ile Lys Asp Gly Leu Lys Pro 第340位 第345位 第350位 Gln Ser Asp Cys Met Leu Ala Arg Tyr Asn Asp Pro Gly Arg Thr Trp 第355位 第360位 第365位 Lys Phe Ile Met Arg Asn Glu Val Leu Ile Trp Leu Glu Asp Phe Lys 第370位 第375位 第380位 Leu Asp 第385位 <210> 11 <211> 6662 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 11 aatggccacc tcacaacttt ccgacctcat tttccggcca tcacttcaca ggcgaaccaa 60 tttccggcaa tgccacccta cttcagtctt tctcactcct ccaaaaaata tcaaaacaaa 180. aactcttaaa ttgacagaa aaattaagtg gttaattaag tttagtttag ttgataaaca gagcccaacc aaaaaacca cagttgatat gaccatta gtggaatttt tatatgagga tttacctcat ctttttgatg atcaaggtat tgatcgtaag gcatatgatg attatgtgaa gtttagggat ccaattacaa aacatgattc aattgatggc tatttgttta atattgccat gttgaaacag ttgtttaagc ctgattttca gctgcattgg gctaaacagg ttagtatttc 420 tacccttctt gttaattggt tagttaaact tccttttgttg ggagcatggc gtaattagta 480 gttgccaacg ggttcaagcc atgaaaaccg actcttgcat aaatgcgagg tagtccaaa tgatcgaatt gtagttctta acaatagtag gtcgagatga tcgaatggaa atgacaattt ttacatggca tattttcata cattctggtc ttcagtctct tacataatt aagaacattg aataatatg tcaaggagtc gtttgaacat tatttttttt ccttctatcc aaaagacgta ttttcaagcc atgtttggtt atcaaattgg actgaacttt tgtgtttttt ttttttttga aaatgagttt taagtttgaa gtaaaaattc actttctctt acaaaactgt aatatttttt 840 caagtgaaat acatgtccaa acataacttt aaatttcaaa tattattttt caacttaact 900 ccgatccaaa cactactttg ttttctttta gaaatcacat tttatgtcc taagcgtcta 960 ctaaatgtct catccgaagc tcatgagact atcaacaact gtgaccatca ccttcatcga 1020 tatatttca cagtgaattt ctacactata aatgaaattt aacaggctat aagatgtcaa 1080 actcgttatg tagatcaatt gttgtaagtc aatataacct aattgttcaa ctatctatac 1140 gttatcagtt tacataatct ttacctgtag tttaacttct attaactgtt ttagtgtata 1200 tagttgtaac gtataactgt ctaaacttca tgttaatgat agcatttact tgtagatgat 1260 attattcatt tatcttgttc gttagtgatt aaattttgat cactcttcta agctttctac 1320 cgttaatgct ggtcgataaa aattccttca aaagttcact agtacgttaa gcacttaagc 1380 ctagacagtt gacattaata gactactaat actcacatga tagacaaaac tattttataa 1440 tttgctgatt atatataata atcatgtcat gaccaattat atatggaatt atcaatacaa 1500 tcacaattca catcattatg tgaggtaat tccacta gaaaactgtc aaaaaagcat 1560 ccagagcata ccgacagaaa ttgattggaa ataataccga ctgacatcgg tcagaaacga 1620 agtaaattgg tcgaaaatt aaaaaca attgtaata aaataccga ccgcaattag 1680 tagaaaaatg tgatcccaca aaaaagaaag tttacttat ctgacactgt taaaattttc 1740 gaccgattac gtcggaaaa tgatcaattt tttaccaag cccgatcaga cttgcatact 1800 atcaatata gttttagaa gtttattttt ctttttagat ttgtatcgaa ttagttaga 1860 tttgtgtcga attagatttg cccggagata atgaattaa aattaatca aaagaagttc 1920 agaatagaaa acttaacca acctcgatat tttaattata acttatactt aaattaaag 1980 aatttaaaaa gaaaaaaaaaaatgagg gacctgcaat aaatcacaac caatcctttc 2040 tatggtcaat attack cgtaagccaa agacatcctt ttacacatg siactaaaa 2100 acatcactac taaaaacaaa tggccacctc acatcactc acatcactt tccggctc 2160 acttcaccgg cgcaccactt tccggcaatg acttcacctc acttcctc acttcctc 2220 aaaaaatatc aaaaaaaa ctcttaaata tgacagaaaa atcaagtgtt taattaagtt 2280 tagtttagtt gataaacaga gcccaaccaa aaaaccaaca gttgatatga accaattagt 2340 ggaattttta tatgaggatt tgcctcatct ttttgatgat caaggtattg atcgtaaggc 2400 atatgatgat tatgtgaagt atagggatcc aattacaaaa catgattcaa ttgatggcta 2460 tttgtttaat attgcaatgt tgaaacagtt gttaggcct gattttcagc tgcattgggc 2520 taaacaggtt agcatttctg cagtttcttg tgaattggtt gtgtggctat gaggtgccta 2580 aagtaaaaat agcacgggct agccaatttt cggattggta attgaaaaat agtcaacgtt 2640 tgcaaagtca ttgaaaaata gccactattt tgctgcaaca tggaaagtta caacataata 2700 tactggagat tagtgcacat gtgtatgaac tttcagcata atatactgga gattggagga 2760 gcacatgtgt atgaactttc agcaccatga tattatgctg gaactccagt atattatgct 2820 ggaagttcat aggtaaaaaa ttcgaactcc agtatattat gctggaatat tttcggatt 2880 ttgaataggg tttcgttca gatttatctt tacatgaaaa gtggctaagt tttgattact 2940 tttgaaaatg tggctatttt ttaattacca cttgtaaatc tgactatttt tgaatttaac 3000 ccgtgcctaa aagatggagt aggatttaag ttatatacgc cgtcatgtaa aggttattat 3060 tatactcagt ggatataaac ttgtgggttg attacaatat ttagcagatt actagttaat 3120 acttactaga gattaacatg taattactag ttaataagtg acctcattgt ttaaatattt 3180 ttataccatt aatggataga atttcaactc taaaggataa ctatacctaa tataggtgac 3240 tattagggtg cctaagaagc caccacacta attgtcctta gaaagttgta ttagtaatct 3300 ggataacaag gcagattacc tattacgaca agttaaaata taccgatagt gtgtgtatat 3360 aagttaaata aattggttgt ttaaactctc tgtttttta tcgcttttcg ttcttcttat 3420 tatgttttcg gttggtttaa ggtgttgcac aaatattcaa gaacatagta tcaccccttat 3480 acatagtaca tttactgctc tttgaatatc agtccactgt ttgttaaatg cagaggcaca 3540 gatgaactga tgaactgctt ctgattgttt aagtgtgtca tttcattaag tgaagaattt 3600 tacatgttga gtattatgta gtttggtatt ggggcagaag ttgcatctaa tctgtttgtt 3660 ttgtgctact aaacttaatt gcgtattagt gtctcatggt tttgcatatc catttaagga 3720 taccgataca tgtagtgtac tatgtaggtt ctctttttca aaatttagtt tggaaacagc 3780 ctctctatcc tcacaagata ggggtaaggt ctgcgtatat gctaccctcc ccagatccca 3840 cggtgtggga taatactggg tatgttattg ttgttgttgt agtttggtat tggagcagaa 3900 gttttattta atcatttaat cagtttgctt tgagctacta aacttgtatt tttaaggaat 3960 gtgatgcata gattatgcat ttgaatcttt tgagtgattg agtttgttgt agacaggacc 4020 ctatgaaata actacaaggt ggacaatggt aatgaagttc attcttcttc catggaaacc 4080 tgaattagtc ttcaccggca cttctgttat gggcgtcaat cctgaaacaa acaaatttaa 4140 cagccatgtg gtttgattct tcactcttcc tcgacgcgaa ttcaaatttt tagattgctt 4200 tgcgaattag tcaccgactc tttgttgaaa tctataggac tactgggatt caattaagaa 4260 taacgattat ttttctgtag aaggtctgct ggaggtcata aaacaggtgc gcgttcgaac 4320 tgttttagtt tatgctttta attaaattct tggactgttg ggataaaccg gcccaatgat 4380 actcttaaca tggtgtgata ttgtccgctt ggggccaagc ccgcacggtt ttcactaaaa 4440 gggctcgtac cattaagaga tccttacacc tatatatataa gctcccaata tttcagcta 4500 ccaatgtcgc cttattatga tttgagata caatcttgg gcagttaagg atttacaga 4560 ctccggattt ggaaacacct agttatcaga tattagaag aacaccact tacgaggtttt 4620 gtagtattta tgttataca tcgtattttc aagctaagaa cttctatatt aggcgtacat 4680 ttttattgg ttgcgctac tttagctga actgatgcac taggacgtttt agatttatga 4740 tgtggaatgg tcacacttt cattggtgca atacaggtca ggaaatatga tccgtttata 4800 gttgtagaaa cagaaggtga caaaccgca ggaatagag gtttcaatga tgttgcgggg 4860 tgagataagt tgactatttt tttaaaga aaaaaacttc tgtgatgttt tctgatttag 4920 attggtagac tgttaaccg tcttatata ggtacatatt tggcaaaat tcagcgacag 4980 agagatacc gatgactact cctgtcttca ctcaggcatt cgatgctgaa aaatctaaag 5040 tgtcaatcca gatagttctt ccatcggata aatcttgag caggtaacac ttacacgtt 5100 gtattaataa tatacaagaa aacatctcac actgagtgaa aacagtatgg aatctttaaa 5220. tattcttagc actgatctga ttatactttt tgacaatatg gtttcacatt ttaacaattt gttgaaactt agtggctttt catatatata agtcatcata agcatcgccc gcctatgtgt 5280. aagagttgga ctttagtcct tttctttgtt caaatgagac ctatggaaat tattgcctta ttcttaacag cttaccagct cccaatcaag aaggtataag cttaaggaa acggaaggag gcatcgctgc tgcattaaaa tttagtggaa aaccaactga tgatgttgtt cgagaaaagg agaaacaact cagatcaagt cttattaagg acggtctcaa acctcaatcg gattgcatgc ttgctcgcta caatgatcct ggtcgaacgt ggaagtttat aatggtatat ttctatgtac 5580. tgataatgtt cctttcattg cattcctgct tagttcttc ttttgcttta gactttaga aaaggaatct tcttttgctc tttctatttc tgttttcaat tatcagtgcc ttcattacca agggcggagc tagtatacta tgtacggttt cggcagaacc cactaacttt agctgaaacc atgtattttg ttaaaaata aaaccactca atgtattataa ttaattttatt cagaacccag taagtcagcc ttttctataa taagacccca taaacttaaa atcttagctc ggcctctgtt 5880 cactactgaa tattttgata ttccaactag tttgagattg aggcataggt gattgattga 5940 tcaataaaat tagttgaca ttatgtttgg tatattatct actgctaata ttttctcatc 6000 tgatggaagg ttttgttgg tgtttgcatt atattttatg atcctttggg tgacaagagt 6060 gggttgctct agtggtgagc accctccact tccaaccaag aggttgtgag ttcgagtcac 6120 cccaagaaca aggtggggag ttcttgtcgg gagggagccg agggcttatc gaaaacagcc 6180 tttctacccc agggtagggg taaggtctgc gtacacacta ccttccccat accccactag 6240 ttccccatac cccactagtg ggattatact gggttgttgt tgttgtagtg ttctttatca 6300 cttcttaaag tcattgtgtg tctaatctat ttgtttttct gtttggattt ttgcagagga 6360 atgaagttct tatatggctt gaggatttca agctggatta agctcccatt ctcaaaattg 6420 ctgagagttt catgagtgaa aaacaggaga gattcacaag caaagacagt ttagaaaaat 6480 gttcttatac tgaacagatc caaaagctat gaaattaggt ttactgtatt ttgtatctca 6540 aaatatttag agattttgac aaatatatgg cttgtacata gcacagaatg cttgtgctga 6600 acactactaa actgatttct cttgtgcact gcaccaaata ttgttagagc tgtagttcta 6660 tg 6662 <210> 12 <211> 1161 <212> DNA <213> Nicotiana tabacum <400> 12 atggccacct cacaactttc cgacctcatt ttccggccat cacttcacag gcgaaccaat 60 ttccggcaat gccaccctac ttcagtcttt ctcactcctc caaaaaatat caaaacaaaa 120 actcttaaat atgacagaaa aattaagtgg ttaattaagt ttagtttagt tgataaacag 180 agcccaacca aaaaaccaac agttgatatg aaccaattag tggaattttt atatgaggat 240 ttacctcatc tttttgatga tcaaggtatt gatcgtaagg catatgatga ttatgtgaag 300 tttagggatc caattacaaa acatgattca attgatggct atttgtttaa tattgccatg 360 ttgaaacagt tgtttaagcc tgattttcag ctgcattggg ctaaacagac aggaccctat 420 gaaataacta caaggtggac aatggtaatg aagttcattc ttcttccatg gaaacctgaa 480 ttagtcttca ccggcacttc tgttatgggc gtcaatcctg aaacaaacaa atttaacagc 540 catgtggact actgggattc aattaagaat aacgattatt tttctgtaga aggtctgctg 600 gaggtcataa aacagttaag gatttacaag actccggatt tggaaacacc tagttatcag 660 atattaagaa gaacagcaac ttacgaggtc aggaaatatg atccgttat agttgtagaa 720 acagaaggtg acaaactcgc aggaataga ggtttcaatg atgttgcggg gtacatattt 780 ggcaaaaatt cagcgacaga gaagataccg atgactactc ctgtcttcac tcaggcattc 840 gatgctgaaa aatctaaagt gtcaatccag atagttcttc catcggataa atctttgagc 900 agcttaccag ctcccaatca agaaggtata agcttaagga aaacggaagg aggcatcgct 960 gctgcattaa aatttagtgg aaaaccaact gatgatgttg ttcgagaaaa ggagaaacaa 1020 ctcagatcaa gtcttattaa ggacggtctc aaacctcaat cggattgcat gcttgctcgc 1080 tacaatgatc ctggtcgaac gtggaagttt ataatgagga atgaagttct tatatggctt 1140 gaggattca agctggatta a 1161 <210> 13 <211> 199 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 13 Met Gly Met Ile Leu Gly Lys Ile Thr Val Glu Thr Pro Lys Tyr Glu 1 5 10 15 Leu Ile Gln Ser Thr Ala Asp Tyr Glu Ile Arg Lys Tyr Pro Ala Ser 20 25 30 Val Ile Ala Gln Val Thr Tyr Asp Pro Thr Gln Phe Lys Gly Asn Lys 35 40 45 Asp Gly Gly Phe Met Leu Leu Ala Asn Tyr Ile Gly Ala Leu Gly Asn 50 55 60 Pro Gln Asn Ala Lys Pro Glu Lys Ile Ala Met Thr Ala Pro Val Ile 65 70 75 80 Thr Lys Ser Ser Glu Lys Ile Ala Met Thr Ala Pro Val Val Thr Lys 85 90 95 Ser Gly Asp Gly Glu Asn Asn Met Val Thr Met Gln Phe Ile Leu Pro 100 105 110 Ala Lys Tyr Thr Lys Ala Glu Glu Ala Pro Lys Pro Leu Asp Glu Arg 115 120 125 Val Val Val Lys Glu Glu Gly Glu Arg Lys Phe Gly Val Val Gln Phe 130 135 140 Ser Gly Thr Ala Ser Asp Lys Val Val Lys Glu Lys Val Glu Asn Leu 145 150 155 160 Arg Lys Cys Leu Glu Arg Asp Gly Tyr Lys Ile Ile Gly Asp Phe Glu 165 170 175 Leu Ala Arg Tyr Asn Pro Pro Trp Thr Ile Pro Pro Phe Lys Thr Asn 180 185 190 Glu Val Met Ile Pro Val Glu 195 <210> 14 <211> 1718 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 14 ctccaccagc ttatggtgca aacgtggcaa aacaaactcc acagcagttc cctcatctac 60 cgccattcat tttgcagcaa tcaaaaaccc ctttcccaag ttctctcttc ctctctttcc 120 gatttcccac aagaaacttc ctcttctctc cccttatatg taacaaattc acagcctcca 180 tttactatca aaacattctc aaattccatt cccaataact aatttttcgt tccatcaaaa 240 tgggcatgat tttggggaaa atcactgtgg aaacaccaaa atacgaattg attcaatcta 300 cagctgacta cgaaatccgc aaatacccag catctgttat agcacaagtc acatatgatc 360 caacccagtt caaaggaac aaagacggtg ggtttatgct attagccaat tacatcggcg 420 cacttggcaa tcctcaaaac gctaagcctg aaaaaatcgc catgacagct cctgtaatca 480 ccaaatcgtc cgaaaaaatc gcgatgactg caccagtggt gactaagagt ggtgatggag 540 agaacaatat ggtgacgatg cagtttattt taccagcaaa gtatacaaaa gctgaagagg 600 cacctaagcc gttggatgaa agggtggtgg ttaaagaaga aggggaaaga aagtttggtg 660 tggtgcagtt tagtgggact gcaagtgata aagtggttaa agaaaagtg gagaatttaa 720 ggaaatgttt ggagagagat gggtataaga taattggtga ttttgagttg gctcggtata 780 atcctccttg gacaattcct ccatttaaga ccaatgaagt tatgattcca gttgagtgag 840 ctgaaaatgg tgtattaaag aaaaatatga gacggctgtg aagctagctt gtgaacctac 900 tttatctggt gtttattagt attgaattgt gtatttcttg tttatatgtt tacgttgttg 960 acaatgtctt tgcataccct agctagtgtc aattatcat agttaattag cacatatttt 1020 agctcaattt tatactttgc catagtaaag tagtaaaacc aatgtggttt tgcaagcacc 1080 taatgtggta agtccttact ctctgttttt catttcacac tcttctctct gtccatttct 1140 gccagtataa gtgcttcttc tgtcaatacc cggtgaatca tgtaagggcc ctgttagttg 1200 ggtaaaaatt ttccttttgc ttcatcttta tgtgataaga ttcgctttag caccaattgc 1260 cccagcatga atttccttga tctgaccttt ttgttgaaaa atcttgccat tctgttctag 1320 tagagttgac cgtgacatac tacattcatt ctcttttcat caatgagagc tagttgttca 1380 tactggcttc gtaccaattc ctcgtcgctg agctcggttt cttgtatgat tcttaaagaa 1440 ggaatttcta cttcggcgga aataacgact tcagtaccat aaactagtag gtagggagtt 1500 gccccatttg atgtacgaac tgtggtacga taccccgagt aagaaaatgg tagattatga 1560<00 extraordinaria 0671>tgccattatt tgtaattgtt caccatttcc ctcaatatct ttttgatgtt cttgttggcg 1620 gcttccacgg cttcgttcat ttgcgacctg tacgctatag agttcctatg cttgatcttg 1680 aatgtttcac acatggcttc atcaaattgc tattgaga 1718 <210> 15 <211> 600 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 15 atgggcatga ttttggggaa aatcactgtg gaaacaccaa aatacgaatt gattcaatct 60 acagctgact acgaaatccg caaataccca gcatctgtta tagcacaagt cacatatgat 120 ccaacccagt tcaaaggaaa caaagacggt gggtttatgc tattagccaa ttacatcggc 180 gcacttggca atcctcaaaa cgctaagcct gaaaaaatcg ccatgacagc tcctgtaatc 240 accaaatcgt ccgaaaaaat cgcgatgact gcaccagtgg tgactaagag tggtgatgga 300 gagaacaata tggtgacgat gcagtttatt ttaccagcaa agtatacaaa agctgaagag 360 gcacctaagc cgttggatga aagggtggtg gttaaagaag aaggggaaag aaagtttggt 420 gtggtgcagt ttagtgggac tgcaagtgat aaagtggtta aagagaaagt ggagaattta 480 aggaaatgtt tggagagaga tgggtataag ataattggtg attttgagtt ggctcggtat 540 aatcctcctt ggacaattcc tccatttaag accaatgaag ttatgattcc agttgagtga 600 <210> 16 <211> 233 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 16 Met Gln Ala Val Met Asn Phe Phe Met Arg Leu Ala Phe Phe Leu Ile 1 5 10 15 Leu Val Ser Lys Leu Ser Asn Gly Tyr Ser Thr Asn His Leu Gln Pro 20 25 30 Gln Lys Leu Gly Phe Tyr Pro Pro Thr Cys Asn Arg Ile Glu Cys Pro 35 40 45 Asn Tyr Asp Leu Ile Gln Ser Gly Lys Asp Tyr Glu Ile Arg Leu Tyr 50 55 60 Asn Ser Ser Met Trp Met Ser Thr Ala Pro Ile Asp Asp Ile Asn Leu 65 70 75 80 Tyr Ser Ala Thr Arg Thr Gly Phe Leu Arg Leu Phe Asp Tyr Ile Gln 85 90 95 Gly Lys Asn Ser Tyr Gln Glu Lys Ile Glu Met Thr Ala Pro Val Ile 100 105 110 Thr Gln Val Lys Pro Ser Asp Gly Pro Phe Cys Ala Ser Ser Phe Val 115 120 125 Val Ser Phe Tyr Val Pro Lys Lys Asn Gln Pro Asn Pro Pro Pro Ala 130 135 140 Lys Gly Leu His Val Gln Ile Trp Ser Asn Thr Tyr Val Ala Val Arg 145 150 155 160 Gln Phe Gly Gly Phe Val Ala Asp Val Asp Val Ala Lys Glu Ala Ala 165 170 175 Ala Leu Ser Ala Ser Ile Ala Asp Thr Lys Trp Ala Ala Ala Val Glu 180 185 190 Lys Ser His Ala Ala Asp Asn Thr Thr Met Tyr Thr Val Ala Gly Tyr 195 200 205 Asn Ser Pro Phe Glu Phe Lys Asp Arg Val Asn Glu Ile Trp Phe Thr 210 215 220 Phe Asp Leu Asp Lys Ala Ser Ala Ile 225 230 <210> 17 <211> 1673 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 17 aaaacctctg gacaccccct cattgatttt ctttttcttt tatgcatgag gaaattagtt 60 cattcgggtc acttcattct ctctccgtat aatttctagg aactaaaaca ctaaaaagaa 120 acaaaagatg caggctgtta tgaatttctt catgaggcta gctttctttc tgatcctcgt 180 ttctaaattg agcaatgggt attcgacaaa ccacttgcag ccacagaagt tgggtttcta 240 tcctccaacc tgtaaccgaa ttgagtgccc aaattatgac ttaattcaat ctggaaaaga 300 ctatgaaatt cgtctttata attcatccat gtggatgtct actgcaccca ttgatgatat 360 taacctttat tccgccacca gaactggtttt cctcaggtta atattatatt ccctctttct 420 cccactagtt cccttttaca tcagttatct gtatctattg tttttttttt ttcaattcct 480 ttctatttgt tgcatatttg ataagttagc tacactgtgc aacaccaact aaagcatgtg 540 tgtttgcttt ccaagattcc tcaaaaaaaa aaaaaaaaa acaagggact taattaaagg 600 tttacttaca tctcaatcaa ttgaaaataa tcgactatta aaaggaaaaa aaaaattatg 660 tcttgccagg ttacattgaa gcaggttgct gccagttacc aatttacaat tatattgcta 720 gttatctgta actgatttga tttatatgta ctgaaaatgc atagtattta atctagattg 780 ttaaagcaaa tacctgcctt atttacaag ttactaatct ccctttttac ggttagttat 840 cgtgttatta ttgagttgac ttgatagtgt aaaagtgatc ttttccactg tcagtattta 900 gaagttatt tatgtgaact taatctttat acttaagatg ttagcctatc catttgcaaa 960 acataaaaaa cagttgttgt aaactttgcc ttggcaggct attcgattac attcaaggga 1020 agaacagtta ccaggagaaa atagagatga cagctccagt tatcactcaa gtaaaaccaa 1080 gtgatggacc attttgtgca tcttcatttg ttgtgagctt ctacgtacca aagaagaacc 1140 aaccaaatcc tcctccagct aaaggccttc acgtccaaat atggagcaat acttatgtcg 1200 ccgtcaggca attcggcgga tttgtagctg atgttgatgt tgcaaaagaa gctgctgctt 1260 tgagtgctag tattgctgac actaaatggg cagcagccgt tgaaaaaagc catgctgcag 1320 ataacactac gatgtataca gtggcgggat acaactctcc ttttgagttc aaggacagag 1380 ttaatgagat ttggtttaca tttgatttgg acaaagcatc tgccatttga ttctggactc 1440 tttaacattt caggagacat gaaactatct tattttcttg gtttgtatta ctattaaaca 1500 acagttttgt atgaccacaa tgctacatta tatagataga aaataagata ggttgtacaa 1560 tacaatttga ttctgaggat ttagtggtta agatacctct ttcaagacct ttcaaaataa 1620 tccaatatat atggaaaagg gcttagacca tattgagttt attgtatgca gtt 1673 <210> 18 <211> 702 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 18 atgcaggctg ttatgaattt cttcatgagg ctagctttct ttctgatcct cgtttctaaa 60 ttgagcaatg ggtattcgac aaaccacttg cagccacaga agttgggttt ctatcctcca 120 acctgtaacc gaattgagtg cccaaattat gacttaattc aatctggaaa agactatgaa 180 attcgtcttt ataattcatc catgtggatg tctactgcac ccattgatga tattaacctt 240 tattccgcca ccagaactgg tttcctcagg ctattcgatt acattcaagg gaagaacagt 300 taccaggaga aaatagatagat gacagctcca gttatcactc aagtaaaacc aagtgatgga 360 ccattttgtg catcttcatt tgttgtgagc ttctacgtac caaagaagaa ccaaccaaat 420 cctcctccag ctaaaggcct tcacgtccaa atatggagca atacttatgt cgccgtcagg 480 caattcggcg gatttgtagc tgatgttgat gttgcaaaag aagctgctgc tttgagtgct 540 agtattgctg acactaaatg ggcagcagcc gttgaaaaaa gccatgctgc agataacact 600 acgatgtata cagtggcggg atacaactct ccttttgagt tcaaggacag agttaatgag 660 atttggttta catttgattt ggacaaagca tctgccattt ga 702 <210> 19 <211> 386 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 19 Met Ala Thr Ser Gln Leu Ser Gly His Ile Phe Arg Ser Ser Leu His 1 5 10 15 Arg Arg Thr Ile Phe Arg Gln Cys His Pro Thr Ser Val Phe Leu Thr 20 25 30 Pro Pro Lys Asn Ile Lys Thr Lys Pro Leu Lys Tyr Asp Arg Lys Phe 35 40 45 Lys Trp Leu Ile Lys Phe Ser Leu Val Asp Lys Gln Thr Pro Thr Lys 50 55 60 Lys Pro Thr Val Asp Met Lys Gln Leu Val Glu Phe Leu Tyr Glu Asp 65 70 75 80 Leu Pro His Leu Phe Asp Asp Gln Gly Ile Asp Arg Lys Ala Tyr Asp 85 90 95 Asp Tyr Val Lys Phe Arg Asp Pro Ile Thr Lys His Asp Ser Ile Asp 100 105 110 Gly Tyr Leu Phe Asn Ile Ala Met Leu Lys Gln Leu Phe Arg Pro Asp 115 120 125 Phe Gln Leu His Trp Ala Lys Gln Thr Gly Pro Tyr Glu Ile Thr Thr 130 135 140 Arg Trp Thr Met Val Met Lys Phe Ile Leu Leu Pro Trp Lys Pro Glu 145 150 155 160 Leu Val Phe Thr Gly Thr Ser Val Met Gly Val Asn Pro Glu Thr Asn 165 170 175 Lys Phe Asn Ser His Val Asp Tyr Trp Asp Ser Ile Lys Asn Asn Glu 180 185 190 Tyr Phe Ser Leu Glu Gly Leu Leu Glu Val Ile Lys Gln Leu Arg Ile 195 200 205 Tyr Lys Thr Pro Asp Leu Glu Thr Pro Ser Tyr Gln Ile Leu Arg Arg 210 215 220 Thr Ala Thr Tyr Glu Val Arg Lys Tyr Asp Pro Phe Ile Val Val Glu 225 230 235 240 Thr Glu Gly Asp Lys Leu Ala Gly Asn Arg Gly Phe Asn Asp Val Ala 245 250 255 Gly Tyr Ile Phe Gly Lys Asn Ala Ala Thr Glu Lys Ile Pro Met Thr 260 265 270 Thr Pro Val Phe Thr Gln Ala Phe Asp Ala Glu Lys Ser Lys Val Ser 275 280 285 Ile Gln Ile Val Leu Pro Ser Asp Lys Ser Leu Asn Ser Leu Pro Ala 290 295 300 Pro Asn Gln Glu Gly Ile Ser Ile Arg Lys Thr Glu Gly Gly Ile Ala 305 310 315 320 Ala Ala Leu Lys Phe Ser Gly Lys Pro Thr Asp Asp Ile Val Arg Glu 325 330 335 Lys Glu Lys Gln Leu Arg Ser Ser Leu Ile Lys Asp Gly Leu Lys Pro 340 345 350 Gln Ser Gly Cys Met Leu Ala Arg Tyr Asn Asp Pro Gly Arg Thr Trp 355 360 365 Lys Phe Ile Met Arg Asn Glu Val Leu Ile Trp Leu Glu Asp Phe Lys 370 375 380 Leu Asp 385 <210> 20 <211> 3736 <212> DNA <213> Nicotiana tabacum <400> 20 actaaaacca aatggccacc tcacaacttt ccggccacat tttccggtca tcacttcacc 60 ggcgcaccat tttccggcaa tgccacccaa cttcagtctt ccttactccc ccaaaaaata 120 tcaaaacaaa acctcttaaa tatgacagaa aattcaagtg gttaataaag tttagtttag 180 ttgataaaca gaccccaaca aaaaaaccaa cagttgatat gaaacaatta gttgaatttt 240 tatatgagga tttacctcat ttatttgatg atcaaggtat tgatcgaaag gcatatgatg 300 attatgtgaa gtttagagat ccaattacaa aacatgattc aattgatggc tatttgttta 360 atattgccat gttgaaacag ttgtttaggc ctgattttca gctgcattgg gctaaacagg 420 ttagcatttc tacagtttct tgttaattgg ttagttaaac tgctgtttgt ttaactttct 480 tacttggatt taagtttacg ggagccttgg cgtaactggt aaagttgatg tcatgtcacc 540 aaaaggtcat gagttcgagc agtggaaaca acctcttgca gaaatgcagt ataaagctgc 600 atacaataga cctttgtggt ccagcccttt tccggacccc gcgcatagcg ggagcttatg 660 cactgggctg tctttttttc ctcggatttt aagttatgtt caatgatggt gtgaaattat 720 tttactctaa ttaatgtgtg tcgtagagat aatgtaatca cctcataatt gacctgactg 780 tgtaaataat tttatatcgt cggtgcataa aacgaaaggt ccacaatcac ttagagtacc 840 atgttacaaa taaatgttta tcatcaagaa tcacttagaa ttaccttgcg atatgattgt 900 gtatttgcac tgtcagtgcc taaacttaa gcgtcacgcc father ttattaatgc ttattatagg gaatcacttg gaattgcctt acgatatgat tgtgtatttg cattgttagt gcctgaaact taaaggtctg cgaacatatt acctttagcc gagggtctat cgaaataact ctctacttgc acaaggtagg ggtaaggtct gcgtatatac taatctccct agaccccgct tgtgggatta cactgggttt attgttgttg ttgtgcctaa agcttaagca ccatatctat fathers caattgaatc acttggacta ccttaattgt gtattagtgt ctcatggttt tgtaatcta tttatggata ccgatacatg tagtgtacta tgtaggttct ctctttcaaa atttagtttg gtgtcagaag tcttattcaa tcatttaatc tatttgcttt ggccgagggt 1380. ccatcggaa cggtctctct accttctcaa ggtaaaggta aggtctgcgt acacactacc cttccgagac cccactcgtg gtattacact gtggtattac actgaattta ttattgttgt tgtagtcagt ttgctttgtg ctattaaact tgtatttcta aggaatgtga tacataaatt atgccttgaa tttttgagtg attgagtttg ttgtagacag ggccctatga aataactaca agatggacta tggtgatgaa gttcattctt cttccatgga aacctgaatt agtcttcact 1680 ggcacttctg ttatgggcgt caatcctgaa acaaacaagt ttaacagcca tgtggtttga 1740 ttcttcactc ttcctcgacg caaattcaaa tttattagat tgctttgcag attagtcacc 1800 gactcttgtt gaaatctata ggactactgg gattcaatta agaataacga atatttttcc 1860 ctggaaggtc tgctcgaggt cataaaacag gtgcgcgctc gaactgtttt agtttatgtt 1920 tcttgaactg ttggactaaa ccagcgcaaa gatactctta tcatggtgtg atattgtctg 1980 ctttgggcca aattcgcgcg gttttcccta tgaggcctca caccattggg agatctctac 2040 accttatata taggctctca atcttatcaa ctaccaatgt ggctttatta taatttgaga 2100 acacaaattt tgggcagtta aggatttaca agactccgga cctggaaacg cctagttatc 2160 agatactaag aagaacagca acttacgagg tttgtcatat ttatgtttac agcgttgtac 2220 ttcaagctaa gaacttctat attaggcgta catttttatt gggtttgcgc tacttttagc 2280 tgaactgatg cactaggacg tttagattta tgatgtggaa tggtcacaat tatcatttat 2340 gtaatacagg tcaggaata tgatccgttt atagttgttg aaacagaagg tgacaactc 2400 gcaggaaata gaggtttcaa tgatgttgca gggtgagata agttgaccct tttctttaa 2460 aatctctgt gtgttttct gatttagatt ggcggaccat taactcgtct taatataggt 2520 acatattgg gaaaaatgct gcaacagaga agataccgat gactactcct gtcttcactc 2580 aggcattga tgctgaaaaa tctaaagtgt caatccagat agttctcca tcggataaat 2640 ctttgaacag gtaacacgtt ttatta atagacacga aacatctca caccgagcga 2700 tcatattgta cttattacaa tatgagttca aatttttaca ttcgtcgaaa cttagtaatt 2760 catatatata attcataagc atcgttcgcc tctgtgtaag agttggattt tagttatttt 2820 ctttgatcaa atgagaatta tggaaattaa agctttattc ttaacagctt accagctccc 2880 atcaagaag gtattagcat aaggagaca gaaggaggaa tcgcggctgc actaaaattt 2940 agtggaaagc cacagatga tattgttcgc gaaaggaga aacaacttag atccagctctt 3000 attaaggacg gtctcaacc tcaatcgggt tgtagcttg ctcgctacaa tgatcttggt 3060 cgaacgtgga agtttataat ggtatacttc tatgtactga atatcgttcg tttcattgca 3120 ttcttgttta agttcttatt ttgttttagt atgaagtgtc tctttagaat gagaaccttg 3180 gcgtaactgg taaagttgct gctatgtgac caggaggtca cgggttcgag ccgtggaaac 3240 accctttgc agaaatgttg ggtaaggctg cgtacaattg acccttgtgg tccggccctt 3300 ccgcggaccc ctcgtatagc aggaacttag tgcaccgggc ttccctttta gtctgaagtg 3360 tctctttatc actttgtcat ctggtgtcta atctatttgt ttttctgttt ggatttttgc 3420 agaggaatga agttcttata tggcttgagg atttcaagct ggattaactc cattccaaa 3480 aattgctgag atttcatgt atgtaaaaca ggagagattc acaagcaaag acagtttaga 3540 aaaattctta tactgaaatg aaattagttc tcaaaatgtt tagagatttt gacaaatata 3600 tgacttgtgc atagccacaa atgcttgtgc tgaactcact ttacactaaa caaacttcta 3660 ttgtgcatta caaagttcga tgtgttgatg gtgtaaataa tatttgtcac ttgtaaggta 3720 attaatggta attaac 3736 <210> 21 <211> 1161 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 21 atggccacct cacaactttc cggccacatt ttccggtcat cacttcaccg gcgcaccatt 60 ttccggcaat gccacccaac ttcagtcttc cttactcccc caaaaaatat caaaacaaaa 120 cctcttaaat atgacagaaa attcaagtgg ttaataaagt ttagtttagt tgataaacag 180 accccaacaa aaaaaccaac agttgatatg aaacaattag ttgaattttt atatgaggat 240 ttacctcatt tatttgatga tcaaggtatt gatcgaaagg catatgatga ttatgtgaag 300 tttagagatc caattacaaa acatgattca attgatggct atttgtttaa tattgccatg 360 ttgaaacagt tgtttaggcc tgattttcag ctgcattggg ctaaacagac agggccctat 420 gaaataacta caagatggac tatggtgatg aagttcattc ttcttccatg gaaacctgaa 480 ttagtcttca ctggcacttc tgttatgggc gtcaatcctg aaacaaacaa gtttaacagc 540 catgtggact actgggattc aattaagaat aacgaatatt tttccctgga aggtctgctc 600 gaggtcataa aacagttaag gatttacaag actccggacc tggaaacgcc tagttatcag 660 atactaagaa gaacagcaac ttacgaggtc aggaaatatg atccgtttat agttgttgaa 720 acagaaggtg acaaactcgc aggaaataga ggtttcaatg atgttgcagg gtacatattt 780 gggaaaaatg ctgcaacaga gaagataccg atgactactc ctgtcttcac tcaggcattt 840 gatgctgaaa aatctaaagt gtcaatccag atagttcttc catcggataa atctttgaac 900 agcttaccag ctcccaatca agaaggtatt agcataagga agacagaagg aggaatcgcg 960 gctgcactaa aatttagtgg aaagccaaca gatgatattg ttcgcgaaaa ggagaaacaa 1020 cttagatcca gtcttattaa ggacggtctc aaacctcaat cgggttgtat gcttgctcgc 1080 tacaatgatc ctggtcgaac gtggaagttt ataatgagga atgaagttct tatatggctt 1140 gaggatttca agctggatta a 1161 <210> 22 <211> 299 <212> PRT <213> Tobacco (Nicotiana tabacum) <400> 22 Met Leu Leu Cys Ser Pro Ser Ser Ile Ser Ala His Asn Leu Ser Arg 1 5 10 15 Asn Arg Thr Arg Pro Ser Pro Ile Asn Ser Met Ala Val Asp Arg Ser 20 25 30 Ser Ser Arg Val Ala Thr Thr Ala Ser Gln Arg Arg Asn Gly Thr Ser 35 40 45 Ala Leu Glu Ala Arg Ile Ser Leu Val Ile Ala Leu Ala Ser Gln Thr 50 55 60 Ser Ser Leu Ser Gln Lys Leu Leu Thr Glu Leu Ala Gly Glu Thr Ala 65 70 75 80 Lys Tyr Val Leu Pro Lys Arg Ile Phe Glu Ser Arg Thr Leu Glu Glu 85 90 95 Ala Leu Met Ser Val Pro Asp Leu Glu Thr Val Lys Phe Asn Val Leu 100 105 110 Lys Arg Ser Asp Gln Tyr Glu Ile Arg Glu Val Glu Pro Tyr Phe Val 115 120 125 Ala Glu Ala Thr Met Pro Gly Lys Tyr Gly Phe Asp Phe Asn Gly Ala 130 135 140 Ser Gln Ser Phe Asn Thr Leu Ala Glu Tyr Leu Phe Gly Lys Asn Thr 145 150 155 160 Light Light Glu Ser Met Ala Met Thr Thr Pro Val Ile Thr Arg Arg Thr 165 170 175 Gln Ser Asp Gly Glu Arg Met Glu Met Thr Thr Pro Val Ile Thr Lys 180 185 190 Arg Val Glu Asp Gln Gly Lys Trp Arg Met Ser Phe Val Met Pro Ser 195 200 205 Lys Tyr Gly Ser Asp Leu Pro Leu Pro Lys Asp Ser Ser Val Thr Ile 210 215 220 Lys Glu Val Pro Arg Lys Thr Val Ala Val Val Ala Phe Ser Gly Phe 225 230 235 240 Val Thr Asp Glu Glu Val Lys Ala Arg Glu Ser Arg Leu Arg Ala Ala 245 250 255 Leu Lys Gly Asp Ala Glu Phe Arg Val Lys Asp Gly Ala Ser Ile Glu 260 265 270 Val Ala Gln Tyr Asn Pro Pro Phe Thr Leu Pro Phe Thr Arg Arg Asn 275 280 285 Glu Ile Ser Leu Glu Val Glu Arg Glu Gln Glu 290 295 <210> 23 <211> 6324 <212> DNA <213> Tobacco (Nicotiana tabacum) <220> <221> misc_feature <222> (3617)..(4958) <223> n is a, c, g or t ​​atgtgacata cacagtatgt gataaggaat taaccaagta attcgggggc gacaaataca 60 aatatagatg aaccacaatc cccttctttg aagcacagaa tttcaaaagg agtgaaacaa 120 acactgtaac gtagttaaag atgcttctgt gctcaccttc ttccatttct gctcacaatc 180 ttagcagaaa cagaaccaga ccttcgccta tcaattctat ggcagttgac agaagcagct 240 cccgcgtgc cacaactgct tcgcagcgga gaaacggcac gtcagctctc gaagctcgca 300 tctccctcgt catcgctctc gcctcccaaa cctctctct ctctcagaaa cgtaatttct 360 tcactccatt tcctaattcc tcattttaat cggattttaa gcatttctct cccaattctt 420 tttttgattt gttaaaaca tttcgtccag tgcttccagg actctttaa gtcagattag 480 agaacttctt agaaaagaat taatttagtt taagagacta attatttacc agtgttggaa 540 tgaacttgtg cttgacggct aaagcggaac ggatataaat gattctatgt agtcctctgg 600 ctagtttagg aatgagaacc tagtcaattg actaattgag taattgctac ttatattgtt 660 aaattttta ttgttcgcag ttctgacgga attggctggt gaaaccgcga aatacgtgtt 720 accgaagagg atattcgaaa gtcggacttt agaggaagct ttgatgtctg gtattctttc 780 tcaatctttt ctatgattgt tatatcctct atttaataa taatgagaat ttagatggtt 840 aaaaataatg actagctccc atcgctgtta tcttcttgaa gtgccggatc tggacacagt 900 gaaattcaat gttttgaaac gcagtgatca gtatgaata agggagttg aggtatttc 960 tctatgattc tatttataaa ttactaatgt tgtcattcct tttttttt tcccagtaga 1020 gtctagattt gtatgcatcc attctgcaac tttatcgggg gagtggttcc agacgagaga 1080 ggagcaatat tgacgggaa aaaaaggtt taaaccttca ttaaaaaat gttgaagata 1140 gaggggcaaac tgttttgag aacacctta gatactact tacatagata caatattag 1200 gcaagatac gttattatta ttgttcaca ccagctaaat attctcaaac aaagattaat 1260 cctttagtta gatttcttg atatatggta gatgctccg tctcagaag taatcaaggg 1320 tactaataca agaagcatt agttggtaaa agttactagg ccatgtatt agatacaaga 1380 gabatttgtg aagattttgt tgtacttga ttcata tggccttag ttcatatttg 1440 tgttttccct tattatggtc attaaaaaaaaaaaacaca agtacctggg gattgaaaat 1500 gcaattctga aaggtacaat acatggaa gtggaactgg cacaactgga gactgagt 1560 tatagtgggg agtcagcatg tctatcaata agaaaaaa agaataac gaacctgaaa 1620 gagatttggt attcaaagt atttggaaa agaatgaat tgacagaat agactagtga 1680 tcacaatca ggattttcct caatttgag gaacatgtta tgactcggt gttaaaacat 1740 gatgagaaag cagaggtgcc agttttttga aaaacata tgatgttcta acttttggtt 1800 tgataccta gattaagca tatcataagc tcttccatg aacattaaag ccctggatcc 1860 tgatacttga aagtcattca ttcacaatt gttttggc agatcact tgtattaaa 1920 attgaattct ggaccatcag tcaccctgtt atttatggtg cacatatttc ttcttgtg 1980 tatatgctct tattctttgc ttttcggaa acagcctacta cccccgggta ggggtaggt 2040 ctgcatacac actaccctcc ccagacccca tacactggtt gttgttgttg ttgtatatgc 2100 tcttaaattc ttgtcctcag ttattcctaa cctcttta tagcattct cttgattata 2160 gatatgctgt attattagaa cttaatcac cgatatctct ttatgttgtc atgttgatag 2220 ttattctttc ccttcttgtc ctcttaacaa taactctgag ttttttatt ttttattttga 2280 taaagttcat tataaactct gagttaattt atctaaagta atattgcgac tttgaacagc 2340 cttactttgt tgctgaggct acaatgcctg ggaagtatgg gtttgacttt aatggtgcat 2400 ctcaatcctt caacacattg gctgagtact tgtttggtaa ggtagttaaa ggtacttata 2460 gagagtcaaa cttagatcgc ctccaaccac attgtaatgg tggtcttctt tggctctgtt 2520 ctatttcttg gtgatttcca gaacacgaag aaggagagca tggcgatgac aacaccagtt 2580 atcactcgta gaactcaatc tgatggggag agatggaaa tgactactcc agtgataact 2640 aaacgggtac agctcttcta ctggttgttc cttttccct ttagttcttt tgcaatgtat 2700 atttactatg tcattcgtga tatatggtgt ggttactaat atgcttgcat ttttgtatga 2760 catggtgtta ctcaaaatat taaactgata atatattgaa gttgagtcac cacataactt 2820 gggaaatggt aaatgaagaa ctactctaaa atgaccacat atcgtcaaat ggaaatactg 2880 actttactac tgattctgtt gaaaagaaac atcgtctgat tacctatcac taggaaaaca 2940 tttaagctct tgatttagaa ttgataaaag atgacatgtg ctttagattg agatttgagg 3000 atctttcaat tgtatgctag gcttatacgc cagcggggt acatacaaaa cgtaaacatt 3060 ctggttgctt agagtttgaa tctgtactac tctaagatag tattgcaaat tctgagagag 3120 tggaaactgt tagattccag gttttatt gttgcatgga tccttcaaac gggtgtgaca 3180 tgggtgtggg tacttcttgc aggttcttca aagtagaaaa gaaatgcacg aactcctatc 3240 aaatacttac atacacacat ggtgtacacc tataggtttg tccatataat gcagatgttt 3300 aatcttagtc cagaacaagt gtcgaacgga ataacagtca caaagaacaa tcttcataat 3360 gataaagatt atggtccgaa taaaatacac ccccatataa atgataacac attttcttgt 3420 aattcttaag tagatctccg agaataacat tttgtaactt aaaagaccaa ttttttgtga 3480 aaatgggccc gccgtattga ttggcatcct ataagaattc cttattcca gaacctttga 3540 aaccagaatt agtccattct agaaaagaaa aactcagttt agcttgatgt ctgatcctat 3600 gttatagtgc ttaacannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnga tcctactcgt tatacgtcgc 4980 cttaacaccc ccccccccca cccccccccc ctcctttctc ccccgcgccg cgcctccacc 5040 accaccaaca aacctagtaa aatcccacaa gtggagtctg gggaggtagc gtgtatgcag 5100 accatacccc tacttgcggg aaggtagaga ggatgtttt ctaatttgga atgtcatgcg 5160 ctcatatttt caccaaaaatt acaccactg gaatctcatt aagaatttca ttaggtggaa 5220 gatcaaggaa agtggaggat gtcctttgtc atgccctcaa agtatggttc agacttgcca 5280 ctaccaaagg attcctccgt aactatcaag gaggtgccta ggaaaactgt cgccgttgtt 5340 gctttttcag gtttgtgatt tcagatttta gcatgtcaat ataaagttct attttgtatg 5400 atgatgatat gagttgagct tacagaaaga agtgaggatt catagcgccg acctcaactt 5460 tgtgggactg aggcgtagtt gttgctgcta atataaaatt ctagacagat ttgcatttta 5520 agttaatttg aaattcaata tcctagtaat cggaaagcag agctctgtct cattggtcta 5580 tggaagtaaa tcttgtttaa gtttacttag tgctttattg atagcttctc ttgttatgac 5640 atgagagaat ttttcatcta gttgttttt ctgtgttttt cctttgatgt cctatatgcc 5700 tgctgtggta gatagcaatt tccattcaga gttcaaaagc aaaaacattt aggaattcta 5760 tttttgaagt tttacatcct aacttagcta gttttctccc attcgatttc aacaaaggtt 5820 ttgtgactga tgaagaagtt aaagcccgag aatcaagact ccgtgctgcg ctaaagggag 5880 atgcagagtt tcgggtaaaa gatggtgcct caatagaagt tgcacaggta tattcgtaga 5940 accatttcaa tagtacttgt aattctcctg gggaacatat tttggcctag tgtaaccccc 6000 aaatgacata ctagttgtgt gttttttata cttctgcttg cagtataatc caccatttac 6060 tcttccgttc acacgtcgga atgagattag cctggaagtt gaaagggaac aggaatagct 6120 agtcagtgtg cagtatattg cagataaatc cgtgcactga tgaaaccaga gaaacaaaac 6180 atatatggtg taaataaatt atatatatat atgtatatat atttggtgat tgcttgacat 6240 tttggtaaca aggttatgta catgcacaga aatgtaaggt catttatgct tcaactctat 6300 aataacacgt tgtctttaat actc 6324 <210> 24 <211> 900 <212> DNA <213> Tobacco (Nicotiana tabacum) <400> 24 atgcttctgt gctcaccttc ttccatttct gctcacaatc ttagcagaaa cagaaccaga 60 ccttcgccta tcaattctat ggcagttgac agaagcagct cccgcgttgc cacaactgct 120 tcgcagcgga gaaacggcac gtcagctctc gaagctcgca tctccctcgt catcgctctc 180 gcctcccaaa cctctctct ctctcagaaa cttctgacgg aattggctgg tgaaaccgcg 240 aaatacgtgt taccgaagag gatattcgaa agtcggactt tagaggaagc tttgatgtct 300 gtgccggatc tggagacagt gaaattcaat gttttgaaac gcagtgatca gtatgaaata 360 agggaagttg agccttactt tgttgctgag gctacaatgc ctgggaagta tgggtttgac 420 tttaatggtg catctcaatc cttcaacaca ttggctgagt acttgtttgg taagaacacg 480 aagaaggaga gcatggcgat gacaacacca gttatcactc gtagaactca atctgatggg 540 gagaggatgg aaatgactac tccagtgata actaaacggg tggaagatca aggaaagtgg 600 aggatgtcct ttgtcatgcc ctcaaagtat ggttcagact tgccactacc aaaggattcc 660 tccgtaacta tcaaggaggt gcctaggaaa actgtcgccg ttgttgcttt ttcaggtttt 720 gtgactgatg aagaagttaa agcccgagaa tcaagactcc gtgctgcgct aaagggagat 780 gcagagtttc gggtaaaaga tggtgcctca atagaagttg cacagtataa tccaccattt 840 actcttccgt tcacacgtcg gaatgagatt agcctggaag ttgaaaggga acaggaatag 900 <210> 25 <211> 300 <212> DNA <213> Artificial sequence <220> <223> 300 - nucleotide cDNA fragment <400> 25 gtaagaacac aaagaaggaa agtatggcga tgacaacacc cgtaatcact cgtagaactc 60 aatctgatgg ggagaagatg gaaatgacta cttcagtgat aactaaaagg gtggaagatc 120 aaggaaagtg gaggatgtcc tttgtcatgc cctcaaagta tggttcggac ttgccactac 180 caaaggattc ctccgtaact atcaaggagg tgcctaggaa aactgtcgcc gttgttgctt 240 tttcaggttt tgtgactgat gaagaagtta aagcccgaga atcaagacta tgtgctgcgt 300 <210> 26 <211> 20150 <212> DNA <213> Artificial sequence <220> <223> TRV RNA1 <400> 26 gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatttc gggaaacctc 60 ctcggattcc attgcccagc tatctgtcac ttcatcgaaa ggacagtaga aaaggaaggt 120 ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga tgcctctgcc 180 gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtc 240 ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt aagggatgac 300 gcacaatccc actatccttc gcaagaccct tcttctatat aaggaagttc atttcatttg 360 gagaggacag cccaagcttt ctagaggatc cataaaacat ttcaatcctt tgaacgcggt 420 agaacgtgct aattggattt tggtgagaac gcggtagaac gtacttatca cctacagttt 480 tattttgttt ttctttttgg tttaatctat ccagcttagt accgagtggg ggaaagtgac 540 tggtgtgcct aaaacctttt ctttgatact ttgtaaaaat acatacagat acaatggcga 600 acggtaactt caagttgtct caattgctca atgtggacga gatgtctgct gagcagagga 660 gtcatttct tgacttgatg ctgactaaac ctgattgtga gatcgggcaa atgatgcaaa 720 gagttgttgt tgataaagtc gatgacatga ttagagaaag aaagactaaa gatccagtga 780 ttgttcatga agttctttct cagaaggaac agaacaagtt gatggaaatt tatcctgaat 840 tcaatatcgt gtttaaagac gacaaaaaca tggttcatgg gtttgcggct gctgagcgaa 900 aactacaagc tttatgctt ttagatagag ttcctgctct gcaagaggtg gatgacatcg 960 gtggtcaatg gtcgttttgg gtaactagag gtgagaaaag gattcattcc tgttgtccaa 1020 atctagatt tcgggatgat cagagaagaa tttctcgaca gatattctt actgctattg 1080 gtgatcaagc tagaagtggt aagacaga tgtcggagaa tgagctgtgg atgtatgacc 1140 aatttcgtga aaatattgct gcgcctaacg cggtaggtg caataataca tatcagggtt 1200 gtacatgtag gggttttct gatggtaaga agaaaggcgc gcagtatgcg atagctcttc 1260 acagcctgta tgacttcaag ttgaaagact tgatggctac tatggttgag aagaaacta 1320 aagtggttca tgctgctatg ctttttgctc ctgaaagtat gttagtggac gaaggtccat 1380 taccttctgt tgacggttac tacatgaaga agaacgggaa gatctatttc ggttttgaga 1440 aagatcctt ctttctctac attcatgact gggaagagta caagaagtat ctactgggga 1500 agccagtgag ttaccaaggg aatgtgttct acttcgaacc gtggcaggtg agaggagaca 1560 caatgcttt ttcgatctac aggatagctg gagttccgag gaggtctcta tcatcgcaag 1620 agtactaccg aagaatatat atcagtagat gggaaagcat ggttgttgtc ccaattttcg 1680 atctggtcga atcaacgcga gagttggtca agaagacct gtttgtagag aacaattca 1740 tggacaagtg ttggattac atagctaggt tatctgacca gcagctgacc atagcaatg 1800 ttaaatcata cttgagttca aaatttggg tcttattcat aaacggggcg gccgtgaaga 1860 acagcaag tgtagattct cgagatttac agttgttggc tcaacttg ctagtgaagg 1920 aacaagtggc gagacctgtc atgaggt tgcgtgaagc aattctgact gagacgaaac 1980 ctatcacgtc attgactgat gtgctggtt taatatcaag aaaactgtgg aagcagtttg 2040 ctaacaagat cgcagtcggc ggattcgttg gcatggttgg tacttatt ggattctatc 2100 caagaaggt actaacctgg gcgaaggaca caccaaatgg tccagaacta tgttacgaga 2160 actcgcacaa aaccaaggtg atagtatttc tgagtgttgt gtagccatt ggaggaatca 2220 cgcttatgcg tcgagacatc cgagatggac tggtgaaaaa actatgtgat atgtttgata 2280 tcaaacgggg ggcccatgtc ttagacgttg agaatccgtg ccgctattat gaaatcaacg 2340 atttctttag cagtctgtat tcggcatctg agtccggtga gaccgtttta ccagatttat 2400 ccgaggtaaa agccaagtct gataagctat tgcagcagaa gaaagaaatc gctgacgagt 2460 ttctaagtgc aaaattctt aactattctg gcagttcggt gagaacttct ccaccatcgg 2520 tggtcggttc atctcgaagc ggactgggtc tgttgttgga agacagtaac gtgctgaccc 2580 aagctagagt tggagtttca agaaaggtag acgatgagga gatcatggag cagtttctga 2640 gtggtcttat tgacactgaa gcagaaattg acgaggttgt ttcagccttt tcagctgaat 2700 gtgaaagagg ggaaacaagc ggtacaaagg tgttgtgtaa acctttaacg ccaccaggat 2760 ttgagaacgt gttgccagct gtcaaacctt tggtcagcaa aggaaaaacg gtcaaacgtg 2820 tcgattactt ccaagtgatg ggaggtgaga gattaccaaa aaggccggtt gtcagtggag 2880 acgattctgt ggacgctaga agagagtttc tgtactactt agatgcggag agagtcgctc 2940 aaaatgatga aattatgtct ctgtatcgtg actattcgag aggagttatt cgaactggag 3000 gtcagaatta cccgcacgga ctgggagtgt gggatgtgga gatgaagaac tggtgcatac 3060 gtccagtggt cactgaacat gcttatgtgt tccaaccaga caaacgtatg gatgattggt 3120 cgggatactt agaagtggct gtttgggaac gaggtatgtt ggtcaacgac ttcgcggtcg 3180 aaaggatgag tgattatgtc atagtttgcg atcagacgta tctttgcaat aacaggtaat 3240 aatcctctct cttgatattt ttaaattata gaattaatta gttacttta ttctttacta 3300 tatgatttaa atagtttaat cttgtttttg agtaaactat tcgattttga tatttgtatt 3360 cgtcctacaa agttggaaat actgatgata ttttctttg aacgtgatac ctaccaatac 3420 taatcttacg gaatctttta atagagcact aatcaacatg gaactaaaga ccaattctta 3480 agtgtctctg ttgtacagtt cattttagta gtgcgtttaa gtattattat ctcccttcat 3540 gcggggcaat tatgtagatt aaaatcgaaa ttatataaaa tttacataag tctaagtcta 3600 gggtctccag ctaattgtta ttttttaac gatgttgact aaagcaataa cgacgttgac 3660 ttgtgttaaa caggttgatc ttggacatt taagtgccct ggatctagga ccagttaact 3720 gttcttttga attagttgac ggtgtacctg gttgtggtaa gtcgacaatg attgtcaact 3780 cagctaatcc tgtgtcgat gtggttctct ctactgggag agcagcacc gacgacttga 3840 tcgagagatt cgcgagcaaa gttttccat gcaaattgaa aaggagtg aagacggttg 3900 attcttttt gatgcattgt gtcgatggtt ctttaaccgg agacgtgttg cattcgacg 3960 aagctctcat ggcccatgct ggtatgtgt acttttgcgc tcagatagct ggtgctaaac 4020 gatgtatctg tcaggagat cagaatcaa ttctttcaa gcctaggta tctcaagttg 4080 atttgaggtt ttctagtctg gtcggaagt ttgacattgt tacagaaaaa agagaaactt 4140 acagaagtcc agcagatgtg gctgccgtat tgaacaagta ctatactgga gatgtcagaa 4200 cacataacgc gactgctaat tcgatgacgg tgaggagat tgtgtctaaa gaacaggttt 4260 ctttgaagcc tggtgctcag tacataactt tccttcagtc tgagagaag gagttgtaa 4320 atttgttggc attgaggaaa gtggcagcta aagtgagtac agtacacgag tcgcaggg 4380 agacattcaa agatgtagtc ctagtcagga cgaaacctac ggatgactca atcgctagag 4440 gtcgggagta cttaatcgtg gcattgtcgc gtcacacaca atcacttgtg tatgaaactg 4500 tgaaagagga cgatgtaagc aaagagatca gggaaagtgc cgcgcttacg aaggcggctt 4560 tggcaagatt ttttgttact gagaccgtct tatgacggtt tcggtctagg tttgatgtct 4620 ttagacatca tgaagggcct tgcgccgttc cagattcagg tacgattacg gacttggaga 4680 tgtggtacga cgctttgttt ccgggaaatt cgttaagaga ctcaagccta gacgggtatt 4740 tggtggcaac gactgattgc aatttgcgat tagacaatgt tacgatcaaa agtggaaact 4800 ggaaagacaa gtttgctgaa aaagaaacgt ttctgaaacc ggttattcgt actgctatgc 4860 ctgacaaaag gaagactact cagttggaga gtttgttagc attgcagaaa aggaaccaag 4920 cggcacccga tctacaagaa aatgtgcacg cgacagttct aatcgaagag acgatgaaga 4980 agctgaaatc tgttgtctac gatgtgggaa aaattcgggc tgatcctatt gtcaatagag 5040 ctcaaatgga gagatggtgg agaaatcaaa gcacagcggt acaggctaag gtagtagcag 5100 atgtgagaga gttacatgaa atagactatt cgtcttacat gtatatgatc aaatctgacg 5160 tgaaacctaa gactgattta acaccgcaat ttgaatactc agctctacag actgttgtgt 5220 atcacgagaa gttgatcaac tcgttgttcg gtccaatttt caaagaaatt aatgaacgca 5280 agttggatgc tatgcaacca cattttgtgt tcaacacgag aatgacatcg agtgatttaa 5340 acgatcgagt gaagttctta aatacggaag cggcttacga ctttgttgag atagacatgt 5400 ctaaattcga caagtcggca aatcgcttcc atttacaact gcagctggag atttacaggt 5460 tatttgggct ggatgagtgg gcggccttcc tttgggaggt gtcgcacact caaactactg 5520 tgagagatat tcaaaatggt atgatggcgc atatttggta ccaacaaaag agtggagatg 5580 ctgatactta taatgcaaat tcagatagaa cactgtgtgc actcttgtct gaattaccat 5640 tggagaaagc agtcatggtt acatatggag gagatgactc actgattgcg tttcctagag 5700 gaacgcagtt tgttgatccg tgtccaaagt tggctactaa gtggaatttc gagtgcaaga 5760 tttttaagta cgatgtccca atgttttgtg ggaagttctt gcttaagacg tcatcgtgtt 5820 acgagttcgt gccagatccg gtaaaagttc tgacgaagtt ggggaaaag agtataaagg 5880 atgtgcaaca tttagccgag atctacatct cgctgaatga ttccaataga gctcttggga 5940 actacatggt ggtatccaaa ctgtccgagt ctgttttcaga ccggtatttg tacaaggtg 6000 attctgttca tgcgctttgt gcgctatgga agcatattaa gagttttaca gctctgtgta 6060 cattattccg agacgaaac gatgaat tgaacccggc taggttt tggagagg 6120 cacagagagc tgtgtcaaac ttttacgact ggtatatgg aagacaagtc attggtcacc 6180 ttgaagaga agacttcga agtctcaaa ttctcaatc taggggccat tgaattgttt 6240 gtggacggta ggaggaagg accgaagtat ttcacagaa gagagaac tgtcctaaat 6300 catgttggtg ggaagag tgacacaag ttagacgtttt ttgaccaag ggattacaaa 6360 atgattaat cttacgcgtt tctaaaggta gtaggtgtac aactagttgt aacatcacat 6420 ctacctgcag atacgcctgg gttcattcaa atcgatctgt tggattcgag acttactgag 6480 aaaagaaga gaggaagac tattcagaga ttcaagctc gagcttgcga taactgttca 6540 gttgcgcagt acaaggttga atacagtatt tccacacagg agaacgtact tgatgtctgg 6600 aaggtgggtt gtatttctga gggcgttccg gtctgtgacg gtacataccc tttcagtatc 6660 gaagtgtcgc taatatgggt tgctactgat tcgactaggc gcctcaatgt ggaagaactg 6720 aacagttcgg attacattga aggcgatttt accgatcaag aggttttcgg tgagttcatg 6780 tctttgaaac aagtggagat gaagacgatt gaggcgaagt acgatggtcc ttacagacca 6840 gctactacta gacctaagtc attattgtca agtgaagatg ttaagagagc gtctaataag 6900 aaaaactcgt cttaatgcat aaagaaattt attgtcaata tgacgtgtgt actcaagggt 6960 tgtgtgaatg aagtcactgt tcttggtcac gagacgtgta gtatcggtca tgctaacaaa 7020 ttgcgaaagc aagttgctga catggttggt gtcacacgta ggtgtgcgga aaataattgt 7080 ggatggtttg tctgtgttgt tatcaatgat tttacttttg atgtgtataa ttgttgtggc 7140 cgtagtcacc ttgaaaagtg tcgtaaacgt gttgaaacaa gaaatcgaga aatttggaaa 7200 caaattcgac gaaatcaagc tgaaaacatg tctgcgacag ctaaaaagtc tcataattcg 7260 aagacctcta agaagaaatt caaagaggac agagaatttg ggacaccaaa aagattttta 7320 agagatgatg ttccttcgg gattgatcgt ttgtttgcttt ttgatttta ttttatattg 7380 ttatctgttt ctgtgtatag actgtttgag attggcgctt ggccgactca ttgtcttacc 7440 ataggggaac ggactttgtt tgtgttgtta ttttattgtgt atttattaa aattctcaat 7500 gatctgaaaa ggcctcgagg ctaagagatt attggggggt gagtaagtac ttttaaagtg 7560 atgatggtta caaaggcaaa aggggtaaaa cccctcgcct acgtaagcgt tattacgccc 7620 ggatcccccg gggagctcga attcgctgaa atcaccagtc tctctctaca aatctatctc 7680 tctctattt ttccataaat aatgtgtgag tagttcccg ataagggaaa ttagggttct 7740 tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt 7800 aaaatcttc tattatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat 7860 ccagatctcc taaagtccct atagatcttt gtcgtgaata taaaccagac acgagacgac 7920 taaacctgga gcccagacgc cgttcgaagc tagaagtacc gcttaggcag gaggccgtta 7980 gggaaaagat gctaggcag ggttggttac gttgactccc ccgtaggtt ggttaataa 8040 tgatgagtg gcggaagg agggag tgatgagtg taggttgca ggccctgtgc 8100 aaggtagaa gatgaattt tgatagaggt acgctactat acttatacta tacgctagg 8160 gatgcttgt atttaccc tataccccct ataacccct tatcaattta agaataatc 8220 cgcataagcc cccgcttaa aattggtatc agagccatga ataggtctat gaccaaaact 8280 aaaatacgaa aaacctcacc aaaatacgaa agagttctta actctaaga windowagatct 8340 ttcagatca aaactagttc cctcacaccg gagcatgcga tatcctcgac ctgcaggcat 8400 gcaagcttgg cgtaatcatg gtcatagctg ttcctgtgt gaaattgtta tccgctcaca 8460 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 8520 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcgggg aaacctgtcg 8580 tgccagctgc attaatgaat cggccaacgc gcggggag gcggttttgcg tattgggcca 8640 aagacaaag ggcgacattc aaccgattga gggagggag gtaaatattg acggaatta 8700 ttcattaaag gtgaattatc accgtcaccg acttgagcca tttgggaatt agagccagca aaatcaccag tagcaccatt accattagca aggccggaa cgtcaccaat gaaccatcg atagcagcc cgtaatcagt agcgacagaa tcaagtttgc ctttagcgtc agactgtagc gcgttttcat cggcattttc ggtcatagcc cccttattag cgtttgccat cttttcataa 8940 tcaaaatcac cggaaccaga gccaccaccg gaaccgcctc cctcagagcc gccaccctca gaccgccac cctcagagcc accacccctca gagccgccac cagaaccacc accagagccg ccgccagcat tgacaggagg cccgatctag taacatagat gacaccgcgc gcgataattt atcctagttt gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac 9180. tctaatcata aaaacccatc tcataaata cgtcatgcat tcatgttaa ttattacatg cttaacgtaa ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa tcttaagaaa ctttattgcc aaatgtttga acgatcgggg atcatccggg tctgtggcgg 9420. gaactccacg aaaatatccg aacgcagcaa gatatcgcgg tgcatctcgg tcttgcctgg gcagtcgccg ccgacgccgt tgatgtggac gccgggcccg atcatattgt cgctcaggat 9480 cgtggcgttg tgcttgtcgg ccgttgctgt cgtaatgata tcggcacctt cgaccgcctg 9540 ttccgcagag atcccgtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat 9600 ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga 9660 atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt 9720 cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg 9780 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata 9840 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg 9900 atgaatccag aaaagcggcc atttccacc atgatattcg gcaagcaggc atcgccatgg 9960 gtcacgacga gatcatcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct 10020 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc 10080 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga 10140 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca 10200 aggtgagatg agaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc 10260 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat 10320 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa 10380 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc 10440 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc 10500 aatccatctt gttcaatcat gcgaaacgat ccagatccgg tgcagattat ttggattgag 10560 agtgaatatg agactctaat tggataccga ggggaattta tggaacgtca gtggagcatt 10620 tttgacaaga aatatttgct agctgatagt gaccttaggc gacttttgaa cgcgcaataa 10680 tggtttctga cgtatgtgct tagctcatta aactccagaa acccgcggct gagtggctcc 10740 ttcaacgttg cggttctgtc agttccaaac gtaaaacggc ttgtcccgcg tcatcggcgg 10800 gggtcataac gtgactccct taattctccg ctcatgatca gattgtcgtt tcccgcttc 10860 agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 10920 tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 10980 tatgtgcatg ccaaccacag ggttccccag atctggcgcc ggccagcgag acgagcaaga 11040 ttggccgccg cccgaaacga tccgacagcg cgcccagcac aggtgcgcag gcaaattgca 11100 ccaacgcata cagcgccagc agaatgccat agtgggcggt gacgtcgttc gagtgaacca 11160 gatcgcgcag gaggcccggc agcaccggca taatcaggcc gatgccgaca gcgtcgagcg 11220 cgacagtgct cagaattacg atcaggggta tgttgggttt cacgtctggc ctccggacca 11280 gcctccgctg gtccgattga acgcgcggat tctttatcac tgataagttg gtggacatat 11340 tatgtttatc agtgataaag tgtcaagcat gacaaagttg cagccgaata cagtgatccg 11400 tgccgccctg gacctgttga acgaggtcgg cgtagacggt ctgacgacac gcaaactggc 11460 ggaacggttg ggggttcagc agccggcgct ttactggcac ttcaggaaca agcgggcgct 11520 gctcgacgca ctggccgaag ccatgctggc ggagaatcat acgcattcgg tgccgagagc 11580 cgacgacgac tggcgctcat ttctgatcgg gaatgcccgc agcttcaggc aggcgctgct 11640 cgcctaccgc gatggcgcgc gcatccatgc cggcacgcga ccgggcgcac cgcagatgga 11700 aacggccgac gcgcagcttc gcttcctctg cgaggcgggt ttttcggccg gggacgccgt 11760 caatgcgctg atgacaatca gctacttcac tgttggggcc gtgcttgagg agcaggccgg 11820 cgacagcgat gccggcgagc gcggcggcac cgttgaacag gctccgctct cgccgctgtt 11880 gcgggccgcg atagacgcct tcgacgaagc cggtccggac gcagcgttcg agcagggact 11940 cgcggtgatt gtcgatggat tggcgaaaag gaggctcgtt gtcaggaacg ttgaaggacc 12000 gagaaagggt gacgattgat caggaccgct gccggagcgc aacccactca ctacagcaga 12060 gccatgtaga caacatcccc tccccctttc caccgcgtca gacgcccgta gcagcccgct 12120 acgggctttt tcatgccctg ccctagcgtc caagcctcac ggccgcgctc ggcctctctg 12180 gcggccttct ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgcttttc cgctgcataa ccctgcttcg gggtcattat 12600 agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt 12660 tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg 12720 cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa 12780 cgggaatcct gctctgcgag gctggccggc taccgccggc gtaacagatg agggcaagcg 12840 gatggctgat gaaaccaagc caaccaggaa gggcagccca cctatcaagg tgtactgcct 12900 tccagacgaa cgaagagcga ttgaggaaaa ggcggcggcg gccggcatga gcctgtcggc 12960 ctacctgctg gccgtcggcc agggctacaa aatcacgggc gtcgtggact atgagcacgt 13020 ccgcgagctg gcccgcatca atggcgacct gggccgcctg ggcggcctgc tgaaactctg 13080 gctcaccgac gacccgcgca cggcgcggtt cggtgatgcc acgatcctcg ccctgctggc 13140 gaagatcgaa gagaagcagg acgagcttgg caaggtcatg atgggcgtgg tccgcccgag 13200 ggcagagcca tgactttttt agccgctaaa acggccgggg ggtgcgcgtg attgccaagc 13260 acgtccccat gcgctccatc aagaagagcg acttcgcgga gctggtgaag tacatcaccg 13320 acgagcaagg caagaccgag cgcctttgcg acgctcaccg ggctggttgc cctcgccgct 13380 gggctggcgg ccgtctatgg ccctgcaaac gcgccagaaa cgccgtcgaa gccgtgtgcg 13440 agacaccgcg gccgccggcg ttgtggatac ctcgcggaaa acttggccct cactgacaga 13500 tgaggggcgg acgttgacac ttgaggggcc gactcacccg gcgcggcgtt gacagatgag 13560 gggcaggctc gatttcggcc ggcgacgtgg agctggccag cctcgcaaat cggcgaaaac 13620 gcctgatttt acgcgagttt cccacagatg atgtggacaa gcctggggat aagtgccctg 13680 cggtattgac acttgagggg cgcgactact gacagatgag gggcgcgatc cttgacactt 13740 gaggggcaga gtgctgacag atgaggggcg cacctattga catttgaggg gctgtccaca 13800 ggcagaaaat ccagcatttg caagggtttc cgcccgtttt tcggccaccg ctaacctgtc 13860 ttttaacctg cttttaaacc aatatttata aaccttgttt ttaaccaggg ctgcgccctg 13920 tgcgcgtgac cgcgcacgcc gaaggggggt gccccccctt ctcgaaccct cccggcccgc 13980 taacgcgggc ctcccatccc cccaggggct gcgcccctcg gccgcgaacg gcctcacccc 14040 aaaaatggca gcgctggcag tccttgccat tgccgggatc ggggcagtaa cgggatgggc 14100 gatcagcccg agcgcgacgc ccggaagcat tgacgtgccg caggtgctgg catcgacatt 14160 cagcgaccag gtgccgggca gtgagggcgg cggcctgggt ggcggcctgc ccttcacttc 14220 ggccgtcggg gcattcacgg acttcatggc ggggccggca atttttacct tgggcattct 14280 tggcatagtg gtcgcgggtg ccgtgctcgt gttcgggggt gcgataaacc cagcgaacca 14340 tttgaggtga taggtaagat tataccgagg tatgaaaacg agaattggac ctttacagaa 14400 ttactctatg aagcgccata tttaaaaagc taccaagacg aagaggatga agaggatgag 14460 gaggcagatt gccttgaata tattgacaat actgataaga tatatatct tttatataga 14520 agatatcgcc gtatgtaagg atttcagggg gcaaggcata ggcagcgcgc ttatcaatat 14580 atctatagaa tgggcaaagc ataaaaactt gcatggacta atgcttgaaa cccaggacaa 14640 taaccttata gcttgtaaat tctatcataa ttgggtaatg actccaactt attgatagtg 14700 tttatgttc agataatgcc cgatgacttt gtcatgcagc tccaccgatt ttgagaacga 14760 cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga ttcaggttat gccgctcaat 14820 tcgctgcgta tatcgcttgc tgattacgtg cagctttccc ttcaggcggg attcatacag 14880 cggccagcca tccgtcatcc atatcaccac gtcaaagggt gacagcaggc tcataagacg 14940 ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca acaaccgtct tccggagact 15000 gtcatacgcg taaaacagcc agcgctggcg cgatttagcc ccgacatagc cccactgttc 15060 gtccatttcc gcgcagacga tgacgtcact gcccggctgt atgcgcgagg ttaccgactg 15120 cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc caacgcccat aatgcgggct 15180 gttgcccggc atccaacgcc attcatggcc atatcaatga ttttctggtg cgtaccgggt 15240 tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac ggcagtgaga gcagagatag 15300 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag 15360 15420 cattaagttg gcagcatcac ccataattgt ggtttcaaaa tcggctccgt cgatactatg 15480 ttatacgcca actttgaaaa caactttgaa aaagctgttt tctggtattt aaggttttag 15540 15600 aaatactgta gaaaagagga aggaataat aaatggctaa aatgagaata tcaccggaat 15660 tgaaaaaact gatcgaaaaa taccgctgcg taaagatac ggaaggatg tctcctgcta aggtatataa gctggtgggga gaaaatgaa acctatatttt aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat gatgctatgg ctggaagga agctgcctgt tccaaaggtc ctgcactttg aacggcatga tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga agatgaacaa agccctgaaa attachment gctgtatgcg gagtgcatca ggctctttca ctccatcgac atatcggatt gtccctatac gacagccgct tagccgatt ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg aagaagacac tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt ctgcgtccgg tcgatcaggg 16320. aggatatcgg ggaagaacag tatgtcgagc tattttttga cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt gttttagtac ctagatgtgg 16440 cgcaacgatg ccggcgacaa gcaggagcgc accgacttct tccgcatcaa gtgttttggc 16500 tctcaggccg aggcccacgg caagtatttg ggcaaggggt cgctggtatt cgtgcagggc 16560 aagattcgga ataccaagta cgagaaggac ggccagacgg tctacgggac cgacttcatt 16620 gccgataagg tggattatct ggacaccaag gcaccaggcg ggtcaaatca ggaataaggg 16680 cacattgccc cggcgtgagt cggggcaatc ccgcaaggag ggtgaatgaa tcggacgttt 16740 gaccggaagg catacaggca agaactgatc gacgcggggt tttccgccga ggatgccgaa 16800 accatcgcaa gccgcaccgt catgcgtgcg ccccgcgaaa cttccagtc cgtcggctcg 16860 atggtccagc aagctacggc caagatcgag cgcgacagcg tgcaactggc tcccctgcc 16920 ctgcccgcgc catcggccgc cgtggagcgt tcgcgcgtc tcgaacagga ggcggcaggt 16980 ttggcgaagt cgatgaccat cgacacgcga ggaactatga cgaccaagaa gcgaaaacc 17040 gccggcgagg acctggcaaa acaggtcagc gaggccaagc aggccgcgtt gctgaaacac 17100 acgaagcagc agatcaagga aatgcagctt tccttgttcg atattgcgcc gtggccggac 17160 acgatgcgag cgatgccaaa cgacacggcc cgctctgccc tgttcaccac gcgcaacaag 17220 aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc acgtcaacaa ggacgtgaag 17280 atcacctaca ccggcgtcga gctgcgggcc gacgatgacg aactggtgtg gcagcaggtg 17340 ttggagtacg cgaagcgcac ccctatcggc gagccgatca ccttcacgtt ctacgagctt 17400 tgccaggacc tgggctggtc gatcaatggc cggtattaca cgaaggccga ggaatgcctg 17460 tcgcgcctac aggcgacggc gatgggcttc acgtccgacc gcgttgggca cctggaatcg 17520 gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca agaaaacgtc ccgttgccag 17580 gtcctgatcg acgaggaaat cgtcgtgctg tttgctggcg accactacac gaaattcata 17640 tgggagaagt accgcaagct gtcgccgacg gcccgacgga tgttcgacta tttcagctcg 17700 caccgggagc cgtacccgct caagctggaa accttccgcc tcatgtgcgg atcggattcc 17760 acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct gcgaagagtt gcgaggcagc 17820 ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc attgcaaacg ctagggcctt 17880 gtggggtcag ttccggctgg gggttcagca gccagcgctt tactggcatt tcaggaacaa 17940 gcgggcactg ctcgacgcac ttgcttcgct cagtatcgct cgggacgcac ggcgcgctct 18000 acgaactgcc gataaacaga ggattaaaat tgacaattgt gattaaggct cagattcgac 18060 ggcttggagc ggccgacgtg caggatttcc gcgagatccg attgtcggcc ctgaagaaag 18120 ctccagagat gttcgggtcc gtttacgagc acgaggagaa aaagcccatg gaggcgttcg 18180 ctgaacggtt gcgagatgcc gtggcattcg gcgcctacat cgacggcgag atcattgggc 18240 tgtcggtctt caaacaggag gacggcccca aggacgctca caaggcgcat ctgtccggcg 18300 ttttcgtgga gcccgaacag cgaggccgag gggtcgccgg tatgctgctg cgggcgttgc 18360 cggcgggttt attgctcgtg atgatcgtcc gacagattcc aacgggaatc tggtggatgc 18420 gcatcttcat cctcggcgca cttaatattt cgctattctg gagcttgttg tttatttcgg 18480 tctaccgcct gccgggcggg gtcgcggcga cggtaggcgc tgtgcagccg ctgatggtcg 18540 tgttcatctc tgccgctctg ctaggtagcc cgatacgatt gatggcggtc ctgggggcta 18600 tttgcggaac tgcgggcgtg gcgctgttgg tgttgacacc aaacgcagcg ctagatcctg 18660 tcggcgtcgc agcgggcctg gcgggggcgg tttccatggc gttcggaacc gtgctgaccc 18720 gcaagtggca acctcccgtg cctctgctca cctttaccgc ctggcaactg gcggccggag 18780 gacttctgct cgttccagta gctttagtgt ttgatccgcc aatcccgatg cctacaggaa 18840 ccaatgttct cggcctggcg tggctcggcc tgatcggagc gggtttaacc tacttccttt 18900 ggttccgggg gatctcgcga ctcgaaccta cagttgtttc cttactgggc tttctcagcc 18960 ccagatctgg ggtcgatcag ccggggatgc atcaggccga cagtcggaac ttcgggtccc 19020 cgacctgtac cattcggtga gcaatggata ggggagttga tatcgtcaac gttcacttct 19080 aaagaaatag cgccactcag cttcctcagc ggctttatcc agcgatttcc tattatgtcg 19140 gcatagttct caagatcgac agcctgtcac ggttaagcga gaaatgaata agaaggctga 19200 taattcggat ctctgcgagg gagatgatat ttgatcacag gcagcaacgc tctgtcatcg 19260 ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt 19320 gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc 19380 ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc 19440 tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt aaaaattg 19500 acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgggg tggttttct 19560 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg 19620 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttcc 19680 gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt gagtgttgtt 19740 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 19800 accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg 19860 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 19920 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgcc 19980 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 20040 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 20100 gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccccc 20150 <210> 27 <211> 20150 <212> DNA <213> Artificial Sequence <220> <223> TRV RNA2 <400> 27 gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatttc gggaaacctc 60 ctcggattcc attgcccagc tatctgtcac ttcatcgaaa ggacagtaga aaaggaaggt 120 ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga tgcctctgcc 180 gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtc 240 ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt aagggatgac 300 gcacaatccc actatccttc gcaagaccct tcttctatat aaagttc atttcatttg 360 gagagcag cccaagcttt ctagaggatc cataaaacat ttcaatcctt tgaacgcggt 420 agaacgtgct aattggattt tggtgagaac gcggtagaac gtacttatca cctacagttt 480 tattttgttt ttctttttgg tttaatctat ccagcttagt accgagtggg ggaaagtgac 540 tggtgtgcct aaaacctttt ctttgatact ttgttaaaat acatacagat acaatggcga 600 660 gtcatttctt tgacttgatg ctgactaaac ctgattgtga gatcgggcaa atgatgcaaa 720 780 840 tcaatatcgt gtttaaagac gacaaaaaca tggttcatgg gtttgcggct gctgagcgaa 900 aactacaagc tttattgctt ttagatagag ttcctgctct gcaagaggtg gatgacatcg 960 gtggtcaatg gtcgttttgg gtaactagag gtgagaaaag gattcattcc tgttgtccaa 1020 atctagatat tcgggatgat cagagagaaa tttctcgaca gatatttctt actgctattg 1080 gtgatcaagc tagaagtggt aagagacaga tgtcggagaa tgagctgtgg atgtatgacc 1140 aatttcgtga aaatattgct gcgcctaacg cggttaggtg caataataca tatcagggtt 1200 gtacatgtag gggtttttct gatggtaaga agaaaggcgc gcagtatgcg atagctcttc 1260 acagcctgta tgacttcaag ttgaaagact tgatggctac tatggttgag aagaaaacta 1320 aagtggttca tgctgctatg ctttttgctc ctgaaagtat gttagtggac gaaggtccat 1380 taccttctgt tgacggttac tacatgaaga agaacgggaa gatctatttc ggttttgaga 1440 aagatccttc cttttcttac attcatgact gggaagagta caagaagtat ctactgggga 1500 agccagtgag ttaccaaggg aatgtgttct acttcgaacc gtggcaggtg agaggagaca 1560 caatgcttt ttcgatctac aggatagctg gagttccgag gaggtctcta tcatcgcaag 1620 agtactaccg aagaatatat atcagtagat gggaaagcat ggttgttgtc ccaattttcg 1680 atctggtcga atcaacgcga gagttggtca agaaagacct gtttgtagag aaacaattca 1740 tggacaagtg tttggattac atagctaggt tatctgacca gcagctgacc ataagcaatg 1800 ttaaatcata cttgagttca aataattggg tcttattcat aaacggggcg gccgtgaaga 1860 acaagcaaag tgtagattct cgagatttac agttgttggc tcaaactttg ctagtgaagg 1920 aacaagtggc gagacctgtc atgagggagt tgcgtgaagc aattctgact gagacgaaac 1980 ctatcacgtc attgactgat gtgctgggtt tatatcaag aaaactgtgg aagcagtttg 2040 ctaacaagat cgcagtcggc ggattcgttg gcatggttgg tactctaatt ggattctatc 2100 caaagaaggt actaacctgg gcgaaggaca caccaaatgg tccagaacta tgttacgaga 2160 actcgcacaa aaccaaggtg atagtatttc tgagtgttgt gtatgccatt ggaggaatca 2220 cgcttatgcg tcgagacatc cgagatggac tggtgaaaaa actatgtgat atgtttgata 2280 tcaaacgggg ggcccatgtc ttagacgttg agaatccgtg ccgctattat gaaatcaacg 2340 atttctttag cagtctgtat tcggcatctg agtccggtga gaccgtttta ccagatttat 2400 ccgaggtaaa agccaagtct gataagctat tgcagcagaa gaaagaaatc gctgacgagt 2460 ttctaagtgc aaaattctt aactattctg gcagttcggt gagaacttct ccaccatcgg 2520 tggtcggttc atctcgaagc ggactgggtc tgttgttgga agacagtaac gtgctgaccc 2580 aagctagagt tggagtttca agaaaggtag acgatgagga gatcatggag cagtttctga 2640 gtggtcttat tgacactgaa gcagaaattg acgaggttgt ttcagccttt tcagctgaat 2700 gtgaaagagg ggaaacaagc ggtacaaagg tgttgtgtaa acctttaacg ccaccaggat 2760 ttgagaacgt gttgccagct gtcaaacctt tggtcagcaa aggaaaaacg gtcaaacgtg 2820 tcgattactt ccaagtgatg ggaggtgaga gattaccaaa aaggccggtt gtcagtggag 2880 acgattctgt ggacgctaga agagagtttc tgtactactt agatgcggag agagtcgctc 2940 aaaatgatga aattatgtct ctgtatcgtg actattcgag aggagttatt cgaactggag 3000 gtcagaatta cccgcacgga ctgggagtgt gggatgtgga gatgaagaac tggtgcatac 3060 gtccagtggt cactgaacat gcttatgtgt tccaaccaga caaacgtatg gatgattggt 3120 cgggatactt agaagtggct gtttgggaac gaggtatgtt ggtcaacgac ttcgcggtcg 3180 aaaggatgag tgattatgtc atagtttgcg atcagacgta tctttgcaat aacaggtaat 3240 aatcctctct cttgatattt ttaaattata gaattaatta gtttacttta ttctttacta 3300 tatgatttaa atagtttaat cttgtttttg agtaaactat tcgattttga tatttgtatt 3360 cgtcctacaa agttggaaat actgatgata ttttcttttg aacgtgatac ctaccaatac 3420 taatcttacg gaatctttta atagagcact aatcaacatg gaactaaaga ccaattctta 3480 agtgtctctg ttgtacagtt cattttagta gtgcgtttaa gtattattat ctcccttcat 3540 gcggggcaat tatgtagatt aaaatcgaaa ttatataaaa tttacataag tctaagtcta 3600 gggtctccag ctaattgtta tttttttaac gatgttgact aaagcaataa cgacgttgac 3660 ttgtgttaaa caggttgatc ttggacaatt taagtgccct ggatctagga ccagttaact 3720 gttcttttga attagttgac ggtgtacctg gttgtggtaa gtcgacaatg attgtcaact 3780 cagctaatcc ttgtgtcgat gtggttctct ctactgggag agcagcaacc gacgacttga 3840 tcgagagatt cgcgagcaaa gttttccat gcaaattgaa aaggagtg aagacggttg 3900 attcttttt gatgcattgt gtcgatggtt ctttaaccgg agacgtgttg cattcgacg 3960 aagctctcat ggcccatgct ggtatgtgt acttttgcgc tcagatagct ggtgctaaac 4020 gatgtatctg tcaggagat cagaatcaa ttctttcaa gcctaggta tctcaagttg 4080 atttgaggtt ttctagtctg gtcggaagt ttgacattgt tacagaaaaa agagaaactt 4140 acagaagtcc agcagatgtg gctgccgtat tgaacaagta ctatactgga gatgtcagaa 4200 cacataacgc gactgctaat tcgatgacgg tgaggagat tgtgtctaaa gaacaggttt 4260 ctttgaagcc tggtgctcag tacataactt tccttcagtc tgagagaag gagttgtaa 4320 atttgttggc attgaggaaa gtggcagcta aagtgagtac agtacacgag tcgcaggg 4380 agacattca agatgtagtc ctagtcagga cgaaacctac ggatgactca atcgctagag 4440 gtcgggagta cttaatcgtg gcattgtcgc gtcacaca atcacttg tatgaaactg 4500 tgaaagagga cgatgtaagc aaagagatca gggaaagtgc cgcgcttacg aaggcggctt 4560 tggcaagatt ttttgttact gagaccgtct tatgacggtt tcggtctagg tttgatgtct 4620 ttagacatca tgaagggcct tgcgccgttc cagattcagg tacgattacg gacttggaga 4680 tgtggtacga cgctttgttt ccgggaaatt cgttaagaga ctcaagccta gacgggtatt 4740 tggtggcaac gactgattgc aatttgcgat tagacaatgt tacgatcaaa agtggaaact 4800 ggaaagacaa gtttgctgaa aaagaaacgt ttctgaaacc ggttattcgt actgctatgc 4860 ctgacaaaag gaagactact cagttggaga gtttgttagc attgcagaaa aggaaccaag 4920 cggcacccga tctacaagaa aatgtgcacg cgacagttct aatcgaagag acgatgaaga 4980 agctgaaatc tgttgtctac gatgtgggaa aaattcgggc tgatcctatt gtcaatagag 5040 ctcaaatgga gagatggtgg agaaatcaaa gcacagcggt acaggctaag gtagtagcag 5100 atgtgagaga gttacatgaa atagactatt cgtcttacat gtatatgatc aaatctgacg 5160 tgaaacctaa gactgattta acaccgcaat ttgaatactc agctctacag actgttgtgt 5220 atcacgagaa gttgatcaac tcgttgttcg gtccaatttt caaagaaatt aatgaacgca 5280 agttggatgc tatgcaacca cattttgtgt tcaacacgag aatgacatcg agtgatttaa 5340 acgatcgagt gaagttctta aatacggaag cggcttacga ctttgttgag atagacatgt 5400 ctaaattcga caagtcggca aatcgcttcc atttacaact gcagctggag atttacaggt 5460 tatttgggct ggatgagtgg gcggccttcc tttgggaggt gtcgcacact caaactactg 5520 tgagagatat tcaaaatggt atgatggcgc atatttggta ccaacaaaag agtggagatg 5580 ctgatactta taatgcaaat tcagatagaa cactgtgtgc actcttgtct gaattaccat 5640 tggagaaagc agtcatggtt acatatggag gagatgactc actgattgcg tttcctagag 5700 gaacgcagtt tgttgatccg tgtccaaagt tggctactaa gtggaatttc gagtgcaaga 5760 tttttaagta cgatgtccca atgttttgtg ggaagttctt gcttaagacg tcatcgtgtt 5820 acgagttcgt gccagatccg gtaaaagttc tgacgaagtt ggggaaaaag agtataaagg 5880 atgtgcaaca tttagccgag atctacatct cgctgaatga ttccaataga gctcttggga 5940 actacatggt ggtatccaaa ctgtccgagt ctgtttcaga ccggtatttg tacaaaggtg 6000 attctgttca tgcgctttgt gcgctatgga agcatattaa gagttttaca gctctgtgta 6060 cattattccg agacgaaac gatgaat tgaacccggc taggttt tggagagg 6120 cacagagagc tgtgtcaaac ttttacgact ggtatatgg aagacaagtc attggtcacc 6180 ttgaagaga agacttcga agtctcaaa ttctcaatc taggggccat tgaattgttt 6240 gtggacggta ggaggaagg accgaagtat ttcacagaa gagagaac tgtcctaaat 6300 catgttggtg ggaagag tgacacaag ttagacgtttt ttgaccaag ggattacaaa 6360 atgattaat cttacgcgtt tctaaaggta gtaggtgtac aactagttgt aacatcacat 6420 ctacctgcag atacgcctgg gttcattcaa atcgatctgt tggattcgag acttactgag 6480 aaaagaaga gaggaagac tattcagaga ttcaagctc gagcttgcga taactgttca 6540 gttgcgcagt acaagttga atacagtatt tccacacagg agaacgtact tgatgtctgg 6600 aaggtgggtt gtatttctga gggcgttccg gtctgtgacg gtacataccc ttcagtatc 6660 gaagtgtcgc taatatgggt tgctactgat tcgactaggc gcctcaatgt ggaagaactg 6720 aacagttcgg attacattga aggcgatttt accgatcaag aggttcgg tgttcatg 6780 tctttgaaac aagtggagat gagacgatt gaggcgaagt acgatggtcc tcagacca 6840 gctactacta gacctaagctc attattgtca agtgagatg ttaagagc gtctaataag 6900 aaaaactcgt cttaatgcat aaagaaattt attgtcaata tgacgtgtgt actcaagggt 6960 tgtgtgaatg aagtcactgt tcttggtcac gagacgtgta gtatcggtca tgctaacaaa 7020 ttgcgaaagc aagttgctga catggttggt gtcacacgta ggtgtgcgga aaataattgt 7080 ggatggttg tctgtgttgt tatcaatgat tttactttg atgtgtataa tgttgtggc 7140 cgtagtcacc ttgaaagtg tcgtaaacgt gttgaacaa gaatcgaga aatttggaaa 7200 haaattcgac gaaatcaagc tgaaacatg tctgcgacag ctaaaaagtc tcaatattcg 7260 aagaccctta agaagaattt haagaggac agagaatttg gacaccaa aagattttta 7320 agagatgatg ttccttcgg gattgatcgt tgtttgctt ttgatttta ttttattg 7380 ttatctgttt ctgtgtatag actgtttgag attggcgctt ggccgactca ttgtcttacc 7440 ataggggaac ggactttgtt tgtgttgtta ttttattgtgt atttattaa aattctcaat 7500 gatctgaaaa ggcctcgagg ctaagagatt attggggggt gagtaagtac ttttaaagtg 7560 atgatggtta caaaggcaaa aggggtaaaa cccctcgcct acgtaagcgt tattacgccc 7620 ggatcccccg gggagctcga attcgctgaa atcaccagtc tctctctaca aatctatctc 7680 tctctattt ttccataaat aatgtgtgag tagttcccg ataagggaaa ttagggttct 7740 tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt 7800 aaaatcttc tattatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat 7860 ccagatctcc taaagtccct atagatcttt gtcgtgaata taaaccagac acgagacgac 7920 taaacctgga gcccagacgc cgttcgaagc tagaagtacc gcttaggcag gaggccgtta 7980 gggaaaagat gctaaggcag ggttggttac gttgactccc ccgtaggttt ggtttaaata 8040 tgatgaagtg gacggaagga aggaggaaga caaggaagga taaggttgca ggccctgtgc 8100 aaggtaagaa gatggaaatt tgatagaggt acgctactat acttatacta tacgctaagg 8160 gaatgcttgt atttataccc tatacccct aataacccct tatcaattta agaataatc 8220 cgcataagcc cccgcttaaa aattggtatc agagccatga ataggtctat gaccaaaact 8280 caagaggata aaacctcacc aaaatacgaa agagttctta actctaaaga taaaagatct 8340 ttcaagatca aaactagttc cctcacaccg gagcatgcga tatcctcgac ctgcaggcat 8400 gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 8460 attccacaca acatacgagc cggaagcata aaggttaaag cctggggtgc ctaatgagtg 8520 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 8580 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcca 8640 aagacaaaag ggcgacattc aaccgattga gggagggaag gtaaatattg acggaaatta 8700 ttcattaaag gtgaattatc accgtcaccg acttgagcca tttgggaatt agagccagca 8760 8820 atagcagcac cgtaatcagt agcgaagaa tcaagtttgc ctttagcgtc agactgtagc 8880 gcgttttcat cggcattttc ggtcatagcc cccttattag cgtttgccat cttttcataa 8940 tcaaaatcac cggaaccaga gccaccaccg gaaccgcctc cctcagagcc gccaccctca 9000 gaaccgccac cctcagagcc accaccctca gagccgccac cagaaccacc accagagccg 9060 ccgccagcat tgacaggagg cccgatctag taacatagat gacaccgcgc gcgataattt 9120 atcctagttt gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac 9180 tctaatcata aaaacccatc tcataaataa cgtcatgcat tacatgttaa ttattacatg 9240 cttaacgtaa ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa 9300 tcttaagaaa ctttattgcc aaatgtttga acgatcgggg atcatccggg tctgtggcgg 9360 gaactccacg aaaatatccg aacgcagcaa gatatcgcgg tgcatctcgg tcttgcctgg 9420 gcagtcgccg ccgacgccgt tgatgtggac gccgggcccg atcatattgt cgctcaggat 9480 cgtggcgttg tgcttgtcgg ccgttgctgt cgtaatgata tcggcacctt cgaccgcctg 9540 ttccgcagag atcccgtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat 9600 ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga 9660 atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt 9720 cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg 9780 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata 9840 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg 9900 atgaatccag aaaagcggcc atttccacc atgatattcg gcaagcaggc atcgccatgg 9960 gtcacgacga gatcatcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct 10020 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc 10080 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga 10140 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca 10200 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc 10260 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat 10320 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa 10380 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc 10440 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc 10500 aatccatctt gttcaatcat gcgaaacgat ccagatccgg tgcagattat ttggattgag 10560 agtgaatatg agactctaat tggataccga ggggaattta tggaacgtca gtggagcatt 10620 tttgacaaga aatatttgct agctgatagt gaccttaggc gacttttgaa cgcgcaataa 10680 tggtttctga cgtatgtgct tagctcatta aactccagaa acccgcggct gagtggctcc 10740 ttcaacgttg cggttctgtc agttccaaac gtaaaacggc ttgtcccgcg tcatcggcgg 10800 gggtcataac gtgactccct taattctccg ctcatgatca gattgtcgtt tcccgccttc 10860 agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 10920 tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 10980 tatgtgcatg ccaaccacag ggttccccag atctggcgcc ggccagcgag acgagcaaga 11040 ttggccgccg cccgaaacga tccgacagcg cgcccagcac aggtgcgcag gcaaattgca 11100 ccaacgcata cagcgccagc agaatgccat agtgggcggt gacgtcgttc gagtgaacca 11160 gatcgcgcag gaggcccggc agcaccggca taatcaggcc gatgccgaca gcgtcgagcg 11220 cgacagtgct cagaattacg atcaggggta tgttgggttt cacgtctggc ctccggacca 11280 gcctccgctg gtccgattga acgcgcggat tctttatcac tgataagttg gtggacatat 11340 tatgtttatc agtgataaag tgtcaagcat gacaaagttg cagccgaata cagtgatccg 11400 tgccgccctg gacctgttga acgaggtcgg cgtagacggt ctgacgacac gcaaactggc 11460 ggaacggttg ggggttcagc agccggcgct ttactggcac ttcaggaaca agcgggcgct 11520 gctcgacgca ctggccgaag ccatgctggc ggagaatcat acgcattcgg tgccgagagc 11580 cgacgacgac tggcgctcat ttctgatcgg gaatgcccgc agcttcaggc aggcgctgct 11640 cgcctaccgc gatggcgcgc gcatccatgc cggcacgcga ccgggcgcac cgcagatgga 11700 aacggccgac gcgcagcttc gcttcctctg cgaggcgggt ttttcggccg gggacgccgt 11760 caatgcgctg atgacaatca gctacttcac tgttggggcc gtgcttgagg agcaggccgg 11820 cgacagcgat gccggcgagc gcggcggcac cgttgaacag gctccgctct cgccgctgtt 11880 gcgggccgcg atagacgcct tcgacgaagc cggtccggac gcagcgttcg agcagggact 11940 cgcggtgatt gtcgatggat tggcgaaaag gaggctcgtt gtcaggaacg ttgaaggacc 12000 gagaaagggt gacgattgat caggaccgct gccggagcgc aacccactca ctacagcaga 12060 gccatgtaga caacatcccc tccccctttc caccgcgtca gacgcccgta gcagcccgct 12120 acgggctttt tcatgccctg ccctagcgtc caagcctcac ggccgcgctc ggcctctctg 12180 gcggccttct ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgcttttc cgctgcataa ccctgcttcg gggtcattat 12600 agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt 12660 tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg 12720 cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa 12780 cgggaatcct gctctgcgag gctggccggc taccgccggc gtaacagatg agggcaagcg 12840 gatggctgat gaaaccaagc caaccaggaa gggcagccca cctatcaagg tgtactgcct 12900 tccagacgaa cgaagagcga ttgaggaaaa ggcggcggcg gccggcatga gcctgtcggc 12960 ctacctgctg gccgtcggcc agggctacaa aatcacgggc gtcgtggact atgagcacgt 13020 ccgcgagctg gcccgcatca atggcgacct gggccgcctg ggcggcctgc tgaaactctg 13080 gctcaccgac gacccgcgca cggcgcggtt cggtgatgcc acgatcctcg ccctgctggc 13140 gaagatcgaa gagaagcagg acgagcttgg caaggtcatg atgggcgtgg tccgcccgag 13200 ggcagagcca tgactttttt agccgctaaa acggccgggg ggtgcgcgtg attgccaagc 13260 acgtccccat gcgctccatc aagaagagcg acttcgcgga gctggtgaag tacatcaccg 13320 acgagcaagg caagaccgag cgcctttgcg acgctcaccg ggctggttgc cctcgccgct 13380 gggctggcgg ccgtctatgg ccctgcaaac gcgccagaaa cgccgtcgaa gccgtgtgcg 13440 agacaccgcg gccgccggcg ttgtggatac ctcgcggaaa acttggccct cactgacaga 13500 tgaggggcgg acgttgacac ttgaggggcc gactcacccg gcgcggcgtt gacagatgag 13560 gggcaggctc gatttcggcc ggcgacgtgg agctggccag cctcgcaaat cggcgaaaac 13620 gcctgatttt acgcgagttt cccacagatg atgtggacaa gcctggggat aagtgccctg 13680 cggtattgac acttgagggg cgcgactact gacagatgag gggcgcgatc cttgacactt 13740 gaggggcaga gtgctgacag atgaggggcg cacctattga catttgaggg gctgtccaca 13800 ggcagaaaat ccagcatttg caagggttttc cgcccgtttt tcggccaccg ctaacctgtc 13860 ttttaacctg cttttaaacc aatatttata aaccttgttt ttaaccaggg ctgcgccctg 13920 tgcgcgtgac cgcgcacgcc gaaggggggt gccccccct ctcgaaccct cccggcccgc 13980 taacgcgggc ctcccatccc cccaggggct gcgcccctg gccgcgaacg gcctcacccc 14040 aaaaatggca gcgctggcag tccttgccat tgccgggatc ggggcagtaa cgggatgggc 14100 gatcagcccg agcgcgacgc ccggaagcat tgacgtgccg caggtgctgg catcgacatt 14160 cagcgaccag gtgccgggca gtgagggcgg cggcctgggt ggcggcctgc ccttcacttc 14220 ggccgtcggg gcattcacgg acttcatggc ggggccggca atttttacct tgggcattct 14280 tggcatagtg gtcgcgggtg ccgtgctcgt gttcgggggt gcgataaacc cagcgaacca 14340 tttgaggtga taggtaagat tataccgagg tatgaaaacg agaattggac ctttacagaa 14400 ttactctatg aagcgccata tttaaaaagc taccaagacg aagaggatga agaggatgag 14460 gaggcagatt gccttgaata tattgacaat actgataaga tatatatct tttatataga 14520 agatatcgcc gtatgtaagg atttcagggg gcaaggcata ggcagcgcgc ttatcaatat 14580 atctatagaa tgggcaaagc ataaaaactt gcatggacta atgcttgaaa cccaggacaa 14640 taaccttata gcttgtaaat tctatcataa ttgggtaatg actccaactt attgatagtg 14700 tttatgttc agataatgcc cgatgacttt gtcatgcagc tccaccgatt ttgagaacga 14760 cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga ttcaggttat gccgctcaat 14820 tcgctgcgta tatcgcttgc tgattacgtg cagctttccc ttcaggcggg attcatacag 14880 cggccagcca tccgtcatcc atatcaccac gtcaaagggt gacagcaggc tcataagacg 14940 ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca acaaccgtct tccggagact 15000 gtcatacgcg taaaacagcc agcgctggcg cgatttagcc ccgacatagc cccactgttc 15060 gtccatttcc gcgcagacga tgacgtcact gcccggctgt atgcgcgagg ttaccgactg 15120 cggcctgagt ttttaagtg acgtaaaatc gtgttgaggc caacgcccat aatgcgggct 15180 gttgcccggc atccaacgcc attcatggcc atatcaatga ttttctggtg cgtaccgggt 15240 tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac ggcagtgaga gcagagatag 15300 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag 15360 15420 cattaagttg gcagcatcac ccataattgt ggtttcaaaa tcggctccgt cgatactatg 15480 ttatacgcca actttgaaaa caactttgaa aaagctgttt tctggtattt aaggttttag 15540 15600 aaatactgta gaaaagagga aggaataat aaatggctaa aatgagaata tcaccggaat 15660 tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac ggaaggaatg tctcctgcta 15720 aggtataaa gctgggtggga gaaaatgaaa acctatattt aaaaatgacg gacagccggt 15780 ataaagggac cacctatgat gtggaacggg aaaaggacat gatgctatgg ctggaaggaa 15840 agctgcctgt tccaaaggtc ctgcactttg aacggcatga tggctggagc aatctgctca 15900 tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga agatgaacaa agccctgaaa 15960 agattatcga gctgtatgcg gagtgcatca ggctctttca ctccatcgac atatcggatt 16020 gtccctatac gaatagctta gacagccgct tagccgaatt ggattactta ctgaataacg 16080 atctggccga tgtggattgc gaaaactggg aagaagacac tccattaaa gatccgcgcg 16140 agctgtatga ttttttaaag acggaaaagc ccgaagagga acttgtcttt tcccacggcg 16200 acctgggaga cagcaacatc tttgtgaaag atggcaaagt aagtggcttt attgatcttg 16260 ggagaagcgg cagggcggac aagtggtatg acattgcctt ctgcgtccgg tcgatcaggg 16320 aggatatcgg ggaagaacag tatgtcgagc tattttttga cttactgggg atcaagcctg 16380 attgggagaa aataaaatat tatattttac tggatgaatt gttttagtac ctagatgtgg 16440 cgcaacgatg ccggcgacaa gcaggagcgc accgacttct tccgcatcaa gtgttttggc 16500 tctcaggccg aggcccacgg caagtatttg ggcaaggggt cgctggtatt cgtgcagggc 16560 aagattcgga ataccaagta cgagaaggac ggccagacgg tctacgggac cgacttcatt 16620 gccgataagg tggattatct ggacaccaag gcaccaggcg ggtcaaatca ggaataaggg 16680 cacattgccc cggcgtgagt cggggcaatc ccgcaaggag ggtgaatgaa tcggacgttt 16740 gaccggaagg catacaggca agaactgatc gacgcggggt tttccgccga ggatgccgaa 16800 accatcgcaa gccgcaccgt catgcgtgcg ccccgcgaaa ccttccagtc cgtcggctcg 16860 atggtccagc aagctacggc caagatcgag cgcgacagcg tgcaactggc tccccctgcc 16920 ctgcccgcgc catcggccgc cgtggagcgt tcgcgtcgtc tcgaacagga ggcggcaggt 16980 ttggcgaagt cgatgaccat cgacacgcga ggaactatga cgaccaagaa gcgaaaaaacc 17040 gccggcgagg acctggcaaa acaggtcagc gaggccaagc aggccgcgtt gctgaaacac 17100 acgaagcagc agatcaagga aatgcagctt tccttgttcg atattgcgcc gtggccggac 17160 acgatgcgag cgatgccaaa cgacacggcc cgctctgccc tgttcaccac gcgcaacaag 17220 aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc acgtcaacaa ggacgtgaag 17280 atcacctaca ccggcgtcga gctgcgggcc gacgatgacg aactggtgtg gcagcaggtg 17340 ttggagtacg cgaagcgcac ccctatcggc gagccgatca ccttcacgtt ctacgagctt 17400 tgccaggacc tgggctggtc gatcaatggc cggtattaca cgaaggccga ggaatgcctg 17460 tcgcgcctac aggcgacggc gatgggcttc acgtccgacc gcgttgggca cctggaatcg 17520 gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca agaaaacgtc ccgttgccag 17580 gtcctgatcg acgaggaaat cgtcgtgctg tttgctggcg accactacac gaaattcata 17640 tgggagaagt accgcaagct gtcgccgacg gcccgacgga tgttcgacta tttcagctcg 17700 caccgggagc cgtacccgct caagctggaa accttccgcc tcatgtgcgg atcggattcc 17760 acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct gcgaagagtt gcgaggcagc 17820 ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc attgcaaacg ctagggcctt 17880 gtggggtcag ttccggctgg gggttcagca gccagcgctt tactggcatt tcaggaacaa 17940 gcgggcactg ctcgacgcac ttgcttcgct cagtatcgct cgggacgcac ggcgcgctct 18000 acgaactgcc gataaacaga ggattaaaat tgacaattgt gattaaggct cagattcgac 18060 ggcttggagc ggccgacgtg caggatttcc gcgagatccg attgtcggcc ctgaagaaag 18120 ctccagagat gttcgggtcc gtttacgagc acgaggagaa aaagcccatg gaggcgttcg 18180 ctgaacggtt gcgagatgcc gtggcattcg gcgcctacat cgacggcgag atcattgggc 18240 tgtcggtctt caaacaggag gacggcccca aggacgctca caaggcgcat ctgtccggcg 18300 ttttcgtgga gcccgaacag cgaggccgag gggtcgccgg tatgctgctg cgggcgttgc 18360 cggcgggttt attgctcgtg atgatcgtcc gacagattcc aacgggaatc tggtggatgc 18420 gcatcttcat cctcggcgca cttaatattt cgctattctg gagcttgttg tttatttcgg 18480 tctaccgcct gccgggcggg gtcgcggcga cggtaggcgc tgtgcagccg ctgatggtcg 18540 tgttcatctc tgccgctctg ctaggtagcc cgatacgatt gatggcggtc ctgggggcta 18600 tttgcggaac tgcgggcgtg gcgctgttgg tgttgacacc aaacgcagcg ctagatcctg 18660 tcggcgtcgc agcgggcctg gcgggggcgg tttccatggc gttcggaacc gtgctgaccc 18720 gcaagtggca acctcccgtg cctctgctca cctttaccgc ctggcaactg gcggccggag 18780 gacttctgct cgttccagta gctttagtgt ttgatccgcc aatcccgatg cctacaggaa 18840 ccaatgttct cggcctggcg tggctcggcc tgatcggagc gggtttaacc tacttccttt 18900 ggttccgggg gatctcgcga ctcgaaccta cagttgtttc cttactgggc tttctcagcc 18960 ccagatctgg ggtcgatcag ccggggatgc atcaggccga cagtcggaac ttcgggtccc 19020 cgacctgtac cattcggtga gcaatggata ggggagttga tatcgtcaac gttcacttct 19080 aaagaaatag cgccactcag cttcctcagc ggctttatcc agcgatttcc tattatgtcg 19140 gcatagttct caagatcgac agcctgtcac ggttaagcga gaaatgaata agaaggctga 19200 taattcggat ctctgcgagg gagatgatat ttgatcacag gcagcaacgc tctgtcatcg 19260 ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt 19320 gccgttcttc cgaatagcat cggtaacatg agcaagtct gccgccttac aacggctctc 19380 ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgatt tgtgccgagc 19440 tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg 19500 acgcttagac aacttaataa cacattgcgg acgtttta tgtactgggg tggttttct 19560 ttcaccagt gagacgggca acagctgat gcctcacc gcctggccct gagagagttg 19620 cagcaagcgg tccacgctgg ttgccccag caggcgaaaa tcctgtttga tggtggttcc 19680 gaatcggca aaatccctta taaatcaaa gatagccccg agataggtt gagtgttgtt 19740 ccagtttgga acagagtcc actattaaag aacgtggact ccaacgtca agggcgaaaa 19800 accgtctatc agggcgatgg cccactacgt gaaccatcac ccaatcaag ttttgggg 19860 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gccccgatt tagagcttga 19920 cgggaaagc cggcgaacgt ggcgagaaag gaaggaag aagcgaagg agcggggcgcc 19980 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 20040 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 20100 gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccccc 20150 BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0079] I don't want to be bound by theory. Figure 1 Depicted are the effects of overexpressing the SOUL heme-binding protein on pyridine alkaloid biosynthesis. (A) Upon sensing wounds or herbivory, surface-localized receptors induce immune signaling via phosphorylation of MAPKs and the production of reactive oxygen species (ROS). This activates jasmonic acid (JA)-responsive genes and upregulates pyridine alkaloid production. Intracellular ROS levels are tightly regulated through heme oxygenase (HO)-mediated heme catabolism. (B) SOUL overexpression reduces the available heme for HO degradation. This leads to an increase in intracellular ROS, which upregulates the nicotine biosynthesis pathway. PM: plasma membrane.

[0080] Figure 2 Shown are vectors for transient overexpression of SOUL heme-binding protein in tobacco.

[0081] Figure 3 Shown are the alkaloid (nicotine, nornicotine, anabasine, PON, and anatabine) contents of 5-week-old TN90 leaves expressing Nitab4.5_0013616g0010.2 (SEQ ID No. 3). Alkaloid contents are expressed relative to the control and include three biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0082] Figure 4Shown are the alkaloid (nicotine, nornicotine, anabasine, PON, and anatabine) contents of 5-week-old TN90 leaves expressing a construct that silenced Nitab4.5_0013616g0010.2 (SEQ ID No. 3) via virus-induced gene silencing (VIGS). Alkaloid contents are expressed relative to controls and include three biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P values ​​≤ 0.001.

[0083] Figure 5 The amino acid sequence of Nitab4.5_0013616g0010.2 is shown - SEQ ID No. 1 - from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0084] Figure 6 The genomic sequence of Nitab4.5_0013616g0010.2 – SEQ ID No. 2 – is shown – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0085] Figure 7 The coding sequence of Nitab4.5_0013616g0010.2 is shown – SEQ ID No. 3 – encoding a protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0086] Figure 8 The amino acid sequence of Nitab4.5_0000652g0130.2 is shown – SEQ ID No. 4 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0087] Figure 9 The genomic sequence of Nitab4.5_0000652g0130.2 – SEQ ID No. 5 – is shown – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0088] Figure 10 The coding sequence of Nitab4.5_0000652g0130.2 is shown - SEQ ID No. 6 - encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0089] Figure 11The amino acid sequence of Nitab4.5_0001140g0220.2 is shown – SEQ ID No. 7 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0090] Figure 12 The genomic sequence of Nitab4.5_0001140g0220.2 is shown – SEQ ID No. 8 – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0091] Figure 13 The coding sequence of Nitab4.5_0001140g0220.2 is shown - SEQ ID No. 9 - encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0092] Figure 14 The amino acid sequence of Nitab4.5_0003235g0060.2 is shown – SEQ ID No. 10 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0093] Figure 15 The genomic sequence of Nitab4.5_0003235g0060.2 – SEQ ID No. 11 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0094] Figure 16 The coding sequence of Nitab4.5_0003235g0060.2 - SEQ ID No. 12 - is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0095] Figure 17 The amino acid sequence of Nitab4.5_0004868g0020.2 is shown – SEQ ID No. 13 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0096] Figure 18 The genomic sequence of Nitab4.5_0004868g0020.2 – SEQ ID No. 14 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0097] Figure 19 The coding sequence of Nitab4.5_0004868g0020.2 - SEQ ID No. 15 - is shown. It encodes a protein derived from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0098] Figure 20 The amino acid sequence of Nitab4.5_0006614g0010.2 is shown – SEQ ID No. 16 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0099] Figure 21 The genomic sequence of Nitab4.5_0006614g0010.2 – SEQ ID No. 17 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0100] Figure 22 The coding sequence of Nitab4.5_0006614g0010.2 – SEQ ID No. 18 – is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0101] Figure 23 The amino acid sequence of Nitab4.5_0006991g0050.2 is shown – SEQ ID No. 19 – from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0102] Figure 24 The genomic sequence of Nitab4.5_0006991g0050.2 – SEQ ID No. 20 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0103] Figure 25 The coding sequence of Nitab4.5_0006991g0050.2 - SEQ ID No. 21 - is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0104] Figure 26 The amino acid sequence of Nitab4.5_0009023g0010.2 is shown – SEQ ID No. 22 – from Nicotiana tabacum ( Nicotiana tabacum)'s SOUL heme-binding protein.

[0105] Figure 27 The genomic sequence of Nitab4.5_0009023g0010.2 – SEQ ID No. 23 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0106] Figure 28 The coding sequence of Nitab4.5_0009023g0010.2 - SEQ ID No. 24 - is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum )'s SOUL heme-binding protein.

[0107] Figure 29 SEQ ID No. 25, the 300-nucleotide cDNA fragment used in Example 2, is shown.

[0108] Figure 30 SEQ ID No. 26, sequence TRV1 used in Example 2, is shown.

[0109] Figure 31 SEQ ID No. 27, the sequence TRV2 used in Example 2, is shown.

[0110] Figure 32 Shown are the nornicotine levels of 5-week-old TN90 leaves transiently expressing the indicated constructs: OE = expressing Nitab4.5_0013616g0010.2; or As = silencing the antisense construct of Nitab4.5_0013616g0010.2. Levels are expressed relative to the control and include three biological replicates analyzed by one-way ANOVA with Tukey's multiple comparison post hoc test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0111] Figure 33 Shown are the nornicotine levels of 5-week-old TN90 leaves transiently expressing constructs encoding the homologous sequences shown (see Example 3). Levels are expressed as a percentage relative to the control (100%) and include two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM.

[0112] Figure 34 Shown are the nornicotine levels of 12-leaf stage greenhouse-grown TN90 plants overexpressing Nitab4.5_0013616g0010.2 or control plants (n=24).

[0113] Figure 35Inset (A) shows the nornicotine content of field-grown control (wild-type WT) TN90 leaves and Nitab4.5_0013616g0010.2 overexpressors (OX). Content is expressed relative to the control. Results were analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P values ​​≤ 0.001. Inset (B) shows NNN content of field-grown control (WT) TN90 leaves and Nitab4.5_0013616g0010.2 overexpressors. Content is expressed relative to the control. Results were analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance of P values ​​≤ 0.01.

[0114] Sequence Listing

[0115] A summary of sequence identifiers used throughout the subject specification and the corresponding sequence listing is provided, wherein:

[0116] SEQ ID No. 1 corresponds to the amino acid sequence of Nitab4.5_0013616g0010.2.

[0117] SEQ ID No. 2 corresponds to the genomic sequence of Nitab4.5_0013616g0010.2.

[0118] SEQ ID No. 3 corresponds to the coding sequence of Nitab4.5_0013616g0010.2.

[0119] SEQ ID No. 4 corresponds to the amino acid sequence of Nitab4.5_0000652g0130.2.

[0120] SEQ ID No. 5 corresponds to the genomic sequence of Nitab4.5_0000652g0130.2.

[0121] SEQ ID No. 6 corresponds to the coding sequence of Nitab4.5_0000652g0130.2.

[0122] SEQ ID No. 7 corresponds to the amino acid sequence of Nitab4.5_0001140g0220.2.

[0123] SEQ ID No. 8 corresponds to the genomic sequence of Nitab4.5_0001140g0220.2.

[0124] SEQ ID No. 9 corresponds to the coding sequence of Nitab4.5_0001140g0220.2.

[0125] SEQ ID No. 10 corresponds to the amino acid sequence of Nitab4.5_0003235g0060.2.

[0126] SEQ ID No. 11 corresponds to the genomic sequence of Nitab4.5_0003235g0060.2.

[0127] SEQ ID No. 12 corresponds to the coding sequence of Nitab4.5_0003235g0060.2.

[0128] SEQ ID No. 13 corresponds to the amino acid sequence of Nitab4.5_0004868g0020.2.

[0129] SEQ ID No. 14 corresponds to the genomic sequence of Nitab4.5_0004868g0020.2.

[0130] SEQ ID No. 15 corresponds to the coding sequence of Nitab4.5_0004868g0020.2.

[0131] SEQ ID No. 16 corresponds to the amino acid sequence of Nitab4.5_0006614g0010.2.

[0132] SEQ ID No. 17 corresponds to the genomic sequence of Nitab4.5_0006614g0010.2.

[0133] SEQ ID No. 18 corresponds to the coding sequence of Nitab4.5_0006614g0010.2.

[0134] SEQ ID No. 19 corresponds to the amino acid sequence of Nitab4.5_0006991g0050.2.

[0135] SEQ ID No. 20 corresponds to the genomic sequence of Nitab4.5_0006991g0050.2.

[0136] SEQ ID No. 21 corresponds to the coding sequence of Nitab4.5_0006991g0050.2.

[0137] SEQ ID No. 22 corresponds to the amino acid sequence of Nitab4.5_0009023g0010.2.

[0138] SEQ ID No. 23 corresponds to the genomic sequence of Nitab4.5_0009023g0010.2.

[0139] SEQ ID No. 24 corresponds to the coding sequence of Nitab4.5_0009023g0010.2.

[0140] SEQ ID No. 25 is the 300-nucleotide cDNA fragment used in Example 2.

[0141] SEQ ID No. 26 is the sequence TRV1 used in Example 2.

[0142] SEQ ID No. 27 is the sequence TRV2 used in Example 2.

[0143] Some of the sequences disclosed herein contain "X" or "N" in the nucleotide sequence. An "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"s or "N"s is shown. The number of "X"s or "N"s does not necessarily correlate with the actual number of nucleotides at that position. More or fewer nucleotides than indicated by "X"s or "Ns" may be present in the sequence.

[0144] Details

[0145] The present inventors have shown for the first time that by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein in a plant (eg a tobacco plant), the alkaloid and / or TSNA content of the plant (or processed plant) can be modulated.

[0146] The present invention provides a method of 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 a SOUL heme-binding protein.

[0147] Also provided are methods of modulating (e.g., reducing) the level of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part thereof, the method comprising modifying the plant by modulating (e.g., reducing) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0148] The at least one gene encoding a SOUL heme-binding protein may be selected from at least one gene encoding a SOUL heme-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein the at least one gene encoding a SOUL heme-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 or 24, or a functional variant or functional fragment or ortholog 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 or 24, 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 or 24, or a nucleic acid sequence that is at least 80% identical to SEQ ID No.

[0149] Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

[0150] Suitably, more than one SOUL heme-binding protein may be modified. In one embodiment, at least one SOUL hemoglobin-binding protein gene is modified and is 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 or 22, 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 or 22; or wherein the at least one SOUL hemoglobin-binding protein gene 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 or 24, or a functional variant or functional fragment or ortholog 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 or 24 nucleic acid sequences with at least 80% identity.

[0151] In one embodiment, at least two SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding the SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 nucleic acid sequences with at least 80% identity.

[0152] In one embodiment, at least three SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one gene encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 nucleic acid sequences with at least 80% identity.

[0153] In one embodiment, at least four SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 nucleic acid sequences with at least 80% identity.

[0154] In one embodiment, at least five SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog 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 or 24 nucleic acid sequences with at least 80% identity.

[0155] In one embodiment, at least six SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 nucleic acid sequences with at least 80% identity.

[0156] In one embodiment, at least seven SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog 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 or 24 nucleic acid sequences with at least 80% identity.

[0157] In one embodiment, at least eight SOUL hemoglobin-binding protein genes are modified and are selected from genes encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein at least one of the genes encoding a SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog 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 or 24 nucleic acid sequences with at least 80% identity.

[0158] Suitably, the SOUL heme-binding proteins of the invention may comprise a SOUL motif.

[0159] In one aspect, the at least one SOUL hemoglobin-binding protein gene encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 1, or a functional variant, functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID No. 1; or wherein the at least one gene encoding the SOUL hemoglobin-binding protein comprises the nucleotide sequence as shown in SEQ ID No. 2 or 3, or a functional variant, 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.

[0160] Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

[0161] 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) further genes selected from SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, 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 or 24, or a nucleic acid 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 or 24 may also be modulated. Suitably, the SOUL haemoglobin-binding protein may comprise a SOUL motif.

[0162] The "activity" of a gene encoding a SOUL heme-binding protein may refer to the ability of the SOUL heme-binding protein produced by the gene to bind to other molecules, such as other proteins. Suitably, the "activity" of a gene encoding a SOUL heme-binding protein refers to the ability of the SOUL heme-binding protein produced by the gene to bind to heme.

[0163] "Expression" of a gene encoding SOUL heme-binding protein may refer to the level of transcription, translation, ie, protein expression.

[0164] 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 not modified according to the invention.

[0165] As defined herein, the term "comparable product" will be a product derived from a plant (e.g., a tobacco plant) that has not been modified according to the present invention, but wherein all other relevant characteristics are the same (e.g., plant species, growing conditions, methods of processing the plant, such as tobacco, etc.). A comparable product according to the present invention may mean a plant (e.g., a tobacco plant) or part thereof, such as 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), or plant propagation material (e.g., tobacco plant propagation material), or a product, such as a tobacco product, or a combination thereof, comprising said plant or part thereof. In one embodiment, a comparable product is a product that does not comprise a gene encoding a SOUL heme-binding protein, the activity or expression of which has been modulated.

[0166] As used herein, the term "modification" or "modified" refers to a plant (e.g., a tobacco plant) or nucleic acid sequence that has been altered or changed. The present invention encompasses plant modifications 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, transgenic, 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.

[0167] As defined herein, the term "unmodified plant" will be a plant (e.g., a tobacco plant) that has not been modified according to the present invention, for example, to modulate the activity or expression of a gene encoding a SOUL heme-binding protein or to modify the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein; and wherein all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing tobacco, etc.). In one embodiment, an unmodified plant is a plant that does not comprise a gene encoding a SOUL heme-binding protein whose activity or expression has been modulated. In one embodiment, an unmodified plant is a plant that does not comprise a modified nucleic acid sequence encoding at least one gene encoding a SOUL heme-binding protein.

[0168] SOUL heme-binding protein

[0169] As used herein, "SOUL heme-binding protein" has its ordinary meaning in the art and refers to a protein comprising a SOUL motif, which is a hydrophobic cleft flanked by α-helices and β-loops.

[0170] SOUL heme-binding proteins are found in bacteria, plants, and animals. SOUL heme-binding proteins bind tetrapyrroles such as heme (Takahashi et al. (2008) Photochem. Photobiol. Sci. 7, 1216-1224). It has been proposed that SOUL proteins in plants may be involved in transporting heme to other heme-containing proteins such as cytochrome P450 and in binding free heme (Freire et al. (2009) Acta Cryst. F65, 723-726).

[0171] The three-dimensional structure of the mouse SOUL heme-binding protein has been determined. The mouse SOUL structure consists of nine twisted β-barrels, two α-helices, and a hydrophobic cleft. The hydrophobic cleft is flanked by one of the α-helices and the β8-9 loop, forming a conserved "SOUL motif," which is crucial for heme binding (Dias et al. (2006) J. Biol. Chem. 276, 18161-18168). A similar structure has been predicted for the Arabidopsis SOUL heme-binding protein (Takahashi et al. (2008) Photochem. Photobiol. Sci. 7, 1216-1224).

[0172] In one embodiment, the SOUL heme-binding protein comprises a SOUL motif. As used herein, the term "SOUL motif" refers to a conserved structural motif. SOUL heme-binding protein and the SOUL motif can be identified by comparing the predicted protein structure to known protein structures. For example, the SOUL heme-binding protein and the SOUL motif can be identified by comparing the predicted protein structure to the predicted structure of SEQ ID No. 1, wherein the presence of a hydrophobic cleft flanked by α-helices and β-loops identifies the protein as a SOUL heme-binding protein.

[0173] In one embodiment, the SOUL motif is a region of the protein corresponding to a hydrophobic cleft flanked by α-helices and β-loops.The SOUL motif may be involved in heme-binding.

[0174] Without wishing to be bound by theory, it is hypothesized that increasing the content of SOUL heme-binding protein in plant cells or increasing the activity of SOUL heme-binding protein in plants, such as heme-binding activity, will result in an increase in the binding of free heme. A reduction in the level of free heme will mean a reduction in the degradation of heme by heme oxygenase, resulting in a reduction in the removal of reactive oxygen species (ROS). ROS stimulates the activation of nicotine biosynthesis genes. An increase in the activity or expression of SOUL heme-binding protein thus results in an increase in pyridine alkaloid levels. Conversely, according to this hypothesis, a reduction in the level or activity (such as heme-binding activity) of SOUL heme-binding protein in plant cells will increase the amount of free heme, resulting in an increase in ROS removal, and therefore reducing the activation of nicotine biosynthesis genes, thereby reducing the level of pyridine alkaloids in the cell. This hypothesis is graphically depicted in Figure 1 middle.

[0175] In one embodiment, the SOUL heme-binding protein comprises the amino acid sequence shown in SEQ ID No. 1, or a sequence having at least 80% identity thereto, or a homologue thereof. Suitably, the SOUL heme-binding protein may comprise a SOUL motif. Suitably, a homologue of SEQ ID No. 1 may be selected from the group consisting of: SEQ ID No. 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% identity thereto. Suitably, a homologue of SEQ ID No. 1 may be selected from the group consisting of: SEQ ID No. 4, 7, 10, 13, 16, 19 or 22, wherein the sequence comprises a SOUL motif, or a sequence having at least 80% identity thereto and comprising a SOUL motif.

[0176] In one embodiment, the SOUL heme-binding protein comprises an amino acid sequence as set forth in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence that is at least 80% identical thereto (preferably at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto). In one embodiment, the SOUL heme-binding protein comprises an amino acid sequence as set forth in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence that is at least 80% identical thereto (preferably at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto) and comprises a SOUL motif.

[0177] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0178] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0179] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0180] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0181] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0182] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0183] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0184] Suitably, the SOUL hemoglobin-binding protein according to the present invention may comprise the amino acid sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0185] In one embodiment, the SOUL hemoglobin-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 or 22.

[0186] Suitably, the protein may be derived from tobacco.

[0187] In one embodiment, the SOUL hemoglobin-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 or 24; or a sequence having at least 80% sequence identity thereto. Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0188] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0189] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0190] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0191] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0192] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0193] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0194] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0195] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0196] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0197] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0198] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0199] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0200] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0201] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0202] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0203] Suitably, the SOUL hemoglobin-binding protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence 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 97% or at least 99% identity thereto). Suitably, the SOUL hemoglobin-binding protein may comprise a SOUL motif.

[0204] In one embodiment, the SOUL hemoglobin-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 or 24.

[0205] Suitably, the protein for use according to the present invention may be encoded by a polynucleotide sequence from Nicotiana tabacum.

[0206] In one aspect, 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), comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0207] 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 a SOUL heme-binding protein, the SOUL heme-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a SOUL heme-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 or 24, 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 or 24, 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 or 24. Suitably, the SOUL haemoglobin-binding protein may comprise a SOUL motif.

[0208] In one aspect, the present invention provides a method for reducing the level of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a plant or 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 a SOUL heme-binding protein.

[0209] In one aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a plant or 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 a SOUL heme-binding protein, the SOUL heme-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 or 24, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a SOUL heme-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 or 24, or a sequence having at least 80% identity thereto. No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, a functional variant or functional fragment or ortholog, 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 or 24.

[0210] As used herein, the term "reduce" or "inhibit" (e.g., inhibit the activity or expression of a gene encoding a SOUL heme-binding repeat protein) means that the activity or expression of a gene encoding a SOUL heme-binding repeat protein is lower or reduced compared to the activity or expression of the gene in a comparable product.

[0211] In one aspect, the present invention provides a method of 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 a SOUL heme-binding protein.

[0212] In one aspect, the present invention provides a method for increasing the alkaloid content of 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 a SOUL heme-binding protein, the SOUL heme-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a SOUL heme-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 or 24, 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 or 24, 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 or 24. Suitably, the SOUL haemoglobin-binding protein may comprise a SOUL motif.

[0213] In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamines (TSNAs) or 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 a SOUL heme-binding protein.

[0214] In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamines (TSNAs) or 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 a SOUL heme-binding protein, the SOUL heme-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, or 24, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a SOUL heme-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, or 24, or a sequence having at least 80% identity thereto. No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, a functional variant or functional fragment or ortholog, 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 or 24.

[0215] As used herein, the term "increase" or "enhance" (e.g., increase the activity or expression of a gene encoding a SOUL heme-binding repeat protein) means that the activity or expression of a gene encoding a SOUL heme-binding repeat protein is higher or increased compared to the activity or expression of the gene in a comparable product.

[0216] According to the present invention, the activity or expression of the gene encoding the SOUL heme-binding protein is modulated.

[0217] 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 a SOUL heme-binding protein.

[0218] The term "activity" refers to any function of the SOUL heme-binding protein encoded by at least one gene. Examples of activity include enzymatic activity or localization of the SOUL heme-binding protein. Suitably, the activity is the ability of the SOUL heme-binding protein to interact with another molecule or molecules. In some embodiments, the present 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 SOUL heme-binding protein to interact with another molecule.

[0219] Suitably, the ability of the SOUL heme-binding protein to interact with other molecules is the ability to bind to other molecules. The other molecules may be proteins. The other molecules may be tetrapyrroles. The other molecules may be heme. In some embodiments, the present invention provides methods for modulating (i.e., increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), the methods comprising modifying the plant by modulating (i.e., increasing or decreasing) the ability of the SOUL heme-binding protein to bind heme.

[0220] Suitably, the other molecules are more than one molecule, such as one or more molecules, such as two or more molecules, such as three or more molecules. In the case where the other molecules are more than one molecule, the other molecules may be the same molecule or may be different molecules. In the case where the other molecules are more than one molecule, at least one of the other molecules may be heme.

[0221] Modulation of the activity of a gene encoding a SOUL heme-binding protein may entail increasing or decreasing the activity of the SOUL heme-binding protein.

[0222] Increasing the activity of a SOUL heme-binding protein refers to enhancing or improving the ability of the SOUL heme-binding protein to carry out a specific function compared to the SOUL heme-binding protein in a plant that has not been modified according to the present invention.

[0223] Reducing the activity of a SOUL heme-binding protein means reducing, inhibiting or disrupting the ability of the SOUL heme-binding protein to perform a specific function compared to the SOUL heme-binding protein in a plant that has not been modified according to the present invention. The activity of the SOUL heme-binding protein can be reduced to such an extent that the activity is prevented or eliminated.

[0224] In some embodiments, the activity of the SOUL heme-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 a gene encoding the SOUL heme-binding protein in a plant (e.g., a tobacco plant) not modified according to the invention.

[0225] In some embodiments, the modulated SOUL heme-binding protein exhibits an increased or decreased activity compared to unmodified SOUL heme-binding protein. The modulated SOUL heme-binding protein can 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 unmodified SOUL heme-binding protein.

[0226] Suitably, modulation of activity is an increase or decrease in the ability of the SOUL heme-binding protein to interact with (such as bind to) another molecule or molecules (such as heme).

[0227] In some embodiments, the present invention provides a method for regulating the alkaloid content of a plant, or a part or cell thereof, the method comprising modifying the plant by modulating the ability of a SOUL heme-binding protein to bind heme. Suitably, the ability of a SOUL heme-binding protein to bind heme is increased. Suitably, the ability of a SOUL heme-binding protein to bind heme is decreased. An increase or decrease in the ability of a SOUL heme-binding protein to bind another molecule can be expressed as an increase or decrease in the binding affinity of the SOUL heme-binding protein. In some embodiments, the present invention provides a method for regulating the alkaloid content of a plant, or a part or cell thereof, the method comprising modifying the plant by modulating the binding affinity of a SOUL heme-binding protein for heme.

[0228] Techniques for measuring protein activity are known in the art. For example, assays for measuring the enzymatic activity of proteins are known, and microscopy techniques can be used to identify the localization of proteins.

[0229] Specifically, the ability of a SOUL heme-binding protein 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 microlayer thermophoresis. For example, the ability of a modulated or mutated SOUL heme-binding protein to bind to another molecule can be determined by performing a co-immunoprecipitation experiment using a modulated or mutated SOUL heme-binding protein and a corresponding unmodified or unmutated SOUL heme-binding protein. If the modulation or mutation of the SOUL heme-binding protein reduces, inhibits, or eliminates the ability of the SOUL heme-binding protein to bind to another molecule, the co-immunoprecipitation will show that the modulated or mutated SOUL heme-binding protein binds less to the other molecule.

[0230] According to the present invention, the activity or expression of SOUL heme-binding protein is modulated.

[0231] 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), comprising modifying the plant by modulating (i.e., increasing or decreasing) the expression of at least one gene encoding a SOUL heme-binding protein.

[0232] "Expression" of a gene refers to the degree to which the information encoded in the gene is converted into function. The expression level of a gene can be equated with the amount of the gene product present in a cell or organism. Modifications that modulate (i.e., increase or decrease) gene expression are modifications that increase the amount of the gene product in a plant or cell compared to an unmodified plant or cell.

[0233] In some embodiments, expression of the SOUL heme-binding protein is modulated (ie, increased or decreased) compared to expression of a gene encoding the SOUL heme-binding protein in a plant (eg, a tobacco plant) that has not been modified according to the invention.

[0234] In some embodiments, expression of the SOUL heme-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 a gene encoding the SOUL heme-binding protein in a plant (e.g., a tobacco plant) not modified according to the invention.

[0235] In some embodiments, the modulated SOUL heme-binding protein exhibits increased or decreased expression compared to unmodified SOUL heme-binding protein. The modulated SOUL heme-binding protein can exhibit 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 unmodified SOUL heme-binding protein.

[0236] Typically, genes are transcribed into mRNA, which is translated into proteins, the ultimate gene products. Proteins can be sequestered in cells for storage and / or degraded. Gene expression can be modulated by regulating any or all of these steps. Thus, in some embodiments, the modification modulates expression of at least one SOUL hemoglobin-binding protein gene in one of the following ways:

[0237] regulating transcription from at least one SOUL heme-binding protein gene;

[0238] regulating translation of mRNA from at least one SOUL heme-binding protein gene;

[0239] Modulating the release of SOUL heme-binding protein from intracellular stores; and / or

[0240] Regulates the degradation rate of SOUL heme-binding protein.

[0241] The expression of a specific gene encoding the SOUL heme-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, among others, northern blotting, RNA-Seq, in situ hybridization, DNA microarrays, and RT-PCR. Alternatively, the expression of a gene can be measured indirectly by measuring the level of a gene product, such as a protein encoded by the gene. For example, the expression of the SOUL heme-binding protein can be determined by measuring the presence of the protein by western blotting using an antibody specific for the SOUL heme-binding protein (e.g., an antibody specific for the SOUL motif).

[0242] Modification

[0243] As used herein, the term "modification" refers to an alteration of the genetic material of a plant or cell. The plant or cell can be modified in any manner that modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein. The types of modifications to plants and cells that modulate the activity or expression of genes, as well as techniques for achieving these modifications, are known in the art.

[0244] 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), comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0245] In some embodiments, the present invention provides a method for reducing the level of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part thereof, the method comprising modifying the plant or cell or cell culture by reducing the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0246] Any method known in the art for reducing or inhibiting the activity or expression of a gene can be used in the methods according to the present invention.

[0247] Suitably, the activity or expression of the SOUL heme-binding protein gene may be reduced, partially inactivated, inhibited, eliminated, knocked out or lost such that the protein activity, expression or function of the SOUL heme-binding protein gene is undetectable.

[0248] In one aspect, at least one SOUL hemoglobin-binding protein gene is knocked out. In other words, the SOUL hemoglobin-binding protein gene has been rendered completely ineffective.

[0249] For example, the method may include:

[0250] ● 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto;

[0251] ● 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto;

[0252] ● 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto.

[0253] Each of the above methods results in a reduction or inhibition 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto, or wherein at least one gene encoding a SOUL heme-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 or 24, 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 or 24, or a nucleic acid sequence having at least 80% identity thereto.

[0254] As used herein, the term "mutation" includes natural genetic variants or engineered variants. Specifically, 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 or 22, 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.

[0255] 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 or TSNA precursor in tobacco.

[0256] 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 said nucleic acid results in a reduction or elimination of the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0257] 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 the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein.

[0258] 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 a SOUL heme-binding protein.

[0259] In a preferred embodiment, each copy of the nucleic acid sequence encoding a protein comprising the sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22 or a sequence having at least 80% sequence identity thereto, or wherein the at least one gene encoding the SOUL heme-binding protein comprises the nucleotide sequence as shown in SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, 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 or 24, 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 or 24 nucleic acid sequences with at least 80% identity (which are present in the plant) are modified, e.g., mutated as defined herein (e.g., each genomic copy of the gene encoding the protein in the plant is mutated). For example, each copy of the gene in the allotetraploid genome of tobacco may be mutated.

[0260] In a preferred embodiment, some or all of the homologues of the SOUL hemoglobin-binding proteins described herein are modified, e.g., inhibited or mutated. Suitably, some or all of SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or corresponding sequences having at least 80% sequence identity thereto, are modified, e.g., inhibited or mutated.

[0261] In some embodiments, the plant or plant cell according to the present invention is homozygous. Suitably, the plant or plant cell may be homozygous for a modification such as an inhibition or mutation.

[0262] In some embodiments, plants or plant cells according to the present invention express only modified, e.g., mutated, nucleic acids encoding SOUL heme-binding protein. In other words, in some embodiments, no endogenous (or endogenous and functional) protein is present in the plants according to the present invention. In other words, if any endogenous protein is present, it is preferably in an inactive form.

[0263] In one embodiment, the method may comprise 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 or 24, or a nucleic acid sequence having at least 80% identity thereto.

[0264] The mutation may alter the plant genome such that the nucleic acid sequence encoding a protein comprising an amino acid sequence as set forth in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, is completely or partially deleted or otherwise modified to inhibit or eliminate the ability of the SOUL heme-binding protein to interact with other molecules, such as to bind to other molecules, compared to the protein set forth in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% sequence identity thereto. In some embodiments, the mutation does not alter the level or expression of the protein, but reduces, inhibits or eliminates the ability of the SOUL heme-binding protein to interact with other molecules, such as to bind to other molecules, compared to the protein set forth in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% sequence identity thereto. Suitably, the mutation inhibits or eliminates the ability of the SOUL heme-binding protein to interact with other proteins. Suitably, the mutation inhibits or eliminates the ability of SOUL heme-binding protein to bind to other molecules. Suitably, the mutation inhibits or eliminates the ability of SOUL heme-binding protein to bind to other proteins. Suitably, the mutation inhibits or eliminates the ability of SOUL heme-binding protein to bind to tetrapyrroles or tetrapyrroles. Suitably, the mutation inhibits or eliminates the ability of SOUL heme-binding protein to bind to heme. The expression "inhibits or eliminates" means reducing the interaction between SOUL heme-binding protein and other molecules, suitably to the extent that the interaction is prevented (i.e., the SOUL heme-binding protein does not interact with the other molecule at all). Suitably, the inhibition or elimination of the binding of SOUL heme-binding protein to another molecule means that the binding affinity of SOUL heme-binding protein to the other molecule is reduced. In some embodiments, the mutation reduces the binding affinity of SOUL heme-binding protein to heme.

[0265] Suitably, the mutation may be in a domain of the SOUL heme-binding protein that mediates interactions with other molecules. The mutation may be in a domain of the SOUL heme-binding protein that mediates binding to heme. The mutation may be in the SOUL motif of the SOUL heme-binding protein. In some embodiments, the SOUL motif may be mutated to thereby alter the ability of the SOUL heme-binding protein to interact with other molecules. In other words, the SOUL motif may be mutated to alter the binding affinity of the SOUL heme-binding protein for other molecules. Suitably, the SOUL motif is mutated to increase the binding affinity of the SOUL heme-binding protein for other molecules. Suitably, the SOUL motif is mutated to decrease the binding affinity of the SOUL heme-binding protein for other molecules.

[0266] The mutation may interrupt the nucleic acid sequence encoding a protein comprising the amino acid sequence shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto.

[0267] The interruption may result in the nucleic acid sequence not being transcribed and / or translated.

[0268] 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.

[0269] The nucleotide sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or influence protein transport. For example, protein expression may be reduced or prevented by introducing one or more premature stop codons, frameshifts, splice mutations, or non-tolerant amino acid substitutions into the open reading frame.

[0270] 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 ("opal" or "umber") codon.

[0271] Frameshift mutations (also known as framing errors or frame shifts) are mutations 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 mutated codon to be read to encode a different amino acid. Frameshift mutations will generally result in the introduction of premature stop codons.

[0272] Splicing mutations insert, delete, or change a number of 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 the mature mRNA and may lead to the production of abnormal proteins.

[0273] 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.

[0274] Any method known in the art for providing mutations in nucleic acid sequences can be used in the methods according to the present invention. For example, homologous recombination can be used to generate vectors in which one or more related nucleic acid sequences are mutated and used to transform plants or plant cells. Recombinant plants or plant cells expressing the mutated sequences can then be selected.

[0275] 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 or 22, or a sequence having at least 80% sequence identity thereto.

[0276] In some embodiments, the SOUL motif can contain a mutation that reduces expression of at least one gene encoding a SOUL heme-binding protein.

[0277] The mutation may be a deletion, a splice mutant, or a codon encoding a non-tolerant amino acid substitution.

[0278] In one embodiment, the nucleic acid sequence encoding the SOUL heme-binding protein may be deleted in whole or in part. The deletion may be continuous or may comprise multiple segments of the sequence. The deletion preferably removes a sufficient amount of nucleotide sequence such that the nucleic acid sequence no longer encodes a functional SOUL heme-binding protein. The deletion may be complete, in which case 100% of the coding portion of the nucleic acid sequence is absent when compared to the corresponding genome of a comparable unmodified plant. The deletion may, 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 may be deleted. The deletion may, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the coding portion of the protein.

[0279] The deletion may remove at least a portion of the SOUL motif.

[0280] The deletion may, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the SOUL motif.

[0281] Suitably, the deletion may 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 from the SOUL motif. Suitably, the deletion may 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 from the SOUL motif.

[0282] In one embodiment, the deletion can remove at least 100 amino acids, at least 150, at least 200, at least 250, at least 300, at least 350 amino acids from the C-terminus of the SOUL heme-binding protein.

[0283] Suitably, the mutant protein may be a truncated protein lacking at least about 100 amino acids, at least about 150 amino acids corresponding to the amino acids from the C-terminus of SEQ ID No. 1, or a sequence having at least 80% (preferably at least 85%, at least, at least 90%, at least 95%, at least 98%) sequence identity to a truncated protein lacking at least about 100 amino acids, at least about 150 amino acids corresponding to the amino acids from the C-terminus of SEQ ID No. 1.

[0284] Methods for deleting nucleic acid sequences in plants are known in the art. For example, homologous recombination can be used to produce a vector in which one or more related nucleic acid sequences are lost and used to transform a plant or plant cell. Recombinant plants or plant cells expressing the new sequence portion can then be selected.

[0285] Plant cells transformed with the vectors described herein can be grown and maintained according to well-known tissue culture methods, such as by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0286] Targeted mutagenesis methods (also referred to as targeted nucleotide exchange (TNE) or oligonucleotide directed mutagenesis (oligo-directed mutagenesis) (ODM)) can be used to modify nucleotide sequences. Targeted mutagenesis methods include, but are not limited to, those employing zinc finger nucleases, TALENs (see WO2011 / 072246 and WO2010 / 079430), Cas9-samples, Cas9 / crRNA / tracrRNA, Cas9 / gRNA or other CRISPR systems (see WO2014 / 071006 and WO2014 / 093622), meganucleases (see WO2007 / 047859 and WO2009 / 059195), or employing the targeted mutagenesis methods of mutagenic oligonucleotides, which may contain chemically modified nucleotides (such as Or TALENs) for enhancing mutagenesis with sequence complementarity to genes that enter plant protoplasts.

[0287] Alternatively, 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 incorporated herein by reference) can be used to generate plant lines comprising genes encoding proteins with mutations. TILLING uses traditional chemical mutagenesis (e.g., ethyl methanesulfonate (EMS) mutagenesis to generate random mutations) followed by high-throughput screening of mutations. Thus, plants, seeds, cells, and tissues comprising genes with desired mutations can be obtained.

[0288] Said method may comprise the following steps: mutagenesis plant seeds (for example EMS mutagenesis), the merging of plant individual or DNA, the pcr amplification of target region, heteroduplex formation and high throughput detection, the evaluation of mutant plant, the order-checking of mutant pcr product.Should be understood that other mutagenesis and selection methods can be used to generate the plant of this type of modification equally.Seed can for example be irradiated or chemically treated, and can be for the phenotypic screening plant of modification.

[0289] 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.

[0290] In another approach, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of duplicated loci. See, e.g., Kusaba et al. (2003) Plant Cell 15: 1455-1467 (incorporated herein by reference).

[0291] 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.

[0292] 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto, does not comprise treating the plant with chemicals (e.g., agrochemicals).

[0293] Other ways of reducing or preventing 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).

[0294] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by virus-induced gene silencing.

[0295] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by microRNAs.

[0296] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by RNAi.

[0297] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by antisense inhibition.

[0298] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by sense suppression.

[0299] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by tDNA insertion.

[0300] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a dominant negative construct (or a retro-allele mutation).

[0301] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a targeted mutagenesis-based system.

[0302] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a CRISPR-based system.

[0303] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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.

[0304] In some embodiments, the present invention provides a method of 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 a SOUL heme-binding protein.

[0305] Any method known in the art for increasing or enhancing the activity or expression of a gene can be used in the methods according to the present invention.

[0306] In some embodiments, the method may comprise overexpressing at least one gene encoding a SOUL heme-binding protein. Suitably, the method may comprise expressing one or more additional copies of at least one gene encoding a SOUL heme-binding protein in a plant or cell. Suitably, the method may comprise modifying an endogenous copy of at least one gene encoding a SOUL heme-binding protein such that its expression is increased. The method may comprise mutating the coding sequence of at least one gene encoding a SOUL heme-binding protein. The method may comprise mutating a regulatory sequence that regulates expression of at least one gene encoding a SOUL heme-binding protein.

[0307] Suitably, the method may comprise transforming cells of a plant (e.g., a tobacco plant) with a genetic construct encoding at least one SOUL heme-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein the at least one gene encoding a SOUL heme-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 or 24, or a functional variant or functional fragment or ortholog thereof. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid 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, or 24; or a nucleotide sequence encoding a protein capable of promoting or enhancing at least one endogenous SOUL heme-binding protein gene. It will be understood that each of these options will result in increased activity and expression of a polypeptide encoded by at least one SOUL heme-binding protein gene. The method may include 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 SOUL heme-binding protein gene for increasing alkaloid content (e.g., nicotine content) and / or TSNA content (or precursors thereof) in a plant, or part or cell thereof, transformed with the construct.

[0308] The gene construct may encode a polypeptide comprising the amino acid sequence of SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein the at least one gene encoding the SOUL hemoglobin-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 or 24, or a functional variant or functional fragment or ortholog thereof to SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 nucleic acid sequences with at least 80% identity.

[0309] In another embodiment, the present invention relates to a method for increasing the alkaloid content of a plant or a part thereof and / or the TSNA content (or a precursor thereof) in a plant or a part thereof, comprising modifying said plant by increasing the activity of at least one gene encoding a SOUL heme-binding protein.

[0310] In one embodiment, the activity of at least one gene encoding a SOUL heme-binding protein may be increased by introducing (or providing) a mutation into at least one gene encoding a SOUL motif.

[0311] Suitably, the activity of at least one gene encoding a SOUL hemoglobin-binding protein may be increased by introducing a mutation into at least one gene encoding a SOUL hemoglobin-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22; or a functional variant or functional fragment or orthologue thereof, or a sequence having at least 80% identity to SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22; or wherein the at least one gene encoding a SOUL hemoglobin-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 or 24, 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 or 24, 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 or 24, 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 or 24, or a nucleic acid sequence that is at least 80% identical to SEQ ID No.

[0312] In some embodiments, the modification that increases the activity or expression of at least one SOUL heme-binding protein gene and thereby increases the alkaloid content and / or TSNA content (or precursors thereof) is by one of the following:

[0313] regulating transcription from at least one SOUL heme-binding protein gene;

[0314] regulating translation of mRNA from at least one SOUL heme-binding protein gene;

[0315] Modulating the release of SOUL heme-binding protein from intracellular stores; and / or

[0316] Regulates the degradation rate of SOUL heme-binding protein.

[0317] Alkaloid content

[0318] In one embodiment, the present invention provides a method of modulating the alkaloid content of a plant (eg, a tobacco plant) or a part thereof, comprising modifying the plant by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0319] The term "modulate" is used herein to mean to increase or decrease.

[0320] The term "increased alkaloid content" is used herein to mean that the alkaloid content in a product of the invention (e.g. a plant, a part thereof (e.g. leaves), a processed leaf or a product prepared from the plant (e.g. a tobacco product)) is higher than in a comparable product which has not been modified according to the invention.

[0321] 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 the plant (e.g., a tobacco product)) is lower than in a comparable product which has not been modified according to the invention.

[0322] In some embodiments, modulation of alkaloid levels refers to an increase in alkaloid levels, wherein the activity or expression of at least one SOUL heme-binding protein gene is increased (or in other words, the protein is overexpressed).

[0323] In some embodiments, modulation of alkaloid levels refers to a decrease in alkaloid levels, wherein expression of at least one gene encoding a SOUL heme-binding protein is reduced or inhibited or eliminated.

[0324] 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.

[0325] The term "alkaloid content" is used herein to refer to the concentration and / or total amount of the entire group of compounds classified as alkaloids or the concentration and / or total amount of one or more compounds classified as alkaloids. Alkaloids commonly present in tobacco include nicotine, nornicotine, PON, anatabine, anabasine 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 increases. In some embodiments, the content of 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 all six alkaloids, is reduced. In some embodiments, the total alkaloid content of the plant or cell is modulated. In some embodiments, the total alkaloid content is increased. In some embodiments, the total alkaloid content is increased.

[0326] Suitably, the nicotine content can be adjusted. In one embodiment, the nicotine content is reduced. In another embodiment, the nicotine content is not adjusted, but the content of one or more alkaloids selected from nornicotine, PON, anatabine, anabasine, and mithosamine is adjusted. In another embodiment, the nicotine content is not adjusted, but the content of one or more alkaloids selected from nornicotine, PON, anatabine, and anabasine is adjusted.

[0327] Any method known in the art for determining the concentration and / or total amount of alkaloids may be used.One preferred method for analyzing alkaloid content involves analysis by gas chromatography-flame ionization detection (GC-FID).

[0328] 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, plant propagation material (e.g., tobacco plant propagation material), cell (e.g., tobacco cell), 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), product (e.g., tobacco product) comprising said plant or part thereof, or a combination thereof, said plant of the present invention having a modulated alkaloid content, said method comprising modifying said plant to modulate the activity or expression of a gene encoding a SOUL heme-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 such as tobacco products comprising plants or parts thereof of the present invention, or combinations thereof, to comparable products.

[0329] Suitably, alkaloid content can be in plant, tobacco plant for example, for example regulate in the tobacco plant of modification. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate in the results. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the results of the cutting of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in processing blade (tobacco leaf for example, for example from the processing tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the cutting of the tobacco plant of modification) regulate in the processing. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate in the modulation. Suitably, alkaloid content can be in the extract of green leaf (for example from the green tobacco leaf of the tobacco plant of modification) regulate. Suitably, the alkaloid content can be regulated in a product comprising the plant of the present invention or its part (e.g., a tobacco product, e.g., a tobacco product produced from a modified tobacco plant or its part). Suitably, the alkaloid content can be regulated in any of the above-mentioned products or a combination thereof. Suitably, the regulation of the above-mentioned alkaloid content can be an increase in the alkaloid content. Suitably, the regulation of the above-mentioned alkaloid content can be a reduction in the alkaloid content (e.g., a reduction in nicotine content).

[0330] In one embodiment, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine, and mithosamine is reduced. In one embodiment, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, and anabasine is reduced.

[0331] Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in nicotine content. Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine, anabasine and mithosamine, rather than a reduction in nicotine content. Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine and anabasine, rather than a reduction in nicotine content.

[0332] In one embodiment, the nicotine content of the modified plant (e.g., tobacco plant), plant propagation material (e.g., tobacco plant propagation material), 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), or tobacco products from the modified tobacco plant is reduced.

[0333] In one embodiment, when compared to 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 SOUL heme-binding protein and has been grown under similar growth conditions, the alkaloid content of the plant (e.g., tobacco plant) or its part can be adjusted to at least 0.5, 1.5, or 2 times. Suitably, the alkaloid content can be adjusted to about 0.5 times to about 2 times. Suitably, the modification can be an increase or decrease 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, nicotine content is adjusted.

[0334] In one embodiment of the invention, compared with plant (for example tobacco plant) or its part that are not 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, described regulation can be the increase or reduction of alkaloid content.Suitably, regulation can be the regulation of total alkaloid content. Suitably, the regulation may be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and milostramine. Suitably, the regulation may be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine. Suitably, the regulation is the regulation of nicotine content, such as a reduction in nicotine content. Suitably, the regulation is the regulation of nornicotine content, such as a reduction in nornicotine content. Suitably, the regulation is the regulation of anabasine content, such as a reduction in anabasine content. Suitably, the regulation is the regulation of PON content, such as a reduction in PON content. Suitably, the regulation is the regulation of anatabine content, such as a reduction in anatabine content. Suitably, the modulation is of more than one alkaloid, 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 all six alkaloids, selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine.

[0335] In some embodiments, the alkaloid content of the plant can be modulated by about 5% to about 100%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, about 40% to 60%, or about 40% to 50%.

[0336] In one embodiment, the alkaloid content of the plant may be adjusted by about 50% to about 100%, preferably about 60% to 90%, preferably 80% or more.

[0337] Tobacco-specific nitrosamine (TSNA) content

[0338] In one embodiment, the present invention provides a method for modulating (i.e., increasing or decreasing) the level of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a plant (e.g., a tobacco plant) or part thereof. Suitably, the method may comprise modifying the plant by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein. Suitably, the level of TSNAs or TSNA precursors may be reduced. Suitably, the total TSNA content of the plant or part thereof may be modulated.

[0339] TSNAs can be measured in processed tobacco, such as cured tobacco or reconstituted tobacco. In one embodiment, TSNA content is measured and / or modified (e.g., reduced) in cured tobacco plants or parts thereof (e.g., cured tobacco leaves).

[0340] As used herein, the term "tobacco-specific nitrosamine" or "TSNA" has its ordinary meaning in the art, i.e., a nitrosamine found only in tobacco products or other nicotine-containing products. Suitably, the at least one tobacco-specific nitrosamine may be 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) or N'-nitrosoanatabine (NAB).

[0341] When used in relation to at least one tobacco-specific nitrosamine, the term "precursor thereof" refers to one or more chemicals or compounds of the tobacco plant that cause the formation of tobacco-specific nitrosamines or are associated with nitrosative reactions that lead to the production of tobacco-specific nitrosamines. Suitably, the term "precursor thereof" may refer to nitrates, nitrites or nitric oxide.

[0342] The term "modulate" is used herein to mean to increase or decrease.

[0343] In one embodiment, the TSNA is N'nitrosonornicotine (NNN) and / or the precursor is nornicotine.

[0344] In one embodiment, the TSNA may be one or more selected from the group consisting of N'-nitrosonornicotine (NNN), N'nitrosonicotine (NAT), N'-nitrosoanatabine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Suitably, the at least one tobacco-specific nitrosamine may be NNK or NNN. In one embodiment, the tobacco-specific nitrosamine is NNN.

[0345] In one embodiment, the precursor of TSNA is one or more selected from the group of nornicotine, anabasine, anatabine and an oxidized derivative of nicotine such as pseudo-oxidized nicotine (PON).

[0346] In a preferred embodiment, the precursor of the TSNA is nornicotine.

[0347] In one embodiment, the precursor of TSNAs can be PON. Precursors of TSNAs (e.g., NNN, NNK, 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., NNN, NNK, 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.

[0348] In one embodiment, performance of the methods and or uses of the present 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) not modified according to the invention.

[0349] The term "reduction of at least one TSNA or precursor thereof" or "reduction of at least one TSNA or precursor thereof" is used herein to mean that the concentration and / or total content of at least one TSNA or precursor thereof is lower in the product, method, or use of the present 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 present invention, but in which all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing the tobacco, etc.).

[0350] 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, 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; being developed as a CRM for 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.

[0351] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can be reduced by implementing the methods and / or uses 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 methods and / or uses of the present invention) when compared to the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor in a tobacco plant not modified according to the present invention.

[0352] The concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can 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 cells or cell culture) of the present invention (or part of a tobacco plant or tobacco cells or 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 cells or cell culture) that has not been modified according to the present invention.

[0353] 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.

[0354] 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.

[0355] 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%.

[0356] 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.

[0357] Biomass production

[0358] In one aspect, the present invention provides a method of producing biomass, comprising:

[0359] Cells that have been engineered to modulate (eg, reduce) the activity or expression of a gene encoding a SOUL heme-binding protein are grown under conditions that produce biomass.

[0360] In one embodiment, the present invention provides a method for producing biomass having an altered (e.g., reduced) concentration and / or total content of nicotine, comprising growing cells that have been engineered to reduce the activity or expression of at least one gene encoding a SOUL heme-binding protein, the SOUL heme-binding protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or

[0361] wherein the at least one gene encoding the SOUL hemoglobin-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 or 24, 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 or 24, 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 or 24.

[0362] Cells may be engineered to alter the activity or expression of at least one gene encoding a SOUL heme-binding protein by any method known in the art. Suitably, cells may be engineered to express an exogenous gene encoding a SOUL heme-binding protein. Suitably, cells may be engineered to overexpress a gene encoding a SOUL heme-binding protein. Suitably, cells may be engineered to reduce the activity or expression of a gene encoding a SOUL heme-binding protein.

[0363] Suitably, the biomass may contain a lower concentration and / or total content of nicotine than biomass produced from comparable cells which have not been modified according to the invention.

[0364] Suitably, the cells used for biomass production may be plant cells, such as tobacco cells.

[0365] Suitably, the cells used for biomass production may be yeast cells.

[0366] In one embodiment, the cell (e.g., yeast cell) can be further modified to include one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, these one or more sequences can be incorporated into a nucleic acid construct suitable for transformation of the cell (e.g., yeast cell). The one or more sequences can be overexpressed in the cell (e.g., yeast cell). The sequence can 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 can be taught, for example, in US2016032299, which is incorporated herein by reference.

[0367] Commercially desirable traits

[0368] In one embodiment, the plants of the present invention have 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 selected from nicotine, nornicotine, anabasine, mithofamine, and anatabine, and / or decreased nicotine, while at least maintaining the flavor profile and / or other commercially desirable traits. In one embodiment, the plants of the present invention produce leaves of similar grade and / or quality to plants that have not been modified according to the present invention.

[0369] In one embodiment, the plants of the invention have reduced nicotine content without a significant change in the flavor profile of the plant (eg, compared to the same plant that has not been modified according to the invention).

[0370] In one embodiment, the plants of the present invention have a modified (i.e., increased or decreased) alkaloid and / or TSNA content (e.g., compared to the same plant that has not been modified according to the present 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 present invention.

[0371] Thus, in one embodiment, the methods and uses of the present invention relate to modifying (i.e. increasing or decreasing) the total alkaloid content, and / or modifying (i.e. increasing or decreasing) one or more alkaloids selected from nicotine, nornicotine, anabasine and anatabine, and / or modifying (i.e. increasing or decreasing) the nicotine content and / or TSNA content, while maintaining the flavor profile and / or other commercially desirable traits (e.g. yield).

[0372] 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.

[0373] Leaf quality can be measured based on the color, texture, and aroma of the cured leaves, for example according to United States Department of Agriculture (USDA) grades and standards.

[0374] 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 shine of the leaf), hygroscopicity (the ability of the tobacco leaf to absorb and retain moisture from the environment), and green nuance or cast.

[0375] Leaf grade can be determined using standard methods known in the art, for example, using the Official Standard Grades (7 USC §511) published by the Agricultural Marketing Service of the US Department of Agriculture. 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 11719). 11926); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 FR 17061); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for Georgia and Florida Shade-Grown 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 entireties).

[0376] 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. Alternatively, the blade grade can be determined via hyper-spectral imaging. See, for example, WO 2011 / 027315 (which is incorporated herein by reference).

[0377] In one embodiment, the tobacco plants of the present invention provide commercially acceptable grades of tobacco.

[0378] Suitably, the tobacco plants of the present invention provide commercially acceptable grades of cured tobacco.

[0379] In one embodiment, when growing under similar growth conditions, tobacco plant of the present invention can produce the leaf blade of at least about 70% the USDA grade index value with comparable plant leaves. Suitably, when growing under similar growth conditions, tobacco plant disclosed herein may be able to produce the leaf blade 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% the 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.

[0380] 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 greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater.

[0381] 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.

[0382] 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%.

[0383] 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 greater than the yield of comparable plants when grown under similar field conditions.

[0384] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable flue-cured plants when grown under similar field conditions.

[0385] In one aspect, the tobacco plant of the present 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.

[0386] 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.

[0387] 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.

[0388] Plant breeding

[0389] 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) or TSNA precursor content, comprising:

[0390] a. crossing a donor plant having a modified nicotine content and / or a modified tobacco-specific nitrosamine (TSNA) or TSNA precursor content, and wherein the activity or expression of at least one gene encoding a SOUL heme-binding protein according to the present invention has been modulated in the donor plant, with a recipient tobacco plant that does not have a modified nicotine content or a modified tobacco-specific nitrosamine (TSNA) or TSNA precursor content and has commercially desirable traits;

[0391] b isolating genetic material from the offspring of the donor plant hybridized with the recipient plant; and

[0392] c. Molecular marker-assisted selection using molecular markers, comprising:

[0393] i. Identifying an introgressed region comprising a mutation in a polynucleotide sequence encoding a protein defined in a.

[0394] Suitably, the activity or expression of a protein is modulated in the donor plant when compared to a comparable plant, said protein comprising an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 24; or said protein is encoded by a nucleotide sequence as shown in SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or orthologue thereof. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, 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 or 24, or a nucleic acid sequence that is at least 80% identical to SEQ ID No.

[0395] 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 or 22, or an amino acid sequence having at least 80% sequence identity thereto.

[0396] plant

[0397] Suitable plants according to the present invention include plants of the Solanaceae family, which includes, for example, datura, eggplant, mandrake, deadly nightshade (belladonna), capsicum (chili pepper, capsicum), potato and tobacco.

[0398] In one embodiment, a suitable genus of the Solanaceae family is Nicotiana ( Nicotiana ), such as tobacco or Nicotiana rustica ( Nicotiana rustica ).

[0399] 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.

[0400] tobacco plants

[0401] The present invention provides methods, uses, and cells (eg, tobacco cells), plants (eg, tobacco plants), and plant propagation material for plants (eg, tobacco plants).

[0402] As used herein, the term "tobacco plant" refers to a plant in the genus Nicotiana used in the production of tobacco products. Non-limiting examples of suitable "tobacco" plants include Nicotiana tabacum and Nicotiana rustica (e.g., Nicotiana N. tabacum L.), LAB21, LN KY171, Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico).

[0403] Tobacco material can be derived from or derive from the variant of tobacco type, is commonly referred to as burley tobacco variant, flue-cured tobacco (flue) or bright color (bright) variant and dark (dark) variant.In some embodiments, tobacco material is derived from burley tobacco, Virginia (Virginia) or dark tobacco plant.Tobacco plant can be selected from burley tobacco, rare tobacco, special product tobacco (speciality tobacco), expanded tobacco etc.

[0404] This paper also contemplates the use of tobacco cultivars and original species (elite) tobacco cultivars. Therefore, the tobacco plants used for this paper can be tobacco variants or original species tobacco cultivars. Especially useful tobacco variants include flue-cured tobacco Virginia type, burley type and Oriental type.

[0405] 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.

[0406] 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. Even though not specifically identified herein, the aforementioned low converter subvarieties are also contemplated.

[0407] The tobacco plant may be Burley, Flue-cured Virginia, or Oriental.

[0408] In one embodiment, the plant propagation material may be obtainable from a plant of the invention (eg, a tobacco plant).

[0409] As used herein, "plant propagation material" 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.

[0410] In one embodiment, cells (eg, tobacco cells), tobacco plants and / or plant propagation material may be obtainable (eg, obtained) by a method according to the invention.

[0411] Suitably, a tobacco plant according to the present invention may have a modulated (e.g. reduced) nicotine content when compared to an unmodified tobacco plant, wherein the tobacco plant has been modified to modulate (e.g. reduce) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

[0412] Suitably, tobacco plants according to the present invention may have a modulated (e.g. reduced) tobacco-specific nitrosamine (TSNA) or 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 a SOUL heme-binding protein.

[0413] In one embodiment, a tobacco plant according to the present invention comprises a tobacco cell of the present invention.

[0414] In another embodiment, the plant propagation material may be obtainable (eg, obtained) from a tobacco plant of the present invention.

[0415] In one embodiment, there is provided use of a tobacco plant as described herein for breeding tobacco plants.

[0416] In another embodiment, the present invention also provides use of the tobacco plant of the preceding embodiment for producing tobacco industry products.

[0417] In another embodiment, there is provided use of the tobacco plants of the present invention to grow crops.

[0418] In one embodiment, there is provided use of a cell as provided in the preceding embodiments for producing a tobacco industry product.

[0419] In one embodiment, the invention provides plant cells or cell cultures (eg, in vitro cultures).

[0420] 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 sheets, as an additive component, or both as a substitute and as an additive. Suitably, cell culture can produce nicotine.

[0421] In one embodiment, there is provided the use of a cell culture, eg, a harvested and / or processed cell culture according to the present invention, for producing a tobacco industry product.

[0422] Tobacco cells harvested from in vitro culture can be dried, such as freeze-dried, for example to produce a powder.

[0423] In one embodiment, the plant cell is a tobacco plant cell.

[0424] In one embodiment, cell culture is a tobacco cell culture. The 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 the target tobacco plant, and sterilize its outside to eliminate unwanted biology, plant said seed with the growth target 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.

[0425] Tobacco cells can be harvested by various methods including filtration, such as vacuum filtration. The sample can be washed in a filter by adding water and removing the remaining liquid by filtration, such as vacuum filtration.

[0426] The tobacco cell of results or cell culture can be further processed, for example dry, such as air-drying and / or freeze-drying.The tobacco cell of results or cell culture or the tobacco cell of dry results or cell culture or its extract can be incorporated into tobacco industry product according to the present invention.

[0427] In one embodiment, the invention provides plants (e.g., tobacco plants) or parts thereof for molecular planting. Suitably, plants modified according to the present invention or parts thereof can be used to make proteins, such as therapeutic agents, for example, antibiotics, virus-like particles, nutritional medicines or small molecules.

[0428] 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 SOUL heme-binding protein gene, the at least one SOUL heme-binding protein gene encoding an amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19 or 22, 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 or 22; or wherein the at least one gene encoding the SOUL heme-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 or 24 ... 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or ortholog thereof, 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 or 24; and cultivating the plant under conditions sufficient to allow production of the protein (e.g., a therapeutic protein).

[0429] product

[0430] The present 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 a SOUL heme-binding protein has been modulated are provided.

[0431] In one embodiment, a product may comprise a construct of the invention that modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein as defined herein. In one embodiment, a product may comprise a construct of the invention that modifies the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein as defined herein.

[0432] The invention also provides products obtainable or derived from tobacco according to the invention.

[0433] In one embodiment, there is provided use of the tobacco plant of the present invention to produce tobacco leaves.

[0434] Suitably, the tobacco leaves may be subjected to downstream applications, such as processing.

[0435] Therefore, in one embodiment, the use of the aforementioned embodiments can provide processed tobacco leaves. Suitably, the tobacco leaves can be subjected to conditioning, 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 conditioning, fermentation, pasteurization, or a combination thereof.

[0436] In one embodiment, the present invention provides harvested leaves of the tobacco plant of the present invention.

[0437] In a further embodiment, harvested leaves may be obtainable (eg, obtained) from a tobacco plant propagated from the propagation material of the invention.

[0438] In another embodiment, there is provided harvested leaves obtainable from the methods or uses of the invention.

[0439] Suitably, the harvested leaves may be cut harvested leaves.

[0440] In some embodiments, the harvested leaves may contain living tobacco cells.In other embodiments, the harvested leaves may be subjected to further processing.

[0441] Processed tobacco leaves are also provided.

[0442] Processed tobacco leaves may be obtainable from a tobacco plant of the present invention. Suitably, processed tobacco leaves may be obtainable from a tobacco plant obtained according to any of the methods and / or uses of the present invention.

[0443] In another embodiment, processed tobacco leaves may be obtainable from tobacco plants propagated from tobacco plant propagation material according to the present invention.

[0444] The processed tobacco leaves of the present invention may be obtainable by processing the harvested leaves of the present invention.

[0445] 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.

[0446] 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 living, also known as "non-viable", the cell does not exhibit the characteristics of a living cell.

[0447] The term "substantially free of living cells" means that less than about 5% of all cells are living. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of all cells are living.

[0448] In one embodiment, processed tobacco leaves may be processed by one or more of: curing, fermenting, and / or pasteurizing.

[0449] Suitably, the processed tobacco leaves may be processed by curing.

[0450] 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, flame-curing, flue-curing, and sun-curing.

[0451] Suitably, the tobacco leaves may be air-cured.

[0452] Typically, air-curing is achieved by hanging the tobacco leaves in a well-ventilated barn and allowing them to dry. This is usually done over a period of four to eight weeks. Air-curing is particularly suitable for Burley tobacco.

[0453] Suitably, the tobacco leaves may be open fire cured. Open fire curing is usually achieved by hanging the tobacco leaves in large curing barns where a hardwood fire is kept burning low, either continuously or intermittently, and typically takes from three days to ten weeks, depending on the process and the tobacco.

[0454] In another embodiment, the tobacco leaves can be flue-cured. Flue-curing can include stringing tobacco leaves onto tobacco rods and hanging them on tier-poles in a curing barn. Curing barns typically have flues that operate with an externally supplied firebox. Typically, 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.

[0455] Suitably, the tobacco leaves may be sun-cured. This process generally involves exposing uncovered tobacco to the sun.

[0456] Suitably, the processed tobacco leaves may be processed by fermentation.

[0457] Fermentation can be carried out in any manner known in the art. Usually, during the fermentation, tobacco leaves are piled into a tobacco pile (heap) covered with a modulation in a sack for example to retain moisture. The combination of the residual water in the leaf and the tobacco weight has generated the natural heat that makes tobacco ripe. The temperature in the center of the heap is monitored every day. In some methods, the whole heap is opened weekly. The leaves are then taken out to shake and moisten, and the heap is rotated so that the inner leaves are outside and the bottom leaves are placed on the top of the heap. This ensures the uniform fermentation of the whole heap. The extra moisture on the leaf adds the actual rotation generation heat of the leaf itself, thereby releasing the natural ammonia of the tobacco and reducing nicotine, while also deepening color and improving the aroma of the tobacco. Usually, the fermentation process continues up to 6 months, and this depends on the variety of the tobacco, the handle position on the leaf, the thickness of the leaf and the intended use.

[0458] 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.

[0459] 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).

[0460] During snus production, pasteurization is typically performed 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 virtually sterile product, and without wishing to be bound by theory, one of the consequences of this is believed to be limiting further TSNA formation.

[0461] In one embodiment, the pasteurization process may be steam pasteurization.

[0462] In some embodiments, the processed tobacco leaves can be cut. The processed tobacco leaves can be cut before or after processing. Suitably, the processed tobacco leaves can be cut after processing.

[0463] In one embodiment, use of the foregoing embodiments can provide reconstituted tobacco.

[0464] In one embodiment, reconstituted tobacco is provided.

[0465] As used herein, "reconstituted" may also be referred to as reconstituted, recycled or homogenized sheet tobacco, and refers to tobacco material generated from processed tobacco leaf residues. Reconstituted tobacco allows the production of consistent, high-quality blends and allows the ratios of the various components to be adjusted.

[0466] Reconstituted tobacco can be nanofibre reconstituted (nanofibres can be extracted in solid or liquid form), paper reconstituted (which uses stems, chips and midrib as raw materials), or slurry reconstituted (which uses a mixture of finely ground material and tobacco stems mixed with water and a plant binder; the soluble residue is formed into sheets by extracting the water).

[0467] Any method known in the art can be used to prepare reconstituted tobacco, see, for example, CORESTA Congress, Sapporo, 2012, Smoke Science / Product Technology Groups, SSPT 12 (incorporated herein by reference).

[0468] In some embodiments, the leaves of the results of tobacco plants, tobacco plants and / or processed tobacco leaves can be used to extract nicotine. The extraction of nicotine can be achieved using any method known in the art. For example, the method for extracting nicotine from tobacco is taught in US 2,162,738, which is incorporated herein by reference.

[0469] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0470] In another aspect, the invention provides a tobacco blend comprising a tobacco material made from a tobacco plant according to the invention or its part or from a tobacco cell or cell culture according to the invention. In one aspect, the invention provides a tobacco blend comprising a tobacco material prepared according to the invention.

[0471] Suitably, tobacco blends according to the present invention can comprise 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 according to the present invention or cell culture. Suitably, tobacco blends can comprise about 10% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 20% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 30% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 40% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 50% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 60% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 70% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 80% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 90% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture.

[0472] 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 or cell culture according to the present invention.

[0473] Suitably, the cured tobacco material may be air-cured. Suitably, the cured tobacco material may be flue-cured. Suitably, the cured tobacco material may be sun-cured. Suitably, the cured tobacco material may be flame-cured.

[0474] Tobacco industry products or smoking articles according to the present invention may comprise a tobacco material according to the present invention (eg, a cured tobacco material or a reconstituted tobacco material).

[0475] In another aspect, the present invention provides tobacco industry products.

[0476] In one embodiment, the tobacco industry product according to the present invention may be a blended tobacco industry product. Suitably, a tobacco blend may comprise a cured tobacco material according to the present invention.

[0477] In one embodiment, tobacco industry products can be prepared from the tobacco plants of the present invention or parts thereof.

[0478] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the present invention.

[0479] 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 a flower of tobacco plant. Suitably, " part " can be a leaf, root or stem of tobacco plant.

[0480] Tobacco industry products

[0481] As used herein, the term "tobacco industry product" is intended to include combustible smoking articles such as cigarettes, cigarillos, cigars, tobacco for pipes or roll-your-own tobacco (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 an aerosol from a combination of matrix materials, for example, hybrid systems containing a liquid or gel or solid matrix, and aerosolizable matrix materials used within 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.

[0482] In one embodiment, a tobacco industry product can be prepared from (eg, can comprise) a tobacco plant or part thereof of the invention.

[0483] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the present invention.

[0484] 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.

[0485] In another embodiment, tobacco industry products can be prepared from the harvested leaves of the present invention.

[0486] In a further embodiment, tobacco industry products can be prepared from the processed tobacco leaves of the present invention.

[0487] Suitably, tobacco industry products may be prepared from tobacco leaves processed by one or more of the following: curing, fermentation and / or pasteurisation.

[0488] Suitably, the tobacco industry product may comprise cut tobacco leaves, optionally processed in accordance with the preceding embodiments.

[0489] In another embodiment, tobacco industry products can be prepared from tobacco cells or cell cultures according to the present invention.

[0490] In another embodiment, tobacco industry products can be prepared from (eg, can comprise) a cured tobacco material according to the present invention.

[0491] In another embodiment, a tobacco industry product can be prepared from (eg, can comprise) a tobacco blend according to the present invention.

[0492] In one embodiment, the tobacco industry product may be a smoking article.

[0493] 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.

[0494] In another embodiment, the tobacco industry product can be a smokeless tobacco industry product.

[0495] 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.

[0496] 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, which take any form such as sheets, films, tabs, foams, or beads.

[0497] In one embodiment, smokeless tobacco industry products can include snus, snuff, chewing tobacco, and the like.

[0498] In one embodiment, the tobacco industry product is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0499] 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.

[0500] In one embodiment, the tobacco industry product is a non-combustible gas aerosol delivery system.

[0501] In one embodiment, a tobacco industry product comprises one or more components of a non-combustible gas aerosol delivery system, such as a heater and an aerosolizable substrate.

[0502] In one embodiment, the aerosol delivery system is an electronic cigarette, also known as a vaping device.

[0503] In one embodiment, an 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.

[0504] 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.

[0505] 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 can 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.

[0506] In one embodiment, the heated product is an electronic device.

[0507] 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.

[0508] In one embodiment, the heating product is a non-electronic article.

[0509] 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.

[0510] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0511] 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.

[0512] In one embodiment, the tobacco industry product is a hybrid system, generates aerosol with the combination of the non-burning host material by heating.The host material can comprise for example solid, liquid or gel, which may or may not contain nicotine.In one embodiment, the hybrid system comprises liquid or gel matrix and solid matrix.The 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 comprises liquid or gel matrix and tobacco.

[0513] In further embodiments, the tobacco industry product may be a tobacco heating device or a hybrid device or an electronic cigarette, or the like.

[0514] Typically, in tobacco heating devices or hybrid devices, aerosols are 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 the heat transfer from the heat source and are entrained in the air drawn through the smoking article. As the released compounds cool, they condense to form an aerosol that is inhaled by the user.

[0515] 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.

[0516] Suitably, the tobacco heating device may be an aerosol generating device.

[0517] 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.

[0518] Examples of suitable heat not burn devices may be those taught in WO 2013 / 034459 or GB 2515502, which are incorporated herein by reference.

[0519] In one embodiment, the aerosol-forming substrate of the tobacco heating device may be a tobacco industry product according to the present invention.

[0520] In one embodiment, the tobacco heating device may be a hybrid device.

[0521] Polynucleotide / polypeptide / construct

[0522] In certain embodiments of the present invention, constructs that modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein may be transformed into plant cells, suitably under the direction of a promoter.

[0523] In certain embodiments of the present invention, a construct that reduces (i.e., inhibits) the activity or expression of at least one gene encoding a SOUL heme-binding protein can be transformed into a plant cell under the guidance of a promoter. For example, the genetic construct can be a gene editing construct or can comprise an RNAi molecule, which can comprise a small interfering RNA (siRNA) molecule or a short hairpin (shRNA) molecule.

[0524] In certain embodiments of the invention, a construct that increases the activity or expression of a gene encoding a SOUL heme-binding protein, such as an endogenous SOUL heme-binding protein, can be transformed into a plant cell, suitably under the direction of a promoter.

[0525] 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, a construct sequence targeting a gene encoding the SOUL heme-binding protein in the transcriptional direction 5'-3' 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 described above. The terminator sequence may be derived from a plant, bacterial or viral gene. Suitable terminator sequences are, for example, pea. rbcS E9 terminator sequence, derived from Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) of the nopaline synthase gene nos terminator sequence, and from cauliflower mosaic virus 35S Terminator sequences. Other suitable terminator sequences will be readily known to those skilled in the art.

[0526] The constructs of the present invention may also contain gene expression enhancing mechanisms to increase the strength of the promoter. An example of such an enhancer element is the enhancer element derived from a portion of the promoter of the pea plastocyanin gene and is the subject of International Patent Application No. WO 97 / 20056, incorporated herein by reference. Suitable enhancer elements may be, for example, the enhancer element derived from the nopaline synthase gene of Agrobacterium tumefaciens. nos enhancer element, and from cauliflower mosaic virus 35S Enhancer element.

[0527] 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 the cells of the tissue to be transformed.

[0528] 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, such as the gene encoding the SOUL heme-binding protein according to the present invention, the coding sequence used in the present 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.

[0529] The expression cassette can be incorporated into a basic plant transformation vector such as 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.

[0530] The term "expression vector or plant transformation vector" refers to a construct capable of in vivo or in vitro expression. Preferably, the expression vector is incorporated into the genome of an organism. In one embodiment, the vector of the present invention expresses a protein, such as the SOUL heme-binding protein described herein. The term "incorporated" preferably includes stable incorporation into the genome.

[0531] 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 are incorporated herein by reference.

[0532] Typically, in Agrobacterium-mediated transformation, a binary vector carrying the target exogenous DNA, i.e., a construct according to the present invention, is transferred from an appropriate Agrobacterium strain to the 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 comprising a selective marker and a plant growth hormone. An alternative is the floral dip method (Clough & Bent, 1998 Plant J. December 1998; 16(6): 735-43, incorporated herein by reference), whereby the flower buds of the complete plant are contacted with a suspension of the Agrobacterium strain containing the chimeric gene, and after fruiting, the transformed individuals are germinated and identified by growth on the selective medium. Direct infection of plant tissue by Agrobacterium is a simple technique that has been widely adopted and has been described in Butcher et al. (1980), Tissue Culture Methods for Plant Pathologists , eds. DS Ingrams and JP Helgeson, 203-208, which is incorporated herein by reference.

[0533] Suitable transformation methods include, for example, the use of polyethylene glycol or electroporation, particle bombardment, microinjection, and silicon carbide fibers to transfer genes directly into protoplasts. Ballistic transformation and the production of fertile transgenic maize plants mediated by silicon carbide whiskers are used to transform plants in Frame et al. (1994) The Plant Journal 6: (6): 941-948 (incorporated herein by reference), and viral transformation techniques are taught in, for example, Meyer et al. (1992) Mol. Gen. Genet. 231 (3): 345-352 (incorporated herein by reference). Cassava mosaic virus is taught as a vector system for plants in Meyer et al. (1992) Gene 110: 213-217 (incorporated herein by reference). Further teachings on plant transformation can be found in EP-A-0449375, incorporated herein by reference.

[0534] In a further aspect, the present invention relates to a vector system that carries a construct and introduces it into the genome of an organism such as a plant, suitably a tobacco plant. The vector system may comprise one vector, but it may comprise two vectors. In the case of two vectors, the vector system is generally referred to as a binary vector system. Binary vector systems are described in Gynheung et al., (1980), Binary Vectors, Plant Molecular Biology ManualA3, 1-19, which is incorporated herein by reference.

[0535] A widely adopted system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens or the Ti plasmid 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 Plants Pathologists eds.: DS Ingrams and JP Helgeson, 203-208 (incorporated herein by reference). After each method of introducing the desired foreign gene in a 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 BB, Amsterdam Chapter V; Fraley et al. Crit. Rev. Plant Sci. 4: 1-46; and An et al. (1985) EMBO J 4: 277-284 (incorporated herein by reference).

[0536] Plant cells transformed with one or more constructs that modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein can be grown and maintained according to well-known tissue culture methods, such as by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0537] The term "transgenic plant" in connection with the present invention includes any plant comprising a construct that modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein according to the present invention. Accordingly, a transgenic plant is a plant that has been transformed with a construct according to the present invention. Preferably, according to the present invention, the transgenic plant exhibits modulated alkaloid content and / or modulated TSNA content (or its precursors). 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).

[0538] In one aspect, the gene, construct, plant transformation vector or plant cell encoding the SOUL heme-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.

[0539] In one aspect, the gene, construct, plant transformation vector or plant cell encoding the SOUL heme-binding protein according to the present invention is in a purified form. The term "purified" means in a relatively pure state, for example, at least about 90% pure, or at least about 95% pure, or at least about 98% pure.

[0540] As used herein, the term "nucleotide sequence" refers to an oligonucleotide sequence or a polynucleotide sequence, as well as variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, may be double-stranded or single-stranded, and may represent the sense or antisense strand.

[0541] The term "nucleotide sequence" in relation 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.

[0542] In a preferred embodiment, when relevant to and encompassed by the scope of the present invention itself, the nucleotide sequence, i.e., the gene encoding the SOUL hemoglobin-binding protein, comprises the native nucleotide sequence when in its natural environment and when linked to one or more of its naturally associated sequences that are also in their 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 with which it is naturally associated, said promoter is also in its natural environment.

[0543] Nucleotide sequence for the present invention can be present in a vector, wherein the nucleotide sequence is operably linked to a regulatory sequence that can provide expression of the nucleotide sequence by a suitable host organism. Construct for the present invention can be transformed into a suitable host cell as described herein to provide expression of the polypeptide of the present invention. The selection of carriers such as plasmids, cosmids or phage vectors often depends on the host cell to be introduced therein. Carriers can be for example used for the production of RNA in vitro, or for transfection, transformation, transduction or infection of host cells.

[0544] In some applications, the nucleotide sequences used in the present invention are operably linked to regulatory sequences that are capable of providing expression of the nucleotide sequence, for example, by a selected host cell. For example, the present invention includes vectors comprising a nucleotide sequence encoding a gene for the SOUL hemoglobin-binding protein as described herein operably linked to such regulatory sequences, i.e., the vector is an expression vector.

[0545] 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 ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0546] The term "regulatory sequence" includes promoters and enhancers and other expression regulatory signals. The term "promoter" is used in the ordinary sense of the art, such as an RNA polymerase binding site. The nucleotide sequence within the construct encoding the gene encoding the SOUL heme-binding protein can be operably linked to at least one promoter.

[0547] The term "construct," which is synonymous with terms such as "cassette" or "vector," includes a nucleotide sequence for use in accordance with the present invention attached directly or indirectly to a promoter.

[0548] An example of indirect attachment is to provide a suitable spacer group, such as an intron sequence, such as the Sh1-intron or the ADH intron, between the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in connection with the present invention, which includes direct or indirect attachment. In some cases, the term does not include the natural combination of nucleotide sequences that are typically associated with wild-type gene promoters and the coding proteins when they are all in their natural environment. The construct can even contain or express a marker that allows selection of the genetic construct.

[0549] In some embodiments, the promoter can be operably linked to a nucleotide sequence in a construct or vector for regulating the concentration and / or total content of nicotine in a cell or cell culture or a tobacco plant or part thereof.

[0550] 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.

[0551] In one embodiment, the promoter can be a constitutive promoter.

[0552] Constitutive promoters direct the expression of a gene continuously throughout all 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, such as 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).

[0553] The constitutive promoter may be selected from the group consisting of: 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.

[0554] The promoter can be a tissue-specific promoter. A tissue-specific promoter is a promoter that directs the expression of a gene in one (or several) parts of a plant, typically throughout the life of those plant parts. The category of tissue-specific promoters also generally includes promoters whose specificity is not absolute, i.e., they can 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).

[0555] In another embodiment, the promoter can be a developmentally regulated promoter.

[0556] 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. The gene may be expressed at different (usually lower) levels in that plant part at other times, and may also be expressed in other plant parts.

[0557] In one embodiment, the promoter can be an inducible promoter.

[0558] Inducible promoter can respond to the expression of inducer-directed gene.In the absence of inducer, gene will not express.Inducer can act directly on promoter sequence, or can work by offsetting the effect of repressor molecule.Inducer can be chemical agent such as metabolite, albumen, growth regulator (such as auxin and salicylic acid that activates OCS promoter) or toxic element, physiological stress such as heat, light (such as soybean SSU promoter), damage (for example nos, nopaline synthase promoter), or osmotic pressure, or the indirect consequence of pathogen or pest effect. The promoter of development regulation can be described as the inducible promoter of specific type, and its response is 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, such as described 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, such as described by Gatz ((1995) Methods in Cell Biol. 50 411-424).

[0559] The nucleotide sequence encoding the protein having the specific properties of the gene encoding the SOUL heme-binding protein as defined herein or the protein that is suitable for modification can be identified and / or separated and / or purified from any cell or organism that produces the protein. Various methods for the identification and / or separation and / or purification of nucleotide sequences are well known in the art. For example, once the appropriate sequence has been identified and / or separated and / or purified, more sequences can be prepared using PCR amplification techniques.

[0560] In a still further alternative, the nucleotide sequence encoding the SOUL heme-binding protein may be prepared synthetically by established standard methods, such as those described by Beucage et al. (1981). Tetrahedron Letters 22, 1859-1869 (which is incorporated herein by reference), or by the phosphoramidite method described in Matthes et al., (1984) EMBO J 3, 801-805 (which is incorporated herein by reference). In the phosphoramidite method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, annealed, ligated, and cloned in an appropriate vector.

[0561] 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".

[0562] The present invention also includes the use of sequences that have a certain degree of sequence identity or sequence homology with one or more amino acid sequences of a polypeptide having the specific properties defined herein, or any nucleotide sequence, i.e., a SOUL hemoglobin-binding protein gene encoding such a polypeptide (hereinafter referred to as "one or more homologous sequences"). Here, the term "homolog" means an entity that has a certain degree of homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" can be equivalent to "identity".

[0563] Homologous amino acid sequences and / or nucleotide sequences and / or fragments should provide and / or encode polypeptides that retain the functional activity of the SOUL heme-binding protein gene and / or enhance the activity of the SOUL heme-binding protein gene. Typically, 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 having similar chemical properties / functions), in the context of the present invention, it preferably refers to homology in terms of sequence identity. Homologous sequences typically retain functional domains or motifs. Suitably, homologs of the SOUL heme-binding protein may contain a heme-binding site.

[0564] In one embodiment, homologous sequences are taken to include amino acid sequences or nucleotide sequences that have one, two or several additions, deletions and / or substitutions compared to the subject sequence.

[0565] Sequence identity

[0566] Sequence identity ...

Claims

1. A method for regulating the alkaloid content of a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture, the method comprising modifying the tobacco plant or tobacco plant cell or tobacco plant cell culture by regulating the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the content of one or more alkaloids selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine is adjusted; and The gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID No.

1.

2. A method for regulating the alkaloid content of a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture, said method comprising modifying said tobacco plant or tobacco plant cell or tobacco plant cell culture by regulating the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the content of the alkaloid nornicotine is modulated; and The gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 4, 7, 10, 13, 16, 19 or 22.

3. The method of claim 1 or claim 2, wherein the method is a method for reducing alkaloid content.

4. Use of a gene encoding a SOUL heme-binding protein for regulating the alkaloid content of a tobacco plant cell, or a tobacco plant or part thereof, or a tobacco plant cell culture, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1; and The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

5. Use of a gene encoding a SOUL heme-binding protein for regulating the alkaloid content of a tobacco plant cell, or a tobacco plant or part thereof, or a tobacco plant cell culture, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 4, 7, 10, 13, 16, 19 or 22; and Wherein the alkaloid is nornicotine.

6. A method for producing a tobacco plant or part thereof, a tobacco plant cell or a tobacco plant cell culture, or a tobacco plant propagation material having a modulated alkaloid content, the method comprising modifying the tobacco plant or tobacco plant cell or tobacco plant cell culture to modulate the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1; and The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

7. A method for producing a tobacco plant or part thereof, a tobacco plant cell or a tobacco plant cell culture, or a tobacco plant propagation material having a modulated alkaloid content, the method comprising modifying the tobacco plant or tobacco plant cell or tobacco plant cell culture to modulate the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 4, 7, 10, 13, 16, 19 or 22; and Wherein the alkaloid is nornicotine.

8. The method of claim 6 or claim 7, wherein the modulated alkaloid content is a reduced alkaloid content.

9. The method of any one of claims 1, 2, 6 or 7 or the use of any one of claims 4 or 5, wherein the alkaloid content is modulated compared to a corresponding tobacco plant or tobacco plant cell or tobacco plant cell culture that has not been modified to modulate the activity or expression of a gene encoding SOUL heme-binding protein.

10. A method for producing a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture, which is modified to achieve modulation of alkaloid content compared to a corresponding unmodified tobacco plant or part thereof, or an unmodified tobacco plant cell or an unmodified tobacco plant cell culture, wherein the modification is modulation of the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1; and The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

11. A method for producing a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture, which is modified to achieve modulation of alkaloid content compared to a corresponding unmodified tobacco plant or part thereof, or an unmodified tobacco plant cell or an unmodified tobacco plant cell culture, wherein the modification is modulation of the activity or expression of a gene encoding a SOUL heme-binding protein, wherein the gene encoding the SOUL heme-binding protein encodes a polypeptide consisting of an amino acid sequence as shown in SEQ ID No. 4, 7, 10, 13, 16, 19 or 22; and Wherein the alkaloid is nornicotine.

12. A method of producing plant propagation material obtained from a tobacco plant as defined in claim 10 or claim 11 or a tobacco plant or tobacco plant cell or tobacco plant cell culture produced by the method of any one of claims 6 to 9.

13. The method of any one of claims 1, 2, 6, 7, 10, 11 or 12 or the use of any one of claims 4 or 5, wherein the alkaloid content of the tobacco plant or tobacco plant cell or tobacco plant cell culture is reduced compared to a corresponding tobacco plant or tobacco plant cell or tobacco plant cell culture that has not been modified to modulate the activity or expression of a gene encoding SOUL heme-binding protein.

14. The method or use of claim 13, wherein the activity or expression of the gene encoding the SOUL heme-binding protein is reduced compared to a corresponding tobacco plant or tobacco plant cell or tobacco plant cell culture that has not been modified to modulate the activity or expression of the gene encoding the SOUL heme-binding protein.

15. The method of any one of claims 1, 2, 6, 7, 10, 11 or 12 or the use of any one of claims 4 or 5, wherein the alkaloid content of the tobacco plant or tobacco plant cell or tobacco plant cell culture is increased compared to a corresponding tobacco plant or tobacco plant cell or tobacco plant cell culture that has not been modified to modulate the activity or expression of a gene encoding SOUL heme-binding protein.

16. The method or use of claim 15, wherein the tobacco plant or tobacco plant cell or tobacco plant cell culture is modified to increase the activity or expression of a gene encoding a SOUL heme-binding protein, and the tobacco plant or tobacco plant cell or tobacco plant cell culture exhibits increased alkaloid content compared to a corresponding tobacco plant or tobacco plant cell or tobacco plant cell culture that has not been modified to modulate the activity or expression of a gene encoding a SOUL heme-binding protein.

17. The method according to any one of claims 1-2, 6-7 or claim 10 or claim 11 or the use according to any one of claims 4 or 5, wherein the total alkaloid content of the tobacco plant or tobacco plant cell or tobacco plant cell culture is modulated.

18. The method of any one of claims 1-2, 6-7 or claim 10 or claim 11 or the use of any one of claims 4 or 5, wherein the total alkaloid content of the tobacco plant or tobacco plant cell or tobacco plant cell culture is reduced.

19. The method according to any one of claims 1, 6 or 10 or the use according to claim 4, wherein the content of one or more alkaloids selected from nicotine, nornicotine and / or pseudo-oxidized nicotine is adjusted.

20. The method according to any one of claims 1, 6 or 10 or the use according to claim 4, wherein the content of one or more alkaloids selected from nicotine, nornicotine and / or pseudo-oxidized nicotine is reduced.

21. The method or use of claim 19, wherein the nicotine content is modulated.

22. The method or use of claim 20, wherein the nicotine content is reduced.

23. The method according to any one of claims 1, 6 or 10 or the use according to claim 4, wherein the gene encoding the SOUL heme-binding protein consists of the nucleotide sequence shown in SEQ ID No. 2 or 3.

24. The method according to any one of claims 2, 7 or 11 or the use according to claim 5, wherein the gene encoding the SOUL heme-binding protein consists of a nucleotide sequence as shown in SEQ ID No. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

25. Use of a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture as defined in any one of claims 10, 11 or 13-24, or a tobacco plant produced by the method of any one of claims 6-9 for growing plants.

26. Use of a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture as defined in any one of claims 10, 11 or 13-24, or a plant produced by the method of any one of claims 6-9 for producing a product.

27. Use of a tobacco plant or part thereof as defined in any one of claims 10, 11 or 13 to 24, or a plant produced by the method of any one of claims 6 to 9, for growing crops.

28. Use of a tobacco plant or part thereof according to any one of claims 10, 11 or 13-24, or a plant produced by the method of any one of claims 6-9, for producing leaves.

29. The method of any one of claims 6, 7, 10, 14, 16, or 21-22, further comprising harvesting leaves from the tobacco plant, thereby obtaining harvested tobacco leaves.

30. The method of claim 29, wherein the harvested tobacco leaves are cut harvested leaves.

31. Processed tobacco leaves, which: Obtaining a plant obtained from the use according to any one of claims 4, 5 or 25-27; Obtained by processing a plant as defined in any one of claims 10, 11 or 13-24; Obtained from a plant propagated from a plant propagation material according to claim 12; or Obtained by processing harvested leaves of a plant as defined in any one of claims 10, 11 or 13-24; or Obtained from a plant produced by the method of any one of claims 6, 7, 8, 9, 10, 11 or 13-24.

32. The processed tobacco leaf blade of claim 31, wherein the processed tobacco leaf blade is a non-viable processed tobacco leaf blade.

33. A processed tobacco leaf according to claim 31 or claim 32, wherein the leaf is processed by curing, fermenting, pasteurizing or a combination thereof.

34. A processed tobacco leaf according to claim 31 or claim 32, wherein the processed tobacco leaf is a cut processed leaf.

35. The processed tobacco leaf of claim 33, wherein the processed tobacco leaf is a cut processed leaf.

36. A cured tobacco material made from a plant or part thereof as defined in any one of claims 10, 11 or 13-24, or an extract of said cured tobacco material.

37. A tobacco blend comprising the cured tobacco material of claim 36.

38. Tobacco industry products prepared from: A tobacco plant or part thereof as defined in any one of claims 10, 11 or 13-24, or a tobacco plant cell or tobacco plant cell culture as defined in any one of claims 10, 11 or 13-24; A tobacco plant or part thereof propagated from plant propagation material of a tobacco plant according to claim 12; Harvested leaves of a tobacco plant obtained by the method of claim 29 or claim 30; A processed tobacco leaf according to any one of claims 31-35; or A tobacco plant produced by the method of any one of claims 6, 7, 8, 9, 10, 11 or 13-24.

39. The tobacco industry product of claim 38, wherein the tobacco industry product is a combustible smoking article.

40. The tobacco industry product of claim 38, wherein the tobacco industry product is a smokeless tobacco product.

41. The tobacco industry product of claim 38, wherein the tobacco industry product is a non-combustible gas aerosol supply system.

42. The tobacco industry product of claim 41, wherein the tobacco industry product is a tobacco heating device or an aerosol generating device.

43. A combustible smoking article, a non-combustible gas aerosol delivery system, a smokeless tobacco product or a tobacco heating device comprising a tobacco plant or part thereof as defined in any one of claims 10, 11 or 13-24, or a tobacco plant cell or tobacco plant cell culture as defined in any one of claims 10, 11 or 13-24; or a modulated tobacco material as described in claim 36; or a tobacco blend as described in claim 37.

44. Use of a nucleotide sequence encoding a gene for SOUL heme-binding protein for selecting tobacco plants with modulated alkaloid content, wherein the sequence of the SOUL heme-binding protein is SEQ ID No. 1; or wherein the gene encoding the SOUL heme-binding protein consists of a nucleotide sequence as shown in SEQ ID No. 2 or 3, and The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

45. Use of a nucleotide sequence encoding a gene for SOUL heme-binding protein for selecting tobacco plants with modulated alkaloid content, wherein the sequence of the SOUL heme-binding protein is selected from SEQ ID No. 4, 7, 10, 13, 16, 19 or 22; or wherein the gene encoding the SOUL heme-binding protein consists of a nucleotide sequence as shown in SEQ ID No. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, and wherein the alkaloid is nornicotine.

46. ​​The use of claim 44 or claim 45, wherein the modulated alkaloid content is a reduced alkaloid content.

47. A method for producing a tobacco plant carrying a heritable mutation in a nucleotide sequence of a gene encoding a SOUL heme-binding protein, wherein the nucleotide sequence of the gene is selected from SEQ ID No. 2 or 3; wherein the heritable mutation modulates the activity or expression of the gene encoding the SOUL heme-binding protein, and wherein the mutant tobacco plant has a modulated alkaloid content relative to a comparable plant not carrying the heritable mutation, The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

48. A method for producing a tobacco plant carrying a heritable mutation in a nucleotide sequence of a gene encoding a SOUL heme-binding protein, wherein the nucleotide sequence of the gene is selected from SEQ ID No. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; wherein the heritable mutation modulates the activity or expression of the gene encoding the SOUL heme-binding protein, and wherein the mutant tobacco plant has a modulated alkaloid content relative to a comparable plant not carrying the heritable mutation, Wherein the alkaloid is nornicotine.

49. The method of claim 47 or claim 48, wherein the heritable mutation reduces the activity or expression of a gene encoding the SOUL heme-binding protein.

50. The method of any one of claims 47-48, wherein the modulated alkaloid content is a decreased alkaloid content.

51. A method of producing progeny of a tobacco plant carrying a heritable mutation as defined in claim 47 or claim 48.

52. A method of producing seeds of a tobacco plant carrying a heritable mutation as defined in claim 47 or claim 48.

53. Processed leaves or cured tobacco material produced from a tobacco plant, said tobacco plant comprising a modification in the nucleotide sequence of a gene encoding a SOUL heme-binding protein, wherein the nucleotide sequence of said gene is selected from SEQ ID No. 2 or 3; wherein said modification modulates the activity or expression of said gene encoding a SOUL heme-binding protein, and wherein said tobacco plant has a modulated alkaloid content relative to a comparable plant not carrying said modification in said gene encoding a SOUL heme-binding protein, The alkaloid is one or more selected from nicotine, nornicotine, anabasine, pseudo-oxidized nicotine or anatabine.

54. A processed leaf or cured tobacco material produced from a tobacco plant comprising a modification in the nucleotide sequence of a gene encoding a SOUL heme-binding protein, wherein the nucleotide sequence of the gene is selected from the group consisting of SEQ ID No. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; wherein the modification modulates the activity or expression of the gene encoding the SOUL heme-binding protein, and wherein the tobacco plant has a modulated alkaloid content relative to a comparable plant not carrying the modification in the gene encoding the SOUL heme-binding protein, Wherein the alkaloid is nornicotine.

55. The processed leaf or cured tobacco material of Claim 53 or Claim 54, wherein the modification reduces the activity or expression of a gene encoding a SOUL heme-binding protein.

56. The processed leaf or cured tobacco material of any one of claims 53-54, wherein the modulated alkaloid content is a reduced alkaloid content.

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