bZIP transcription factors regulate the conversion of nicotine to nornicotine and reduce the levels of tobacco-specific nitrosamine (TSNA) precursors

Abstract

A method for reducing the conversion of nicotine to nornicotine is provided herein. The method comprises applying at least one basic region / leucine zipper (bZIP) type transcription factor inhibitor to an organism in need thereof. A method for reducing the conversion of nicotine to nornicotine is also provided herein, the method comprising mutating a bZIP type transcription factor binding site on a nicotine N-demethylase (NND) promoter. A method for reducing the conversion of nicotine to nornicotine is further provided herein, the method comprising mutating a plant genome to knock out at least one bZIP type transcription factor.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 857,718, filed on June 5, 2019, the entire disclosure of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted in ASCII format via EFS-Web, and is incorporated herein by reference in its entirety. The ASCII copy of the sequence listing created on June 5, 2020 is named 13177N-2183W2.txt and is 11.8 kilobytes in size. Technical Field

[0005] The present invention relates to articles and methods for regulating the conversion of nicotine to nornicotine. In particular, the presently disclosed subject matter relates to transcription factors and methods of use thereof for regulating the conversion of nicotine to nornicotine. Background of the Invention

[0007] Nicotiana tabacum (common tobacco) is a natural allotetraploid that originated about 200,000 years ago. The maternal S-genome is derived from an ancestor of N. sylvestris, while the paternal T-genome is from a relative of N. tomentosiformis. Nicotine is the major alkaloid accumulated in most cultivated tobacco varieties. In the past few decades, significant progress has been made in isolating and characterizing the structural genes in the nicotine biosynthetic pathway. Jasmonic acid (JA) is the main elicitor of nicotine biosynthesis. JA-responsive transcription factors (TFs) belong to two major families, APETALA2 / ETHYLENE RESPONSES FACTORS (AP2 / ERFs) and basic HELIX-LOOP-HELIX (bHLH), and are known to induce the expression of genes encoding key enzymes in the nicotine biosynthetic pathway.

[0008] Nicotine and other tropane alkaloids such as scopolamine and scopolamine are synthesized in the root and transported to the leaf by the xylem. Many transporters have been isolated and characterized for their role in the transport of alkaloids in plants and in vacuolar sequestration. In tobacco, many transporters belonging to MULTIDRUG and TOXIC COMPOUND ETRUSION (MATE) families, including the alkaloid transporter (JAT1 / 2) induced by MATE1 / 2 and jasmonic acid, participate in the transport and vacuolar uptake (sequestration) of nicotine. However, the TF involved in the regulation of these transporters has not been thoroughly studied.

[0009] In addition to nicotine, tobacco plants accumulate three other pyridine alkaloids, namely, nornicotine, anabasine, and anatabine. Nornicotine is a demethylated nicotine (without the methyl group) derived from nicotine by an enzymatic process. It is also a precursor to N-nitrosonornicotine (NNN), which is produced during the curing and processing of tobacco materials. More specifically, during post-harvest processing, nornicotine chemically reacts with nitrating agents to form NNN. Because NNN belongs to a class of smoking-related carcinogens called tobacco-specific nitrosamines (TSNAs), there is a great need to reduce TSNAs in tobacco products.

[0010] There are two possible ways to reduce TSNA. One is to reduce the overall nicotine content; the other is to eliminate the conversion of nicotine to nornicotine. The conversion of nicotine to nornicotine is catalyzed by nicotine N-demethylase (NND), which is a small family of cytochrome P450 enzymes. Three NND genes have been identified in the conversion of nicotine to nornicotine in tobacco, CYP82E4v1 (derived from Nicotiana tabacum), CYP82E5v2 (derived from Nicotiana tabacum) and CYP82E10 (derived from Nicotiana tabacum). CYP82E4v1 (E4) plays a major role in the conversion of nicotine to nornicotine in senescent leaves, while CYP82E10 (E10) is reported to be expressed in roots, and CYP82E5 (E5) works in both roots and leaves. However, so far, the transcription factors (TFs) involved in regulating the conversion of nicotine to nornicotine (i.e., transcriptional regulators of E4, 5 and 10 genes) have not been identified. Therefore, although significant progress has been made in the biochemical and molecular characterization of these nicotine transporters and the enzymes involved in nornicotine biosynthesis, the molecular mechanisms of regulation of these genes remain to be elucidated.

[0011] Therefore, there is a need for articles and methods for regulating the conversion of nicotine to nornicotine. SUMMARY OF THE INVENTION

[0013] The presently disclosed subject matter satisfies some or all of the above needs, as will become apparent to one of ordinary skill in the art after studying the information provided in this document.

[0014] This overview describes several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This overview is merely an example of many different embodiments. Mention of one or more representative features of a given embodiment is also exemplary. Such an embodiment may generally exist with or without the features mentioned; likewise, those features may be applied to other embodiments of the presently disclosed subject matter, whether or not listed in this overview. To avoid excessive repetition, this overview does not list or suggest all possible combinations of such features.

[0015] In some embodiments, the presently disclosed subject matter includes methods of reducing the conversion of nicotine to nornicotine and / or methods of reducing the level of at least one tobacco-specific nitrosamine (TSNA) precursor, the methods comprising reducing the activity and / or expression of at least one basic region / leucine zipper (bZIP)-type transcription factor in a tobacco plant, a portion of a tobacco plant, a tobacco plant cell or cell culture, a tobacco plant seed, or another organism that contains nicotine.

[0016] In some embodiments, also provided herein is a method of reducing the activity and / or expression of at least one basic region / leucine zipper (bZIP) type transcription factor in a tobacco plant, a part of a tobacco plant, a tobacco plant cell or cell culture, a tobacco plant seed, or another nicotine-containing organism for reducing the conversion of nicotine to nornicotine and / or for reducing the level of at least one tobacco-specific nitrosamine (TSNA) precursor in a tobacco plant, a part of a tobacco plant, a tobacco plant seed, or a nicotine-containing organism.

[0017] In some embodiments, the at least one bZIP transcription factor includes C bZIP transcription factors, S group bZIP transcription factors, and combinations thereof. In some embodiments, the at least one bZIP transcription factor includes NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and combinations thereof. In some embodiments, the at least one bZIP transcription factor includes NtbZIP1a and NtbZIP1b. In some embodiments, the at least one bZIP transcription factor includes NtbZIP2a and NtbZIP2b. In some embodiments, the at least one bZIP transcription factor includes NtbZIP1a, NtbZIP1b, NtbZIP2a, and NtbZIP2b.

[0018] In some embodiments, the at least one bZIP transcription factor comprises an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by SEQ ID NO: 1; the amino acids encoded by SEQ ID NO: 2; the amino acids encoded by SEQ ID NO: 3; the amino acids encoded by SEQ ID NO: 4; the amino acids encoded by SEQ ID NO: 5; and the amino acids encoded by SEQ ID NO: 6. In one embodiment, the at least one bZIP transcription factor comprises an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by SEQ ID NO: 1 or the amino acids encoded by SEQ ID NO: 2; and an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by SEQ ID NO: 3 or the amino acids encoded by SEQ ID NO: 4. In one embodiment, the at least one bZIP transcription factor comprises an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids of SEQ ID NO:5; and an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids of SEQ ID NO:6. In one embodiment, the at least one bZIP transcription factor comprises an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by SEQ ID NO: 1 or the amino acids of SEQ ID NO: 2; an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by SEQ ID NO: 3 or the amino acids of SEQ ID NO: 4; an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids of SEQ ID NO: 5; and / or an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids of SEQ ID NO: 6.

[0019] In some embodiments, reducing the activity and / or expression of at least one bZIP-type transcription factor comprises: mutating the bZIP-type transcription factor, such as providing a mutation in the binding site of the bZIP-type transcription factor; using a bZIP-type transcription factor inhibitor; or using a gene silencing technique, such as RNAi. In some embodiments, the bZIP transcription factor inhibitor comprises an antisense oligonucleotide, miRNA, siRNA, locked nucleic acid (LNA) nucleotide, or a combination thereof.

[0020] In some embodiments, the mutant bZIP-type transcription factor includes a binding site on a mutant nicotine N-demethylase (NND) promoter. In one embodiment, the NND includes CYP82E4v1, CYP82E5v2 or CYP82E10. In another embodiment, the NND is CYP82E4v1, and the bZIP binding site on the CYP82E4v1 promoter is an A / G box with a pre-mutated sequence of TACGTC. In another embodiment, the mutant binding site has the sequence TGCGTC. In another embodiment, the mutant binding site is formed by site-directed mutagenesis.

[0021] In some embodiments, mutating a bZIP-type transcription factor comprises mutating a plant genome to knock out at least one basic region / leucine zipper (bZIP)-type transcription factor. In one embodiment, the at least one bZIP transcription factor comprises a C bZIP transcription factor, a S group bZIP transcription factor, or a combination thereof. In one embodiment, the at least one bZIP transcription factor comprises NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, or a combination thereof. In one embodiment, the at least one bZIP transcription factor comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acids encoded by SEQ ID NO: 1 or the amino acids of SEQ ID NO: 2; the amino acids encoded by SEQ ID NO: 3 or the amino acids of SEQ ID NO: 4; the amino acids of SEQ ID NO: 5; or the amino acids of SEQ ID NO: 6.

[0022] In some embodiments, the present invention also provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a nucleic acid sequence that encodes a sequence that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof.

[0023] In some embodiments, the present invention also provides an isolated nucleic acid molecule encoding a bZIP-type transcription factor with at least one mutation. In one embodiment, the mutation is in a binding site on a nicotine N-demethylase (NND) promoter. In another embodiment, the NND includes CYP82E4v1, CYP82E5v2 or CYP82E10. In another embodiment, the NND is CYP82E4v1, and the bZIP binding site on the CYP82E4v1 promoter is an A / G box with a pre-mutation sequence of TACGTC. In another embodiment, the mutated binding site has the sequence TGCGTC. In some embodiments, the bZIP transcription factor comprises NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b or a combination thereof. In some embodiments, the bZIP transcription factor comprises a nucleic acid sequence having a sequence of SEQ ID NO:1 or SEQ ID NO:3; a nucleic acid sequence encoding a sequence having a sequence of SEQ ID NO:2, 4, 5 or 6.

[0024] In some embodiments, also provided herein are constructs or vectors comprising the nucleic acid molecules described herein.

[0025] In some embodiments, the present invention also provides tobacco plant cells: a) comprising an exogenous gene, wherein the exogenous gene comprises a nucleic acid molecule described herein; b) comprising a construct or vector described herein; c) obtained or obtainable by the method described herein; and / or d) obtained by reducing the activity and / or expression of at least one bZIP-type transcription factor or knocking out a mutation of at least one bZIP-type transcription factor.

[0026] Also provided herein, in some embodiments, are uses of tobacco cells described herein in producing tobacco industry products.

[0027] In some embodiments, also provided herein are tobacco cell cultures or plants: a) comprising a plant cell described herein; b) which has been modified to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the culture or plant compared to an unmodified culture or plant; c) comprising a construct or vector described herein.

[0028] In some embodiments, also provided herein are plant propagation materials or plant extracts obtainable from the plants described herein or parts of said plants. In one embodiment, the plant propagation material is a plant seed.

[0029] In some embodiments, the purposes of the tobacco plants described herein in cultivating tobacco plants are also provided herein. In some embodiments, the purposes of the tobacco plants described herein are to produce tobacco industry products. In some embodiments, the purposes of the tobacco plants described herein are to plant crops. In some embodiments, the purposes of the tobacco plants described herein are to produce processed tobacco leaves. In one embodiment, the processed tobacco leaves are flue-cured tobacco leaves.

[0030] In some embodiments, also provided herein are harvested leaves of the tobacco plants described herein.In some embodiments, the harvested leaves are cut harvested leaves.

[0031] In some embodiments, also provided herein is a processed tobacco leaf: a) comprising a plant cell described herein; b) can be obtained by processing a tobacco plant described herein; c) can be obtained from a plant propagated from a plant propagation material described herein; and / or d) can be obtained by processing the leaves of the harvest described herein. In some embodiments, the processed tobacco leaf is a non-viable processed tobacco leaf. In some embodiments, the plant or leaf is processed by curing, fermentation, pasteurization, or a combination thereof. In some embodiments, the processed tobacco leaf is a cut processed tobacco leaf.

[0032] Also provided herein, in some embodiments, is a flue-cured tobacco material prepared from the plants, parts thereof, extracts thereof, or cell cultures described herein.

[0033] Also provided herein, in some embodiments, are tobacco blends comprising the flue-cured tobacco material described herein.

[0034] In some embodiments, a tobacco industry product is also provided herein, comprising: a) prepared from a tobacco plant or part thereof described herein; b) prepared from a tobacco plant or part thereof (preferably leaves harvested from a plant) obtained or obtainable by the method described herein; c) prepared from a plant (preferably leaves) propagated from plant propagation materials described herein; d) prepared from leaves harvested as described herein; e) prepared from leaves processed as described herein; f) prepared from plant extracts obtained from modified plants described herein or comprising plant extracts of modified plants described herein; g) flue-cured tobacco materials described herein; and / or h) tobacco blends described herein. In some embodiments, the tobacco industry product is a smoking article. In some embodiments, the tobacco industry product is a smokeless tobacco product. In some embodiments, the tobacco product is a non-flammable aerosol supply system such as a tobacco heating device, such as an aerosol generating device.

[0035] In some embodiments, the present invention also provides a combustible smoking article, a non-flammable aerosol supply system, a smokeless tobacco product, or a tobacco heater, which comprises a plant or part thereof of the species Nicotiana or Nicotiana rustica described herein, or an extract thereof, or a tobacco cell culture described herein, or a flue-cured tobacco material described herein, or a tobacco blend described herein.

[0036] In some embodiments, the present invention also provides the use of a nucleotide sequence encoding at least one gene of a bZIP-type transcription factor having at least one mutation, wherein the sequence of the bZIP-type transcription factor includes a sequence encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least one mutation relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a sequence having at least one mutation relative to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof. In some embodiments, the sequence is used to select plants with reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors.

[0037] Further features and advantages of the presently disclosed subject matter will become apparent to one of ordinary skill in the art after studying the description, drawings, and non-limiting examples in this document.

[0038] Brief description of sequence listing

[0039] This application contains a Sequence Listing attached.

[0040] SEQ ID NO: 1 is the nucleotide sequence encoding NtbZIP1a.

[0041] SEQ ID NO: 2 is the amino acid sequence of NtbZIP1a.

[0042] SEQ ID NO: 3 is the nucleotide sequence encoding NtbZIP1b.

[0043] SEQ ID NO: 4 is the amino acid sequence of NtbZIP1b.

[0044] SEQ ID NO:5 is the amino acid sequence of NtbZIP2a.

[0045] SEQ ID NO:6 is the amino acid sequence of NtbZIP2b. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The presently disclosed subject matter will be better understood when considering the following detailed description, and features, aspects, and advantages other than those set forth above will become apparent. Such detailed description refers to the following drawings, in which:

[0047] Figure 1A diagram illustrating a hierarchical cluster analysis of transcriptome data from eight different tobacco tissues is shown. Each tissue forms a distinct cluster based on expression.

[0048] Figure 2 Shown is a diagram illustrating the distribution of different TF families in tobacco and its ancestors.

[0049] Figure 3 Figures illustrating the co-expression analysis of TF genes and structural genes in the nicotine biosynthetic pathway in different tissues are shown. TF and structural genes were divided into 8 different modules based on their expression (color-coded side bars). The black modules contain most of the structural genes in the nicotine biosynthetic pathway. YF, young flowers; MF, mature flowers; YL, young leaves; ML, mature leaves; SL, senescent leaves.

[0050] Figure 4 Shown are graphs illustrating the expression of NtbZIP1 a / b and NND in different tobacco tissues.

[0051] Figure 5 An image illustrating the bZIP family in tobacco is shown. NtbZIP1a and NtbZIP1b are indicated by *.

[0052] Figure 6 An image comparing the nucleotide and amino acid sequences of NtbZIP1a and 1b is shown.

[0053] Figure 7 Shown is a graph illustrating that transient overexpression of NtbZIP1a in tobacco leaves induces the expression of CYP82E4v1, CYP82E5v2, and CYP82E10.

[0054] Figure 8 Shown are graphs demonstrating that NtbZIP1a significantly activates CYP82E4v1 and CYP82E10 promoters in tobacco cells.

[0055] Fig. 9 Shown is a diagram illustrating that NtbZIP1a activates the CYP82E4v1 promoter in tobacco cells by binding to the A / G box.

[0056] Fig.10 Shown is a graph illustrating that NtbZIP1a and b significantly activate the CYP82E4v1 promoter in tobacco cells.

[0057] Fig.11 Shown is a graph demonstrating that topping of tobacco plants down-regulates the expression of NtbZIP and NND.

[0058] Fig. 12A-C shows graphs and images illustrating overexpression of NtbZIP1a in tobacco plants. (A) Genomic DNA PCR and cDNA PCR of control and three transgenic lines (lines #4, 5 and 9) confirm integration and expression of antibiotic selection marker neomycin phosphotransferase II (npt II; Kan), respectively. (B) Quantitative real-time (qRT-PCR) analysis shows relative expression of NtbZIP1 and E4 in control (EV) and transgenic lines (lines #4, 5 and 9). (C) Metabolic analysis shows conversion of nicotine to nornicotine in control and transgenic lines (T0 or first generation transgenic plants).

[0059] Fig.13 Shown is a graphic illustrating the amino acid sequence alignment of NtbZIP2a and 2b.

[0060] Fig.14 Shown is a graph illustrating that NtbZIP2a and b have similar expression patterns in tobacco tissues.

[0061] Fig.15 Shown is a diagram illustrating that NtbZIP2 acts synergistically with NtbZIP1 to activate the E4 promoter in tobacco cells.

[0062] Fig.16A -B shows images illustrating protein-protein interactions of NtbZIP1 and NtbZIP2 using yeast two-hybrid assay. (A) Yeast two-hybrid assay showing protein-protein interaction between NtbZIP1 and NtbZIP2. Colony growth on synthesis-deficient (SD) medium lacking leucine, tryptophan, histidine and adenine (-leu-trp-his-ade) indicates interaction between proteins (bZIPs). (B) Schematic diagram of NtbZIP1 and NtbZIP2. The bZIP domains are represented by the "shaded" rectangles. Numbers represent amino acids.

[0063] Fig.17 Schematic diagram of RNAi vector comprising a portion of tobacco bZIP1 in sense and antisense orientation with introns. The partial cDNA and introns are expressed under the control of the CaMV 35S promoter and rbcS terminator. RNAi primers (indicated by arrows) are designed to amplify sequences outside the conserved domain.

[0064] Fig.18A -B illustrates (A) NtbZIP1a and NtbZIP1b expression was reduced by 70-80% and E4 expression was reduced by 80-90% in RNAi lines compared to EV controls. (B) Nicotine conversion to nornicotine was reduced by about 50% in RNAi lines compared to EV plants.

[0065] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown in the drawings by way of example and are described in detail below. However, it should be understood that the description of specific embodiments is not intended to limit the present disclosure to cover all modifications, equivalents and substitutes within the spirit and scope of the present disclosure defined by the appended claims.

[0066] definition

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to the methods and materials described herein can be used in the implementation or testing of the present disclosure, including the methods and materials described below.

[0068] Following long-standing patent law convention, the terms "a," "an," and "the" when used in this application, including the claims, mean "one or more." Thus, for example, reference to "a cell" includes a plurality of cells, and so forth.

[0069] The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0070] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties, such as reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0071] As used herein, the term "about" when referring to a value or amount of mass, weight, time, volume, concentration, percentage, and the like, is intended to encompass variations of ±50% in some embodiments, ±40% in some embodiments, ±30% in some embodiments, ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, ±0.1% from the specified amount, and ±0.01% from the specified amount (when such variations are appropriate to perform the disclosed methods).

[0072] As used herein, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. It is also understood that a number of values ​​are disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0073] Certain sequences are described herein with reference to sequence "identity". Sequence identity comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the % identity between two or more sequences.

[0074] % identity can be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is compared directly to the corresponding amino acid in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0075] While this is a very simple and consistent approach, it does not take into account that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the subsequent amino acid residues to be misplaced and therefore may result in a substantial reduction in % identity when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without overly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0076] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher correlation between the two compared sequences - will receive a higher score than a sequence alignment with many gaps. An "affine gap cost" is often used, which charges a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using this type of software for sequence comparison, it is preferred to use the default values.

[0077] Calculation of maximum % identity therefore first requires the generation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed-Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1):187-8 and tatiana@ncbi.nlm.nih.gov ), FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and AlignX. At least BLAST, BLAST 2 and FASTA can be used for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60).

[0078] The % homogeneity of the nucleotide sequence ...

[0079] Alternatively, percent identity can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.) based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

[0080] Once the software has generated an optimal alignment, it can then calculate the % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0081] If gap penalties are used when determining sequence identity, the following parameters can be used for pairwise alignments:

[0082] For BLAST GAP OPEN 0 GAP EXTENSION 0

[0083] For CLUSTAL DNA protein WORD SIZE 2 1 K triple GAP PENALTY 15 10 GAP EXTENSION 6.66 0.1

[0084] In one embodiment, BLAST may be used with the gap penalty and gap extension sets defined above.

[0085] In one embodiment, CLUSTAL may be used with the gap penalty and gap extension sets defined above.

[0086] In some embodiments, the gap penalty for BLAST or CLUSTAL alignments may be different from those detailed above. The skilled person will appreciate that the standard parameters for performing BLAST and CLUSTAL alignments may change periodically, and can select appropriate parameters based on the standard parameters detailed for the BLAST or CLUSTAL alignment algorithm at the time.

[0087] Suitably, the degree of identity with respect to a nucleotide sequence or an amino acid sequence is determined over at least 20 consecutive nucleotides / amino acids, preferably over at least 30 consecutive nucleotides / amino acids, preferably over at least 40 consecutive nucleotides / amino acids, preferably over at least 50 consecutive nucleotides / amino acids, preferably over at least 60 consecutive nucleotides / amino acids, preferably over at least 100 consecutive nucleotides / amino acids.

[0088] Suitably, the degree of identity with respect to nucleotide sequences or amino acid sequences may be determined over the entire sequence.

[0089] The sequence may also have deletions, insertions or substitutions of amino acid residues which produce silent changes and result in functionally equivalent substances. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

[0090] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column and preferably in the same row in the third column can replace each other:

[0091]

[0092] The present invention also encompasses homologous substitutions that may occur (substitution and replacement are used herein to indicate the exchange of existing amino acid residues with alternative residues), i.e., similar substitutions such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions may also occur, i.e., from one type of residue to another, or involving unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0093] Substitution with unnatural amino acids may also be performed, including; α* and α-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halogenated derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, pI-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α-aminoisobutyric acid*, L-ε-aminocaproic acid # , 7-aminoheptanoic acid*, L-methionine sulfone #* , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) # 、L-Tyr(methyl)*、L-Phe(4-isopropyl)*、L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*、L-diaminopropionic acid # and L-Phe(4-benzyl)*. For the purposes of the above discussion (involving homologous or nonhomologous substitution), the symbol * is used to denote the hydrophobic nature of the derivative, while # has been used to denote the hydrophilic nature of the derivative, with #* indicating an amphiphilic character.

[0094] The variant amino acid sequence may include a suitable spacer group that can be inserted between any two amino acid residues of the sequence, including, in addition to amino acid intervals such as glycine or β-alanine, alkyl groups such as methyl, ethyl or propyl. Those skilled in the art will fully appreciate another form of variation, including the presence of one or more amino acid residues in peptidomimetic form. For the avoidance of doubt, "peptidomimetic form" is used to refer to variant amino acid residues in which the α-carbon substituent is on the nitrogen atom of the residue rather than the α-carbon. Methods for preparing peptides in peptidomimetic form are known in the art, such as Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.

[0095] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridizing to the sequences of the present invention or to sequences that are complementary thereto.

[0096] As used herein, the term "hybridization" shall include "the process by which a nucleic acid strand joins with a complementary strand through base pairing" as well as the amplification process performed in polymerase chain reaction (PCR) technology.

[0097] The present invention also relates to nucleotide sequences that can hybridize to the sequences of the present invention, including the complements of those presented herein.

[0098] Preferably, hybridization is determined under stringent conditions (eg, 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015 M Na 3 citrate pH 7.0}).

[0099] More preferably, hybridization is determined under high stringency conditions (eg, 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na 3 citrate pH 7.0}).

[0100] The term "commercially desirable trait" includes traits such as yield, quality (e.g. leaf quality, properly cured leaf quality), abiotic (e.g. drought) stress tolerance, herbicide tolerance and / or biotic (e.g. insect, bacterial or fungal) stress tolerance and / or disease tolerance.

[0101] Leaf quality can be measured based on the color, texture, and aroma of the roasted leaves, for example according to United States Department of Agriculture (USDA) grades and standards.

[0102] Tobacco grades are assessed 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 leaf coloration and glossiness), hygroscopicity (the ability of the tobacco leaf to absorb and retain moisture from the environment), and green nuances or appearance.

[0103] Leaf grade can be determined using standard methods known in the art, for example, using

[0104] Official standard grades published by the USDA

[0105] Department of Agriculture (7 U.S.C. § 511). See, e.g., the official standard grades of burley tobacco.

[0106] Tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55 F.R. 40645);

[0107] The official standard grades for flue-cured tobacco (U.S. types 11, 12, 13, 14 and foreign type 92), effective March 27, 1989 (54 F.R. 7925); the official standard grades for Pennsylvania Seedleaf Tobacco (U.S. type 41), effective January 8, 1965 (29 F.R. 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 F.R. 11926); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for Georgia and Florida ShadeGrown Cigar-Wrapper Tobacco (U.S. Type 62), effective April 1971. USDA grade index values ​​may be determined based on industry-recognized grade indices. See, e.g., Bowman et al, Tobacco Science, 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated herein in their entirety).

[0108] In one aspect, the USDA grade index is a 0-100 numerical representation of the federal grade received, being a weighted average of all stalk positions. A higher grade index indicates higher quality. Alternatively, leaf grade can be determined by hyperspectral imaging. See, e.g., WO 2011 / 027315 (which is incorporated herein by reference).

[0109] In one embodiment, the tobacco plants of the present invention provide commercially acceptable grades of tobacco.

[0110] Suitably, the tobacco plants of the present invention provide commercially acceptable grades of flue-cured tobacco.

[0111] In one embodiment, when growing under similar growth conditions, the USDA grade index value of the leaf that tobacco plant of the present invention can produce is at least about 70% of the USDA grade index value of comparable plant leaves.Suitably, when growing under similar growth conditions, the USDA grade index value of the leaf that tobacco plant disclosed herein can produce is 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% of the USDA grade index value of contrast plant. Suitably, the tobacco plants disclosed herein are capable of producing leaves having a USDA Grade Index value that is 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130% or 120%-130% of the USDA Grade Index value of a comparable plant.

[0112] 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 are capable of producing leaves having a USDA Grade Index value of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more.

[0113] Unless otherwise indicated, as used herein, tobacco yield refers to the cured leaf yield calculated based on the weight of cured tobacco leaf per acre under standard field conditions in accordance with standard agronomic and curing practices.

[0114] In one aspect, tobacco plants of the invention have a yield that is 50%-150%, 55%-145%, 60%-140%, 65%-135%, 70%-130%, 75%-125%, 80%-120%, 85%-115%, 90%-110%, 95%-105%, 50%-100%, 55%-100%, 60%-10 ... -100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 100%-150%, 105%-150%, 110%-150%, 115%-150%, 120%-150%, 125%-150%, 130%-150%, 135%-150%, 140%-150% or 145%-150%.

[0115] In another aspect, the tobacco plants of the invention have a yield that 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.

[0116] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable plants that are cured when grown under similar field conditions.

[0117] In one aspect, the tobacco plant of the invention provides a yield selected from the group consisting of about 1200-3500, 1300-3400, 1400-3300, 1500-3200, 1600-3100, 1700-3000, 1800-2900, 1900-2800, 2000-2700, 2100-2600, 2200-2500, and 2300-2400 pounds per acre.

[0118] In another aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of: about 1200-3500, 1300-3500, 1400-3500, 1500-3500, 1600-3500, 1700-3500, 1800-3500, 1900-3500, 2000-3500, 2100-3500, 2200-3500, 2300-3500, 2400-3500, 2500-3500, 2600-3500, 2700-3500, 2800-3500, 2900-3500, 3000-3500, and 3100-3500 pounds per acre.

[0119] In another aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200-3500, 1200-3400, 1200-3300, 1200-3200, 1200-3100, 1200-3000, 1200-2900, 1200-2800, 1200-2700, 1200-2600, 1200-2500, 1200-2400, 1200-2300, 1200-2200, 1200-2100, 1200-2000, 1200-1900, 1200-1800, 1200-1700, 1200-1600, 1200-1500, and 1200-1400 pounds per acre.

[0120] As used herein, term "tobacco plant" refers to a Nicotiana plant for the production of tobacco industry products. Non-limiting examples of suitable tobacco plants include Nicotiana tabacum and Nicotiana rustica (N. rustica) (e.g., LAB21, LN KY171, T1 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico).

[0121] Thus, in one embodiment the tobacco plant comprises Nicotiana plumbaginifolia.

[0122] Tobacco material can be derived from the kind of tobacco species, is commonly referred to as burley tobacco kind, smoked or bright color kind, dark variety and oriental / Turkey kind.In some embodiments, tobacco material is derived from burley tobacco, Virginia, smoked, air-cured, fire-cured, oriental or dark tobacco plant.Tobacco plant can be selected from Maryland tobacco, rare tobacco, special tobacco, expansion tobacco etc.

[0123] Also contemplated herein are tobacco cultivars and elite tobacco cultivars.Thus, the tobacco plants used herein may be tobacco varieties or elite tobacco cultivars.

[0124] Particularly useful tobacco varieties include Burley, dark, flue-cured, and Oriental types.

[0125] For example, the tobacco plant can be selected from one or more of the following varieties: Nicotiana AA 37-1, Nicotiana B13P, Nicotiana Xanthi (Mitchell-Mor), Nicotiana KT D#3 Hybrid 107, Nicotiana Bel-W3, Nicotiana 79-615, Nicotiana SamsunHolmes NN, F4 from the hybrid Nicotiana BU21 x Nicotiana Hoja Parado, line 97, Nicotiana KTRDC#2 Hybrid 49, Nicotiana KTRDC#4 Hybrid 1 10, Nicotiana Burley 21, Nicotiana PM016, Nicotiana KTRDC#5KY 160SI, Nicotiana KTRDC#7FCA, Nicotiana KTRDC#6TN 86SI, Nicotiana PM021, Nicotiana K 149, Nicotiana K 326, Nicotiana K346, Nicotiana K 358, Nicotiana K 394, Nicotiana K 399, Nicotiana K730, Nicotiana KY 10, Nicotiana KY 14, Nicotiana KY 160, Nicotiana KY 17, Tobacco KY 8959, Tobacco KY 9, Tobacco KY 907, Tobacco MD 609, Tobacco McNair 373, Tobacco NC 2000, Tobacco PG 01, Tobacco PG 04, Tobacco P01, Tobacco P02, Tobacco P03, Tobacco RG 1 1, Tobacco RG 17, Tobacco RG 8, Tobacco Speight G-28, Tobacco TN86, Tobacco TN 90, Tobacco VA509, Tobacco AS44,Tobacco Banket A1, Tobacco Basma Drama B84 / 31, Tobacco Basma I Zichna ZP4 / B, Tobacco Basma Xanthi BX 2A, Tobacco Batek, Tobacco Besuki Jember, Tobacco C104, Tobacco Coker 319, Tobacco Coker 347, Tobacco Criollo Misionero, Tobacco PM092, Tobacco Delcrest, Tobacco Djebel 81, Tobacco DVH 405, Tobacco Galpao Comum, Tobacco HB04P, Tobacco Hicks Broadleaf, Tobacco Kabakulak Elassona, Tobacco PM102, Tobacco Kutsage E1, Tobacco KY14xL8, Tobacco KY171, Tobacco LA BU 21, Tobacco McNair 944, Tobacco NC 2326, Tobacco NC 71, Tobacco NC 297, Tobacco NC 3, Tobacco PVH 03, Tobacco PVH 09, Tobacco PVH 19, Tobacco PVH 21 10, Tobacco Red Russia, Tobacco Samsun, Tobacco Saplak, Tobacco Simmaba, Tobacco Talgar 28, Tobacco PM132, Tobacco Wislica, Tobacco Yayaldag, Tobacco NC 4, Tobacco TR Madole, Tobacco Prilep HC-72, Tobacco Prilep P23, Tobacco Prilep PB 156 / 1, Tobacco Prilep P12-2 / 1, Tobacco Yaka JK-48, Tobacco Yaka JB 125 / 3, Tobacco TI-1068, Tobacco KDH-960, Tobacco TI-1070, Tobacco TW136, Tobacco PM204, Tobacco PM205, Tobacco Basma, Tobacco TKF 4028, Tobacco L8, Tobacco TKF2002, Tobacco TN90, Tobacco GR141, Tobacco Basma xanthi, Tobacco GR149, Tobacco GR153 and Tobacco PetitHavana. ,

[0126] Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD263, DF91 1, DT 538LC Galpao tobacco, GL26H, GL350, GL600, GL 737, GL939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, NarrowLeaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC71, NC 72, NC810, NC BH 129, NC 2002, Neal Smith Madole,OXFORD 207,PD 7302LC, PD 7309LC, PD7312LC 'Periq'e' Tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-1 1, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, 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, Tl 1406, Tl 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (TomRosson)Madole, VA 309, VA359, AA37-1, B 13P, 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, DVH405, Galpao Comum,HB04P,Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU21, NC 2326, NC 297, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, Tl-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. The above low converter subvarieties are also considered even if not specifically identified herein.

[0127] The tobacco plant may be, for example, a Burley-type tobacco plant, suitably Burley Nicotiana tabacum PH2517.

[0128] Plant propagation material may for example be obtained from the tobacco plant of the invention.

[0129] As used herein, "plant propagation material" refers to any plant matter taken from a plant, from which other plants can be produced. Suitably, the plant propagation material may be selected from seeds, plant callus and plant bushes.

[0130] Suitably, the plant propagation material may be a seed. Suitably, the plant propagation material may be a plant callus. Suitably, the plant propagation material may be a plant bush.

[0131] Cells (eg tobacco cells), tobacco plants and / or plant propagation material may eg be obtainable (eg obtained) by a method according to the invention.

[0132] As used herein, the term "processed tobacco leaf" refers to a tobacco leaf that has undergone one or more processing steps performed on tobacco in the art."Processed tobacco leaf" contains no or substantially no living cells.

[0133] The term "living cell" refers to a cell that is capable of growth and / or metabolic activity. Thus, if a cell is not alive, also referred to as "non-viable", the cell does not exhibit the characteristics of a living cell.

[0134] The term "substantially free of living cells" means that less than about 5% of the total cells are living. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of the total cells are living.

[0135] In one embodiment, the processed tobacco leaves may be processed by one or more of the following: curing, fermentation and / or pasteurization.

[0136] As used herein, "reconstituted" may also be referred to as reconstituted, recycled or homogenized sheet tobacco, and refers to tobacco material produced from the remaining portion of tobacco leaves after processing. Reconstituted tobacco allows a consistent, high quality blend to be produced, and allows the proportions of the various components to be adjusted.

[0137] Reconstituted tobacco can be nanofiber reconstituted (nanofibers can be extracted in solid or liquid form), paper reconstituted (which uses stems, chips and midribs etc. as raw materials) or pulp type reconstituted (which uses fine powder and tobacco stems and mixture, ground into powder, mixed with water and vegetable binder. By extracting water, the soluble residue is formed into flakes.)

[0138] As used herein, "tobacco industry product" is intended to include combustible smoking articles such as cigarettes, cigarillos, cigars, tobacco for pipes or roll-your-own cigarettes, (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokable materials), non-flammable aerosol delivery systems such as heating products that release compounds from a substrate material without combustion, such as e-cigarettes, tobacco heating products, and hybrid systems that produce aerosols from a combination of substrate materials, such as hybrid systems containing liquid or gel or solid substrates, and aerosolizable substrate materials used within these aerosol delivery systems; and non-aerosol delivery articles such as lozenges, chewing gum, patches, articles containing respirable powders and smokeless tobacco industry products such as moist snuff and snus, which may or may not deliver nicotine.

[0139] The term " part " used herein in the context of tobacco plant refers to the part of tobacco plant. Suitably, " part " can be leaf, root or stem or flower of tobacco plant. Suitably, " part " can be leaf, root or stem of tobacco plant. Preferably, " part " is the leaf of tobacco plant.

[0140] As used herein, the term "smoking article" may include smokable products such as cigarettes, cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes.

[0141] 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.

[0142] Smokeless tobacco industry products (including heat-not-burn materials) can contain tobacco in any form, including dry particles, fragments, granules, powders or slurries, deposited on, mixed in, surrounded by or combined with other ingredients, the other ingredients being in any form, such as flakes, films, tablets, foams or beads.

[0143] Amino acids are referred to herein using either their names, three letter abbreviations, or their single letter abbreviations.

[0144] As used herein, the term "protein" includes proteins, polypeptides, and peptides.

[0145] 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."

[0146] The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined by the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0147] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one with a general dictionary of many of the terms used in the present disclosure.

[0148] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

[0149] All combinations of method or process steps as used herein may be performed in any order unless otherwise indicated or clearly implied to the contrary by the context in which the referenced combination is performed. DETAILED DESCRIPTION OF THE INVENTION

[0151] This document sets forth the details of one or more embodiments of the subject matter currently disclosed. After studying the information provided in this document, modifications to the embodiments and other embodiments described in this document will be clear to those of ordinary skill in the art. The information provided in this document, particularly the specific details of the exemplary embodiments described, is provided primarily for clear understanding and no unnecessary limitations should be understood therefrom. In the event of a conflict, the specification (including definitions) of this document shall prevail.

[0152] The subject matter disclosed at present relates to articles and methods for regulating the conversion of nicotine to nornicotine and / or regulating the level of tobacco-specific nitrosamine (TSNA) precursors. In some embodiments, the articles include one or more transcription factor (TF) inhibitors. In one embodiment, for example, the articles include one or more basic region / leucine zipper (bZIP) type transcription factor inhibitors. In another embodiment, the bZIP type transcription factor is derived from tobacco. In another embodiment, in 133 kinds of bZIP type transcription factors identified by the inventors in tobacco, they are divided into 10 subgroups: A, B, C, D, E,, F, G, H and S, suitable bZIP type transcription factors include at least one of 27 kinds of bZIPs in subgroup S, at least one of 6 kinds of bZIPs in subgroup C, other bZIP 63 homologues or their combination. In certain embodiments, the S subgroup bZIP transcription factors include but are not limited to NtbZIP1a (SEQ ID NOs: 1 and 2), NtbZIP1b (SEQ ID NOs: 3 and 4), or a combination thereof; and / or the C subgroup bZIP transcription factors include but are not limited to NtbZIP2a (SEQ ID NO: 5), NtbZIP2b (SEQ ID NO: 6), or a combination thereof.

[0153] One or more transcription factor inhibitors include, but are not limited to, antisense oligonucleotides, miRNA, siRNA, locked nucleic acid (LNA) nucleotides, or a combination thereof. In some embodiments, the inhibitor provides RNAi-mediated knockdown / silencing of bZIP-type transcription factors. As will be appreciated by those skilled in the art, the specific sequence / structure of the transcription factor inhibitor is based on the sequence of a specific transcription factor. Therefore, as will be appreciated by those skilled in the art, antisense oligonucleotides and / or LNAs can be formed by any suitable method using the bZIP-type transcription factor sequences provided herein. For example, in one embodiment, the inhibitor includes an antisense oligonucleotide having 100% sequence homology to a complementary bZIP-type transcription factor. In another embodiment, the transcription factor inhibitor includes an antisense oligonucleotide having 100% sequence homology to a bZIP-type transcription factor complementary to NtbZIP1a, NtbZIP1b, NtbZIP2a, and / or NtbZIP2b. In such embodiments, the transcription factor inhibitor provides RNAi-mediated knockdown / silencing of NtbZIP1a, NtbZIP1b, NtbZIP2a and / or NtbZIP2b expression in tobacco.

[0154] In some embodiments, the present invention also provides a method for regulating the conversion of nicotine to nornicotine in tobacco plants or other nicotine-containing organisms. In one embodiment, the method includes applying one or more bZIP inhibitors disclosed herein to an organism containing nicotine. The application of these one or more bZIP inhibitors reduces or eliminates the conversion of nicotine to nornicotine. One or more inhibitors can be applied to a single type of bZIP transcription factor or to a combination of bZIP transcription factors. For example, in one embodiment, the method includes applying an inhibitor of one or more S bZIP transcription factors or C bZIP transcription factors. In another embodiment, the method includes applying an inhibitor of one or more S bZIP transcription factors and a transcription factor of one or more C bZIP transcription factors. In another embodiment, the method includes applying an inhibitor of one or more NtbZIP1a, NtbZIP1b, NtbZIP2a and / or NtbZIP2b to an organism. In certain embodiments, inhibiting S bZIP transcription factors and C bZIP transcription factors has a synergistic effect on reducing or eliminating the conversion of nicotine to nornicotine.

[0155] The methods disclosed herein include administering a single type of inhibitor or any suitable combination of inhibitors, which may be the same or different for each bZIP transcription factor that is inhibited. For example, in one embodiment, the method includes administering antisense oligonucleotides to NtbZIP1a, NtbZIP1b, NtbZIP2a, and / or NtbZIP2b. In another embodiment, the method includes administering antisense oligonucleotides to one bZIP transcription factor, such as NtbZIP1a, and LNA nucleotides to another bZIP transcription factor, such as NtbZIP1b. As will be appreciated by those skilled in the art, although certain combinations of bZIP transcription factors and transcription factor inhibitors are discussed above, the present disclosure is not limited thereto and may include any other suitable combination of TF and TF inhibitors.

[0156] Additionally or alternatively, the method may include a mutation of a bZIP-type transcription factor knockout and / or a bZIP-type transcription factor binding site on a nicotine N-demethylase (NND) promoter. For example, in one embodiment, the method includes editing the plant genome to knock out NtbZIP1a, NtbZIP1b, NtbZIP2a and / or NtbZIP2b. Genome editing can be performed by any suitable process, such as but not limited to CRISPR / Cas9-mediated genome editing. In another embodiment, the method includes mutating the bZIP binding element in the E4 promoter referred to as A / G box (TACGTC) to TGCGTC by site-directed mutagenesis. Although the above is discussed about specific mutations in the E4 promoter, as will be appreciated by those skilled in the art, the present disclosure is not limited thereto, and includes any other mutation in E4, E5 and / or E10 promoters to reduce or eliminate the activation of each NND by a bZIP-type transcription factor.

[0157] Administration of TF inhibitors disclosed herein, TF knockouts and / or binding site mutations reduce or eliminate activation of NND by bZIP-type transcription factors, which reduces or eliminates the conversion of nicotine to nornicotine. In contrast to existing articles including E4, E5, and E10 mutants, the articles disclosed herein control the expression of E4, E5, and E10 to reduce or eliminate the conversion of nicotine to nornicotine. By reducing or eliminating the conversion of nicotine to nornicotine, the articles and methods disclosed herein reduce the harmful effects of products (such as, but not limited to, tobacco products) that typically contain carcinogenic nornicotine.

[0158] The presently disclosed subject matter includes methods of reducing the conversion of nicotine to nornicotine and / or methods of reducing the level of at least one tobacco-specific nitrosamine (TSNA) precursor, the methods comprising reducing the activity and / or expression of at least one basic region / leucine zipper (bZIP)-type transcription factor in a tobacco plant, a portion of a tobacco plant, a tobacco plant seed, or another organism comprising nicotine.

[0159] The presently disclosed subject matter further includes the use of reducing the activity and / or expression of at least one basic region / leucine zipper (bZIP) type transcription factor in a tobacco plant, a portion of a tobacco plant, a tobacco plant cell, a tobacco plant seed, or another nicotine-containing organism, for reducing the conversion of nicotine to nornicotine and / or for reducing the level of at least one tobacco-specific nitrosamine (TSNA) precursor in a tobacco plant, a portion of a tobacco plant, a tobacco plant seed, or a nicotine-containing organism.

[0160] In embodiments of the methods and uses described herein, at least one bZIP transcription factor can be selected from Group C bZIP transcription factors, Group S bZIP transcription factors, and combinations thereof. In some embodiments, the at least one bZIP transcription factor is selected from NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and combinations thereof.

[0161] In some embodiments, the at least one bZIP transcription factor comprises an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with an amino acid selected from the group consisting of amino acids encoded by SEQ ID NO: 1; amino acids encoded by SEQ ID NO: 2; amino acids encoded by SEQ ID NO: 3; amino acids encoded by SEQ ID NO: 4; amino acids encoded by SEQ ID NO: 5; and amino acids encoded by SEQ ID NO: 6.

[0162] In some embodiments, the at least one bZIP transcription factor comprises NtbZIP1a and NtbZIP1b. In some embodiments, the at least one bZIP transcription factor comprises NtbZIP2a and NtbZIP2b. In some embodiments, the at least one bZIP transcription factor comprises NtbZIP1a, NtbZIP1b, NtbZIP2a, and NtbZIP2b.

[0163] In some embodiments, the at least one bZIP transcription factor comprises an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with an amino acid selected from the group consisting of the amino acids encoded by SEQ ID NO: 1 and the amino acids of SEQ ID NO: 2; and an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with an amino acid selected from the group consisting of the amino acids encoded by SEQ ID NO: 3 and the amino acids of SEQ ID NO: 4.

[0164] In some embodiments, the at least one bZIP transcription factor comprises an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acids of SEQ ID NO:5; and an amino acid molecule that is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acids of SEQ ID NO:6.

[0165] In some embodiments, the at least one bZIP transcription factor comprises an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with an amino acid selected from the group consisting of the amino acids encoded by SEQ ID NO: 1 and the amino acids of SEQ ID NO: 2; an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with an amino acid selected from the group consisting of the amino acids encoded by SEQ ID NO: 3 and the amino acids of SEQ ID NO: 4; an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with the amino acids of SEQ ID NO: 5; and an amino acid molecule having at least 90%, 95%, 97%, 98%, 99% or 100% identity with the amino acids of SEQ ID NO: 6.

[0166] In embodiments of the methods and uses described herein, reducing the activity and / or expression of at least one bZIP-type transcription factor can include mutating the bZIP-type transcription factor, such as providing a mutation in a binding site of the bZIP-type transcription factor; using a bZIP-type transcription factor inhibitor; or using gene silencing techniques, such as RNAi.

[0167] In some embodiments, the bZIP transcription factor inhibitor is selected from the group consisting of antisense oligonucleotides, miRNA, siRNA, locked nucleic acid (LNA) nucleotides, and combinations thereof.

[0168] In some embodiments, the mutant bZIP-type transcription factor includes a binding site on a mutant nicotine N-demethylase (NND) promoter. In some embodiments, the NND is selected from CYP82E4v1, CYP82E5v2, and CYP82E10. In some embodiments, the NND is CYP82E4v1, and wherein the bZIP binding site on the CYP82E4v1 promoter is an A / G box with a pre-mutated sequence of TACGTC. In some embodiments, the mutant binding site has the sequence TGCGTC. In some embodiments, the mutant binding site is formed by site-directed mutagenesis.

[0169] In some embodiments, mutating the bZIP-type transcription factor comprises mutating the plant genome to knock out at least one basic region / leucine zipper (bZIP)-type transcription factor.

[0170] The present invention further includes nucleic acid molecules and amino acid molecules as described herein. In some embodiments, the isolated nucleic acid molecule may include a nucleic acid sequence having at least 90%, 95%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a nucleic acid sequence encoding a sequence having at least 90%, 95%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof.

[0171] In some embodiments, the amino acid molecule can include an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acids encoded by the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a sequence that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof.

[0172] In some embodiments, the molecule comprises or encodes a bZIP-type transcription factor with at least one mutation. In some embodiments, the mutation is in a binding site on a nicotine N-demethylase (NND) promoter. In some embodiments, the NND is selected from CYP82E4v1, CYP82E5v2, and CYP82E10. In some embodiments, the NND is CYP82E4v1, and wherein the bZIP binding site on the CYP82E4v1 promoter is an A / G box with a pre-mutation sequence of TACGTC. In some embodiments, the mutated binding site has the sequence TGCGTC.

[0173] The presently disclosed subject matter further includes a construct or vector comprising a nucleic acid molecule as disclosed herein.

[0174] The presently disclosed subject matter further includes tobacco plant cells, which may include an exogenous gene comprising a nucleic acid molecule as disclosed herein, may include a construct or vector as disclosed herein, may be obtained or obtainable by a manner as disclosed herein, and / or may be obtained by mutation to reduce the activity or expression of at least one bZIP-type transcription factor or to knock out at least one bZIP-type transcription factor.

[0175] The presently disclosed subject matter further includes the use of tobacco cells as disclosed herein in the production of tobacco industry products.

[0176] The presently disclosed subject matter further includes tobacco cells, cell cultures, or plants comprising a plant cell as disclosed herein that has been modified to achieve reduced conversion of nicotine to nornicotine and / or a reduced level of at least one tobacco-specific nitrosamine (TSNA) precursor of said culture or plant compared to an unmodified culture or plant; or comprising a construct or vector as disclosed herein.

[0177] Suitably, tobacco plant according to the present invention can have tobacco-specific nitrosamine (TSNA) precursors that for example compare content reduction with unmodified tobacco plant. Suitably, described tobacco plant cell can be sudden change, non-naturally occurring or genetically modified plant.Described plant can comprise one or more sudden changes, and it reduces activity and / or the expression of at least one basic region / leucine zipper (bZIP) type transcription factor.

[0178] In one embodiment, a tobacco plant according to the present invention comprises a tobacco cell according to the present invention.

[0179] In another embodiment, the plant propagation material may be obtainable (eg, obtained) from a tobacco plant of the present invention.

[0180] In one embodiment, there is provided use of a tobacco plant as described herein in growing tobacco plants.

[0181] In another embodiment, the present invention also provides use of the tobacco plant of the preceding embodiment in producing tobacco industry products.

[0182] In another embodiment, there is provided use of the tobacco plant of the present invention in growing crops.

[0183] In one embodiment, there is provided use of a cell as provided in the preceding embodiments in the production of a tobacco industry product.

[0184] In one embodiment, the invention provides a kind of cell (for example tobacco plant cell), it has the activity and / or expression of at least one basic region / leucine zipper (bZIP) type transcription factor of reduction.Suitably, described cell can be the cell of mutation, non-natural existence or genetically modified.Described cell can comprise one or more sudden changes, and it reduces the activity and / or expression of at least one basic region / leucine zipper (bZIP) type transcription factor.

[0185] In one embodiment, the invention provides a cell culture (eg an in vitro culture). Suitably, the cell culture may comprise a plurality of cells according to the invention.

[0186] Tobacco cell culture can be a cell suspension culture. These in vitro cultured cells can be incorporated into the tobacco industry product, for example as a substitute for traditional tobacco particles, fragments, fine cutting or long cutting tobacco leaves, as an additive component or both as a substitute and as an additive. Suitably, the cell culture can produce nicotine.

[0187] In one embodiment, there is provided use of a cell culture according to the invention, such as a harvested and / or processed cell culture, in the production of a tobacco industry product.

[0188] Cells harvested from an in vitro culture may be dried, eg, freeze-dried, eg, to produce a powder.

[0189] In one embodiment, the cell culture is a tobacco cell culture.

[0190] The technician will know the known methods for setting up the in vitro culture of tobacco cell.Only for example, the following method can be used: collect seeds from interested tobacco plants and sterilize their outside to eliminate unwanted organisms, plant said seeds to grow interested tobacco plants, remove tissue from tobacco plants (for example, from tobacco stem) and use as explant, set up callus culture from tobacco explant, set up cell suspension culture from callus culture, and gather in the crops culture material (for example comprising tobacco cells) to produce tobacco cell culture.

[0191] 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 water by filtration, such as vacuum filtration.

[0192] The harvested tobacco cells may be further processed, for example dried, such as air-dried and / or freeze-dried.The harvested tobacco cell culture or the dried harvested tobacco cell culture may be incorporated into a tobacco industry product according to the present invention.

[0193] The presently disclosed subject matter further includes plant propagation material or plant extracts obtainable from the plant or part of the plant according to claim 34. In some embodiments, the plant propagation material is a plant seed.

[0194] The presently disclosed subject matter further includes the use of the tobacco plants as disclosed herein in growing tobacco plants.

[0195] The invention also provides products obtainable or obtained from tobacco according to the invention.

[0196] In one embodiment, there is provided use of the tobacco plant of the present invention in producing tobacco leaves.

[0197] Suitably, the tobacco leaves may be used for downstream applications such as processing.

[0198] Thus, in one embodiment, processed tobacco leaves can be provided using the foregoing embodiments. Suitably, the tobacco leaves can be subjected to curing, 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 curing, fermentation, pasteurization, or a combination thereof.

[0199] The currently disclosed subject matter includes the use of tobacco plants as disclosed herein in producing tobacco industry products. The currently disclosed subject matter further includes the use of tobacco plants as disclosed herein in planting crops. The currently disclosed subject matter further includes the use of tobacco plants as disclosed herein in producing processed tobacco leaves. In some embodiments, the processed tobacco leaves are flue-cured tobacco leaves.

[0200] In one embodiment, the present invention provides the leaf of the results of tobacco plant of the present invention.In another embodiment, the leaf of the results can be that the tobacco plant of breeding material of the present invention can obtain (for example obtain) from breeding.In another embodiment, the leaf of the results that can be obtained from method of the present invention or purposes is provided.Suitably, the leaf of the results can be the leaf of the results of cutting.In some embodiments, the leaf of the results can comprise living tobacco cells.In other embodiments, the leaf of the results can be further processed.

[0201] Also provide processed tobacco leaf.Processed tobacco leaf can be obtained from tobacco plant of the present invention.Suitably, processed tobacco leaf can be obtained from tobacco plant obtained according to any method and / or purposes of the present invention.In another embodiment, processed tobacco leaf can be obtained from tobacco plant bred from tobacco plant propagation material according to the present invention.Processed tobacco leaf of the present invention can be obtained by processing the leaf of results of the present invention.

[0202] Suitably, the processed tobacco leaves can be processed by curing. The tobacco leaves can be cured by any method known in the art. In one embodiment, the tobacco leaves can be cured by one or more curing methods selected from the group consisting of air curing, fire curing, flue curing, and sun curing.

[0203] Suitably, the tobacco leaves may be air-cured. Typically air-curing is achieved by hanging the leaves in a well-ventilated barn and allowing them to dry. This is typically done over a period of 4-8 weeks. Air-curing is particularly suitable for Burley tobacco.

[0204] Suitably, the tobacco leaves may be fire-cured. Fire-curing is usually achieved by hanging the leaves in a large barn where a hardwood fire is kept at a low smoldering rate, either continuously or intermittently, and typically takes between three days and ten weeks, depending on the process and the tobacco.

[0205] In another embodiment, the tobacco leaves can be smoke-cured. Smoking can include stringing the tobacco leaves onto tobacco sticks and hanging them on a tier-pole in a curing room. The curing room usually has a flue extending from an externally supplied firebox. Usually this results in the tobacco being heat-cured without being exposed to smoke. Usually the temperature rises slowly during the curing process, and the whole process takes about 1 week.

[0206] Suitably, the tobacco leaves may be sun-cured. This process generally involves exposing uncovered tobacco to sunlight.

[0207] Suitably, the processed tobacco leaf can be processed by fermentation. Fermentation can be carried out in any manner known in the art. Usually during the fermentation process, the tobacco leaves are piled into a pile (large block) of flue-cured tobacco, covered with, for example, burlap to retain moisture. The weight combination of the remaining water in the leaf and the tobacco produces the natural heat that ripens the tobacco. The temperature at the center of the block is monitored every day. In some methods, the whole block is opened every week. Then the leaves are taken out to shake and moisten, and the block is rotated so that the leaves inside are outside and the leaves at the bottom are placed on the top of the block. This ensures uniform fermentation of the whole block. The extra moisture on the leaves, plus the actual rotation of the leaves themselves, generates heat, releases the natural ammonia of the tobacco and reduces nicotine, while also deepening the color and improving the fragrance of the tobacco. Usually the fermentation process lasts up to 6 months, depending on the type of tobacco, the position of the stalk on the leaf, the thickness and the intended use of the leaf.

[0208] Suitably, the processed tobacco leaves may be processed by pasteurization. Pasteurization may be particularly preferred when the tobacco leaves are to be used in the manufacture of smokeless tobacco industrial products, most preferably moist snuff.

[0209] Tobacco pasteurization can be carried out by any method known in the art. For example, pasteurization can be carried out as described in 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.

[0210] During the production of moist snuff, pasteurization is carried out by a process in which the tobacco is heat treated with steam for 24-36 hours (reaching a temperature of approximately 100° C.). This results in a nearly sterile product and, without wishing to be bound by theory, one of the consequences is believed to be limiting further TSNA formation.

[0211] In one embodiment, the pasteurization may be steam pasteurization.

[0212] 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. In one embodiment, reconstituted tobacco can be provided using the aforementioned embodiments.

[0213] Any method known in the art can be used to make reconstituted tobacco, see, for example, CORESTA Congress, Sapporo, 2012, Smoke Science / Product Technology Groups, SSPT 12 (incorporated herein by reference).

[0214] In some embodiments, tobacco plants, harvested leaves of tobacco plants and / or processed tobacco leaves can be used to extract nicotine. Any method known in the art can be used to achieve the extraction of nicotine. For example, a method for extracting nicotine from tobacco is taught in US 2,162,738, which is incorporated herein by reference.

[0215] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0216] In another aspect, the present invention provides a tobacco blend comprising tobacco material made from a tobacco plant or part thereof according to the present invention, or from a tobacco cell culture according to the present invention. In one aspect, the present invention provides a tobacco blend comprising a flue-cured tobacco material according to the present invention.

[0217] Suitably, tobacco blends according to the present invention may comprise approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 10% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 20% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 30% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 40% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 50% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may comprise approximately 60% from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blend can comprise about 70% from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, tobacco blend can comprise about 80% from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, tobacco blend can comprise about 90% from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention.

[0218] In one aspect, the tobacco blend products of the invention comprise at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% by dry weight of tobacco cured from a tobacco plant or part thereof according to the invention, or a tobacco cell culture according to the invention.

[0219] Suitably, the flue-cured tobacco material may be air-cured. Suitably, the flue-cured tobacco material may be smoke-cured. Suitably, the flue-cured tobacco material may be sun-cured. Suitably, the flue-cured tobacco material may be fire-cured.

[0220] Tobacco industry products or smoking articles according to the present invention may comprise a tobacco material (eg a flue-cured tobacco material or a reconstituted tobacco material) according to the present invention.

[0221] On the other hand, the present invention provides a kind of tobacco industry product.In one embodiment, tobacco industry product according to the present invention can be a mixed tobacco industry product.Suitably, tobacco mixture can include flue-cured tobacco material according to the present invention.In one embodiment, tobacco industry product can be prepared by tobacco plant of the present invention or its part.Suitably, described tobacco plant or its part can be propagated from tobacco plant propagation material according to the present invention.

[0222] In one embodiment, a tobacco industry product can be prepared from (eg, can include) a tobacco plant or part thereof of the present invention.

[0223] Suitably, the tobacco plant or part thereof may be propagated from tobacco plant propagation material according to the present invention.

[0224] In another embodiment, tobacco industry products can be prepared from the harvested leaves of the present invention. In another embodiment, tobacco industry products can be prepared from the processed tobacco leaves of the present invention.

[0225] Suitably, tobacco industry products may be prepared from tobacco leaves processed by one or more of curing, fermentation and / or pasteurization.

[0226] Suitably, the tobacco industry product may comprise cut tobacco leaves, optionally processed in accordance with the preceding embodiments.

[0227] In another embodiment, tobacco industry products can be prepared from tobacco cell cultures according to the present invention.

[0228] In another embodiment, a tobacco industry product can be prepared from (eg, can comprise) a flue-cured tobacco material according to the invention.

[0229] In another embodiment, a tobacco industry product can be prepared from (eg, can include) a tobacco blend according to the present invention.

[0230] In one embodiment, the tobacco industry product may be a smoking article.

[0231] In another embodiment, the tobacco industry product can be a smokeless tobacco industry product.

[0232] In one embodiment, smokeless tobacco industry products can include moist snuff, snuff, chewing tobacco, and the like.

[0233] In one embodiment, the tobacco industry product is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0234] In one embodiment, the tobacco industry product comprises one or more components of a combustible smoking article, such as a filter, a filter plug, a filter plug segment, tobacco, a cigarette stick, a cigarette stick segment, an overflow, 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.

[0235] In one embodiment, the tobacco industry product is a non-combustible aerosol delivery system.

[0236] In one embodiment, the tobacco industry product comprises one or more components of a non-combustible aerosol delivery system, such as a heater and an aerosolizable substrate.

[0237] In one embodiment, the aerosol delivery system is an electronic cigarette, also known as a vaping device.

[0238] In one embodiment, the electronic cigarette comprises a heater, a power source capable of powering the heater, an aerosolizable substrate such as a liquid or gel, a housing, and optionally a mouthpiece.

[0239] In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is combined with or comprises a heater.

[0240] In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating rather than burning a substrate material. The substrate material is an aerosolizable material, which may be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0241] In one embodiment, the heating product is an electronic device.

[0242] 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.

[0243] In one embodiment, the heated product is a non-electronic item.

[0244] In one embodiment, the heating product includes an aerosolizable substrate such as a solid or gel material and a heat source capable of providing thermal energy to the aerosolizable substrate without any electronic means, such as by burning a combustion material such as charcoal.

[0245] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0246] In some embodiments, the aerosolizable substrate material may include a vapor or aerosol generating agent or a humectant, such as glycerin, propylene glycol, triacetin, or diethylene glycol.

[0247] In one embodiment, the tobacco industry product is a hybrid system that produces aerosol by heating rather than burning a combination of substrate materials. The substrate material can include, for example, solid, liquid or gel, which may or may not include nicotine. In one embodiment, the hybrid system includes a liquid or gel substrate and a solid substrate. The solid substrate can be, for example, tobacco or other non-tobacco products, which may or may not include nicotine. In one embodiment, the hybrid system includes a liquid or gel substrate and tobacco.

[0248] In another embodiment, the tobacco industry product can be a tobacco heating device or a mixing device or an electronic cigarette, etc.

[0249] Typically in a tobacco heating device or mixing device, an aerosol is generated by transferring heat from a heat source to a physically separate aerosol-forming substrate or material, which may be located within, around or downstream of the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate by heat transfer from the heat source and are entrained in the air inhaled through the smoking article. When the released compounds cool, they condense to form an aerosol that is inhaled by the user.

[0250] Aerosol-generating articles and devices for consuming or smoking tobacco heating devices are known in the art. For example, they may include 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.

[0251] Suitably, the tobacco heating device may be an aerosol generating device. Preferably, the tobacco heating device may be a heat-not-burn device. Heat-not-burn devices are known in the art and release compounds by heating rather than burning tobacco. Examples of suitable heat-not-burn devices may be those taught in WO2013 / 034459 or GB2515502, which are incorporated herein by reference.

[0252] In one embodiment, the aerosol-forming substrate of the tobacco heating device may be a tobacco industry product according to the present invention.In one embodiment, the tobacco heating device may be a hybrid device.

[0253] In another embodiment, the use of at least one nucleotide sequence encoding a gene of at least one bZIP-type transcription factor having at least one mutation is provided, wherein the sequence of the bZIP-type transcription factor comprises: a sequence encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least one mutation relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a sequence having at least one mutation relative to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof, in order to select plants having a reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors.

[0254] In one embodiment, a plant mutant is provided, which carries at least one heritable mutation in at least one bZIP transcription factor, the mutation being encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least one mutation relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a sequence having at least one mutation relative to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof; wherein the heritable mutation reduces the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in the mutant tobacco plant relative to a comparable plant not carrying the heritable mutation.

[0255] In another embodiment, progeny or seeds of a mutant plant carrying a heritable mutation according to the present invention are provided.

[0256] In another embodiment, a harvested leaf, processed leaf or cured tobacco material produced from a plant is provided, the plant comprising at least one mutation in at least one bZIP transcription factor, the mutation being encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least one mutation relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a sequence having at least one mutation relative to the sequence of SEQ ID NO: 2, 4, 5 or 6; or a combination thereof, and wherein the plant has a reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors relative to a comparable plant that does not carry the mutation in the bZIP transcription factor.

[0257] The presently disclosed subject matter is further illustrated by the following specific but non-limiting examples.The following examples may include a compilation of data representative of data collected at various times during the development and experimentation process associated with the presently disclosed subject matter. Example

[0258] Example 1

[0259] This example describes the analysis of transcriptome datasets from different tobacco tissues, including leaves (young, mature, and senescing), roots, stems, flowers (young and mature), and capsules to generate co-expression networks. First, a hierarchical cluster analysis was performed, which showed that each individual tissue type exhibited a unique expression pattern ( Figure 1 ). Next, we identified genes encoding all major TF families in tobacco and its ancestors. The tobacco genome contains more TF genes than its ancestors, and the number of TFs belonging to the MYB, AP2 / ERF, and bHLH families is significantly higher than that of other families ( Figure 2 ). Given this, TFs and structural genes in the nicotine biosynthesis pathway were then divided into eight different modules (color-coded next to the bars) based on their expression patterns in different tissues ( Figure 3 ). The "black" module is particularly interesting because most of the nicotine biosynthetic genes and many TF genes are found in this module. Many of these TFs belong to MYB, bHLH, bZIP and ERF families. As discussed in Examples 2-4 below, the effects of these TFs in nicotine biosynthesis in tobacco are determined by their separation and functional characterization.

[0260] Example 2

[0261] This example describes two bZIP-type transcription factors from tobacco ( Figure 4 ), which regulates the conversion of nicotine to nornicotine and can be used to reduce smoking-related carcinogens, tobacco-specific nitrosamines (TSNAs).

[0262] bZIP TFs are characterized by a conserved leucine zipper motif that mediates dimer formation for DNA binding. In plants, bZIP TFs regulate processes including pathogen defense, light and stress signaling, seed maturation, and flower development. Many bZIP factors, especially those in tobacco, are not well characterized. Through co-expression and cluster analysis, we identified two bZIP TF genes that are co-expressed with E4, E5, and E10. These tobacco bZIP TFs are called NtbZIP1a and NtbZIP1b.

[0263] NtbZIP1 a / b showed similar expression patterns compared with CYP82E4v1, the major NND enzyme involved in the conversion of nicotine to nornicotine and highly expressed in flowers and senescing leaves ( Figure 4 ).like Figure 5As shown, 133 bZIPs were identified in tobacco and divided into 10 subgroups: A, B, C, D, E, F, G, H, I and S, among which NtbZIP1 a / b belonged to the S subgroup. In maize, the expression of S group bZIPs was induced by wounding, cold and drought stress. Figure 6 It was also found that NtbZIP1a and b were more than 97% identical at the nucleotide and amino acid levels. Without wishing to be bound by theory, it is believed that these two homologous bZIPs originated from two ancestors of tobacco, Nicotiana tabacum and N. tometosiformis.

[0264] After identifying the two bZIP TFs, it was tested whether overexpression of NtbZIP1a resulted in upregulation of E4, 5, and 10. NtbZIP1a was cloned into pCAMBIA2300 (binary vector) under the control of the CaMV 35S promoter and rbcS terminator. The binary vector (empty control and NtbZIP1a) was transferred into Agrobacterium and the transformed Agrobacterium was used to infiltrate tobacco leaves. Total RNA isolated from Agrobacterium-infiltrated leaf discs was used for cDNA synthesis, and real-time quantitative PCR (qRT-PCR) was used to detect the transcriptional levels of NtbZIP1a, NtbZIP1b, E4, 5, and 10. The ubiquitously expressed housekeeping gene tubulin was used as an internal control for qRT-PCR. The results showed that when NtbZIP1a was transiently overexpressed, endogenous NtbZIP1b, E4, 5, and 10 were upregulated (approximately 3-10-fold), indicating that NtbZIP1a induces the expression of NtbZIP1b, E4, 5, and 10 and may therefore be a transcriptional activator of these genes ( Figure 7 ).

[0265] Next, we tested whether NtbZIP1a could bind to the promoters of its potential target genes. The promoters of E4, 5, and 10 (approximately 1.0 kb fragments of the 5' untranslated region of each encoding gene) were isolated, and the individual promoters were fused to the firefly luciferase reporter gene. The NtbZIP1a gene was cloned into the pBlueScript (pBS) vector under the control of the CaMV35S promoter and the rbcS terminator. The plasmids were electroporated into tobacco protoplasts. NtbZIP1a significantly induced the expression of luciferase genes controlled by the E4 and E10 promoters, but not the E5 promoter, indicating that NtbZIP1a can directly activate E4 and E10, possibly by binding to their promoters ( Figure 8). NtbZIP1a does not appear to bind to the 1.0 kb promoter region of E5 used in the activation experiments. However, as described above, overexpression of NtbZIP1a resulted in upregulation of E5 as well as E4 and 10. Promoter activation experiments suggest two possible regulatory relationships between NtbZIP1a and the E5 gene: (1) NtbZIP1a binds to sites outside the 1.0 kb promoter fragment, or (2) NtbZIP1a indirectly activates E5 through another unidentified activator in tobacco (e.g., NtbZIP1a activates another activator, which in turn activates E5).

[0266] To support the possibility that NtbZIP1a directly binds to the E4 promoter to mediate transactivation, the bZIP binding element in the E4 promoter, called the A / G box (TACGTC), was mutated to TGCGTC by site-directed mutagenesis. The mutated promoter was fused to a luciferase reporter gene as described above. Transactivation experiments were then performed using the mutant reporter plasmid and the NtbZIP1a expression vector as described above. The results showed that NtbZIP1a could not activate luciferase gene expression under the control of the mutant promoter ( Fig. 9 ). This experiment confirmed that the E4 promoter is activated by an A / G-box binding factor, most likely NtbZIP1a.

[0267] refer to Fig.10 Transactivation experiments were also performed using the NtbZIP1b expression vector and the E4 promoter-luciferase reporter plasmid as described for NtbZIP1a. NtbZIP1b also activated the E4 promoter at a similar level to NtbZIP1a.

[0268] Finally, because it has been determined that the agronomic practice of tobacco topping (removal of axillary shoots) induces nicotine production, the inventors analyzed transcriptome data from topping or untopping tobacco plants. More specifically, leaf samples were collected from control (untopping) and topping plants 24 hours later. RNA isolated from untopping and topping leaves was used for cDNA synthesis and qRT-PCR analysis. The results showed that topping resulted in a 70-90% reduction in the expression of bZIP1 a / b and E4, 5, and 10 compared to untopping plants, indicating that topping negatively regulates bZIP1 a / b and NND expression in tobacco ( Fig.11 The results also suggest that bZIP1 a / b and NND are coordinately expressed in tobacco, as gene regulators and their target genes usually do.

[0269] Based on the above examples, it is believed that NtbZIP1a and 1b are involved in the regulation of three NND genes as activators. Reduction or inactivation of NtbZIP1a / b can lead to a reduction in nornicotine.

[0270] Example 3

[0271] This example describes the generation of transgenic lines overexpressing NtbZIP1a and the effects of these transgenic lines on endogenous E4 expression.

[0272] To generate transgenic lines, pCAMBIA2300 (a binary vector) containing NtbZIP1a under the control of the CaMV 35S promoter and the rbcS terminator was transferred into Agrobacterium and tobacco leaf discs were infected with the transformed Agrobacterium. More than 20 transgenic lines overexpressing NtbZIP1a were generated from Agrobacterium-infected leaf discs. Genomic DNA isolated from the control and three transgenic lines was used to verify the transgenic status of the plants by PCR amplification of the antibiotic selection marker neomycin phosphotransferase II (nptII; kan). Total RNA isolated from leaves of the control and three transgenic lines was used for cDNA synthesis. RT-PCR was used to verify the expression of the nptII (kan) gene in transgenic plants ( Fig. 12A ). Real-time quantitative PCR (qRT-PCR) was used to detect the transcription levels of NtbZIP1a and E4 ( Fig. 12B ). The ubiquitously expressed housekeeping gene tubulin was used as an internal control for qRT-PCR.

[0273] The results of this example show that NtbZIP1a expression is significantly higher in transgenic plants compared to controls. When NtbZIP1a is highly expressed, endogenous E4 expression is upregulated (about 6-8 times), indicating that NtbZIP1a induces the expression of E4 and may be a transcriptional activator of the E4 gene. In addition, metabolic analysis showed that the conversion rate of nicotine to nornicotine was higher in transgenic tobacco leaves compared to controls ( Fig. 12C ). The formula used to calculate the conversion rate of nicotine to nornicotine is:

[0274]

[0275] Two of the three lines analyzed showed higher conversion of nicotine to nornicotine.Because the metabolic analysis was performed with an independent segregating population of TO (first generation transgenic plants), the metabolic results may vary.

[0276] Example 4

[0277] As described in Example 2 above, the inventors characterized NtbZIP1a and b, two NtbZIPs belonging to the S group bZIP factors. In Arabidopsis, the S group bZIP factors are known to interact with certain C group factors to regulate gene expression. In view of this interaction, the inventors identified a tobacco homolog of Arabidopsis bZIP63, a C group member that interacts with S group factors. In tobacco, there are two bZIP63 homologs, referred to herein as NtbZIP2a and b, which share over 95% amino acid identity ( Fig.13 Without wishing to be bound by theory, it is believed that NtbZIP2a and b are derived from two tobacco ancestors, N. tometosiformis and N. tometosiformis, and function redundantly.

[0278] According to transcriptome analysis, NtbZIP2a and b have similar expression patterns in tobacco flowers, leaves, stems, and roots ( Fig.14 ). Based on the foregoing discussion, the inventors hypothesized that NtbZIP2a and b also regulate E4 / 5 / 10 alone and / or synergistically with NtbZIP1a and b. Therefore, transactivation of the E4 promoter by NtbZIP2a was tested, alone or in combination with NtbZIP1a. More specifically, the E4 promoter was fused to a firefly luciferase reporter gene, and the bZIP TF was cloned into a pBS vector under the control of the CaMV 35S promoter and the rbcs terminator. The results showed that, individually, both NtbZIP1a and NtbZIP2a activated the E4 promoter; however, when NtbZIP1a and 2a were co-expressed, transactivation of the E4 promoter was significantly increased compared to that induced by each factor alone ( Fig.15 ).

[0279] Next, to determine whether NtbZIP1a / b interacts with NtbZIP2a, the inventors performed a yeast hybrid assay. Growth of yeast cells on a deficiency (SD) medium (SD-leu-trp-his-ade) lacking the synthesis of leucine, tryptophan, histidine, and adenine indicated that NtbZIP1a / b interacted with NtbZIP2a ( Fig.16A ; 1 and 2). In addition, NtbZIP2 interacts with itself to form a homodimer ( Fig.16A ; 3) The bZIP domains of NtbZIP1 and NtbZIP2 are Fig. 16B Shown in.

[0280] Although this example only tests the activity of NtbZIP2a on E4, and not E5 or E10, it is believed that both NtbZIP2 factors are activators of the E4 / 5 / 10 genes. That is, this example shows that Group C NtbZIP2a and b are regulators of two previously uncharacterized E4 / 5 / 10 genes. Furthermore, this example shows that NtbZIP1 and NtbZIP2 act synergistically in activating the E4 (and possibly E5 and 10) promoters. In particular, without wishing to be bound by theory, it is believed that NtbZIP2a and / or b interact with NtbZIP1a and / or b to enhance DNA binding ability and significantly increase activation of the E4 / 5 / 10 promoter (compared to NtbZIP1 or NtbZIP2 alone).

[0281] In summary, two S group and two C group NtbZIP factors are characterized herein as positive regulators of the E4 / 5 / 10 genes. The transactivation activities of NtbZIP1a and 2a on the E4 promoter are additive. Therefore, without wishing to be bound by theory, it is believed that knockout approaches that inactivate one or all of these genes will reduce E4 / 5 / 10 gene expression.

[0282] Example 5

[0283] NtbZIP1 RNAi lines (Nicotiana tabacum cv. Samsun NN) were generated and molecular and biochemical analyses were performed.

[0284] Construction of RNAi vectors and transformation of plants. To construct RNAi plasmids, a 200 bp fragment from the 3' end of the NtbZIP1a cDNA was PCR amplified and cloned in the sense and antisense directions into the pKYLX80 vector containing an intron (from the soybean FAD3 gene; Schallet et al. 1987). The sense and antisense fragments with the intron were excised from pKYLX80 and cloned into the binary vector pCAMBIA2300 containing the CaMV35S promoter and the rbcS terminator ( Fig.17 The plasmid was mobilized into Agrobacterium tumefasiens by freeze-thaw method (Weigel and Glazebrook, 2006). Tobacco plants (Nicotiana tabacum cv. Samsun NN) were transformed with Agrobacterium containing the binary vector as described above (Pattanaik et al. 2008), and two independent kanamycin-resistant transgenic lines (lines #3 and 9) were selected for further analysis.

[0285] RNA isolated from leaves of NtbZIP RNAi and empty vector (EV) control plants was used for cDNA synthesis and quantitative RT-PCR (qPCR). NtbZIP1a expression was reduced by approximately 80% in two independent RNAi lines compared with the EV line ( Fig.18A In addition, NtbZIP1b expression was reduced by approximately 70% in both RNAi lines compared with EV ( Fig.18A ). The expression of E4 was significantly reduced in both transgenic lines, for example by up to 90% in line 9 ( Fig.18A ). The results showed that inhibition of NtbZIP1 resulted in downregulation of E4, indicating that NtbZIP1a and NtbZIP1b regulate the E4 gene. In addition, the conversion of nicotine to nornicotine was measured in leaves of RNAi and EV lines. The conversion of nicotine to nornicotine was reduced by about 50% in the RNAi line compared to the EV line ( Fig.18B ).

[0286] In conclusion, RNAi-mediated silencing of NtbZIP1 significantly reduced E4 expression in transgenic tobacco plants compared to EV. In addition, the conversion of nicotine to nornicotine was approximately 50% lower in leaves of transgenic RNAi tobacco compared to EV.

[0287] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference, including those listed in the following list:

[0288] References

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[10] Shoji,T.,and Hashimoto,T.(2011).Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2-locusERF genes.Plant&cell physiology 52,1117-1130.

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[11] Shoji,T.,Kajikawa,M.,and Hashimoto,T.(2010).Clusteredtranscription factor genes regulate nicotine biosynthesis in tobacco.ThePlant cell 22,3390-3409.

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[12] Shoji,T.,Inai,K.,Yazaki,Y.,Sato,Y.,Takase,H.,Shitan,N.,Yazaki,K.,Goto,Y.,Toyooka,K.,Matsuoka,K.,and Hashimoto,T.(2009).Multidrug and toxiccompound extrusion-type transporters implicated in vacuolar sequestration ofnicotine in tobacco roots.Plant physiology 149,708-718.

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[13] Sierro,N.,van Oeveren,J.,van Eijk,M.J.,Martin,F.,Stormo,K.E.,Peitsch,M.C.,and Ivanov,N.V.(2013a).Whole genome profiling physical map andancestral annotation of tobacco Hicks Broadleaf.The Plant journal:for celland molecular biology 75,880-889.

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[14] Sierro,N.,Battey,J.N.,Ouadi,S.,Bovet,L.,Goepfert,S.,Bakaher,N.,Peitsch,M.C.,and Ivanov,N.V.(2013b).Reference genomes and transcriptomes ofNicotiana sylvestris and Nicotiana tomentosiformis.Genome biology 14,R60.

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[0309]

[21] Pattanaik S,Xie CH,Yuan L(2008)The interaction domains of theplant Myc-like bHLH transcription factors can regulate the transactivationstrength.Planta227:707–715.

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[0311]

[23] Weigel D,Glazebrook J(2006)Transformation of Agrobacterium using the freeze–thaw method.Cold Spring Harbor Protocols 2006:Pdb.prot4666.

[0312] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown in the drawings by way of example and are described in detail below. However, it should be understood that the description of specific embodiments is not intended to limit the present disclosure to cover all modifications, equivalents and substitutes within the spirit and scope of the present disclosure defined by the appended claims. Sequence Listing <110> R.J. Reynolds Tobacco Company University of Kentucky Research Foundation <120> bZIP transcription factors regulate the conversion of nicotine to nornicotine and reduce the levels of tobacco-specific nitrosamine (TSNA) precursors <130> 13177N / 2183W2 <150> US 62 / 857,718 <151> 2019-06-05 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 435 <212> DNA <213> Nicotiana tabacum <400> 1 atggctttga cacagcaacc ggctagttca ggttctgatg gccaacgtta tgccacaaat 60 gacgatagaa aacgaaagag aatggagtcc aaccgtgaat ctgcaaggcg gtcacggatg 120 agaaagcagc agcatttgga ggagttgatg agccaaatga cacagctaca gaatcagaac 180 gttctgtggc gcgagaagat tgatgctgtg ggaagaaact acctcaccct cgatgcggag 240 aacaatgtct tgagggctca aatggcagaa ctgactgaac gcttggattc tctcaattcg 300 ctcactcgtt tctgggctga tgctaatgga ctagctgtgg atatccctga aattcctgac 360 actttgcttg agccctggca gcttccttgc ccaattcaac ccatcactgc ttctgctgat 420 atgtttcagt tttga 435 <210> 2 <211> 144 <212> PRT <213> Nicotiana tabacum <400> 2 Asn Ala Leu Thr Gln Gln Pro Ala Ser Ser Gly Ser Asp Gly Gln Arg 1 5 10 15 Tyr Ala Thr Asn Asp Asp Arg Lys Arg Lys Arg Met Glu Ser Asn Arg 20 25 30 Glu Ser Ala Arg Arg Ser Arg Met Arg Lys Gln Gln His Leu Glu Glu 35 40 45 Leu Met Ser Gln Met Thr Gln Leu Gln Asn Gln Asn Val Leu Trp Arg 50 55 60 Glu Lys Ile Asp Ala Val Gly Arg Asn Tyr Leu Thr Leu Asp Ala Glu 65 70 75 80 Asn Asn Val Leu Arg Ala Gln Met Ala Glu Leu Thr Glu Arg Leu Asp 85 90 95 Ser Leu Asn Ser Leu Thr Arg Phe Trp Ala Asp Ala Asn Gly Leu Ala 100 105 110 Val Asp Ile Pro Glu Ile Pro Asp Thr Leu Leu Glu Pro Trp Gln Leu 115 120 125 Pro Cys Pro Ile Gln Pro Ile Thr Ala Ser Ala Asp Met Phe Gln Phe 130 135 140 <210> 3 <211> 435 <212> DNA <213> Nicotiana tabacum <400> 3 atggcttcga tacagcaacc agctagttca ggttctgatg gccaacgata tgctatgaac 60 gacgatagaa aacgaaagag aatggagtcc aaccgtgaat ctgcaaggcg gtcacggatg 120 aggaagcagc agcatttgga agagttgatg agccaaatga cacagctaca gaatcagaac 180 gttctgtggc gtgagaagat tgatgctgtg ggaagaaact acctgaccct tgatgcggag 240 aacaatgtcc tgagggctca aatggcagaa ctgactgaac gcttggattc gctcaattcg 300 ctcgctcgtt tctgggctga tgctaatgga ctagctgtgg ataccctga aattccagac 360 actttgcttg agccgtggca gcttccttgc ccaattcaac ccatcactgc ttctgctaat 420 atgtttcagt tttga 435 <210> 4 <211> 144 <212> PRT <213> Nicotiana tabacum <400> 4 Asn Ala Ser Ile Gln Gln Pro Ala Ser Ser Gly Ser Asp Gly Gln Arg 1 5 10 15 Tyr Ala Met Asn Asp Asp Arg Lys Arg Lys Arg Met Glu Ser Asn Arg 20 25 30 Glu Ser Ala Arg Arg Ser Arg Met Arg Lys Gln Gln His Leu Glu Glu 35 40 45 Leu Met Ser Gln Met Thr Gln Leu Gln Asn Gln Asn Val Leu Trp Arg 50 55 60 Glu Lys Ile Asp Ala Val Gly Arg Asn Tyr Leu Thr Leu Asp Ala Glu 65 70 75 80 Asn Asn Val Leu Arg Ala Gln Met Ala Glu Leu Thr Glu Arg Leu Asp 85 90 95 Ser Leu Asn Ser Leu Ala Arg Phe Trp Ala Asp Ala Asn Gly Leu Ala 100 105 110 Val Asp Ile Pro Glu Ile Pro Asp Thr Leu Leu Glu Pro Trp Gln Leu 115 120 125 Pro Cys Pro Ile Gln Pro Ile Thr Ala Ser Ala Asn Met Phe Gln Phe 130 135 140 <210> 5 <211> 455 <212> PRT <213> Nicotiana tabacum <400> 5 Met Asp Arg Val Phe Ser Val Asp Asp Asp Ile Gly Asp His Phe Trp 1 5 10 15 Ser Thr Pro Pro Thr Ala Asp Leu Gly Val Asp Ser Pro Thr Ala Ala 20 25 30 Ala Ala Val Ser Tyr Ser Lys Met Met Asn Arg Ser Ser Ser Glu Trp 35 40 45 Ala Phe Gln Arg Phe Leu Gln Glu Ala Thr Ala Ala Gly Thr Ser Thr 50 55 60 Ser Ser Pro Pro Gln Pro Pro Thr Met Thr Ala Ser Ser Ser Ser Ser 65 70 75 80 Ser His Gln Asn Asp Val Val Glu Ile Lys Asp Glu Asn Leu Ser Ile 85 90 95 Pro Asn Leu Asn Pro Ser Thr Ala Leu Asn Ser Lys Pro Ala Ser Ser 100 105 110 Phe Gly Leu Ala Pro Pro Pro Asn Ile Ala Val Asp Ser Glu Glu Tyr 115 120 125 Gln Ala Phe Leu Lys Ser Gln Leu His Leu Ala Cys Ala Ala Val Ala 130 135 140 Leu Thr Arg Gly Lys Ser Leu Asn Pro Gln Asp Ser Gly Ser Thr Ala 145 150 155 160 His Asp Lys Gly Ser Glu Thr Ala Ser Ala Ala Gln Ser Gly Ser His 165 170 175 Val Ser Thr Leu Gly Lys Cys Tyr Leu Arg Ser Gly Gln Glu Val Ala 180 185 190 Lys Ile Gln Asp Lys Asp Ala Gly Gly Pro Val Gly Ile Pro Ser Leu 195 200 205 Pro Pro Val Gln Lys Lys Pro Val Val Gln Val Arg Ser Thr Thr Ser 210 215 220 Gly Ser Ser Arg Glu Gln Ser Asp Asp Asp Glu Ala Glu Gly Glu Ala 225 230 235 240 Glu Thr Thr Gln Gly Met Asp Pro Ala Asp Ala Lys Arg Val Arg Arg 245 250 255 Met Leu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Arg Arg Lys Gln 260 265 270 Ala His Leu Thr Glu Leu Glu Thr Gln Val Ser Gln Leu Arg Val Glu 275 280 285 Asn Ser Ser Leu Leu Lys Arg Leu Thr Asp Ile Ser Gln Lys Tyr Asn 290 295 300 Glu Ala Ala Val Asp Asn Arg Val Leu Lys Ala Asp Val Glu Thr Leu 305 310 315 320 Arg Ala Lys Val Lys Met Ala Glu Glu Thr Val Lys Arg Val Thr Gly 325 330 335 Leu Asn Pro Leu Phe Gln Ala Met Ser Glu Ile Ser Ser Met Val Met 340 345 350 Pro Ser Tyr Ser Gly Ser Pro Ser Asp Thr Ser Ala Asp Ala Ala Val 355 360 365 Pro Val Gln Asp Asp Pro Lys His His Tyr Tyr Gln Gln Pro Pro Asn 370 375 380 Asn Leu Met Pro Thr His Asp Pro Arg Ile Gln Asn Gly Met Val Asp 385 390 395 400 Val Pro Pro Ile Glu Asn Val Glu Gln Asn Pro Ala Thr Ala Ala Val 405 410 415 Gly Gly Asn Lys Met Gly Arg Thr Thr Ser Met Gln Arg Val Ala Ser 420 425 430 Leu Glu His Leu Gln Lys Arg Ile Arg Gly Glu Val Ser Ser Cys Gly 435 440 445 Thr Gln Gly Arg Gly Glu Gln 450 455 <210> 6 <211> 442 <212> PRT <213> Nicotiana tabacum <400> 6 Met Asp Arg Val Phe Ser Val Asp Asp Asp Ile Gly Asp His Phe Trp 1 5 10 15 Ser Thr Pro Pro Thr Ala Asp Leu Gly Val Asp Ser Pro Thr Thr Ala 20 25 30 Thr Ala Ala Ser Tyr Ser Lys Met Met Asn Arg Ser Ser Ser Glu Trp 35 40 45 Ala Phe Gln Arg Phe Leu Gln Glu Ala Thr Ala Ala Gly Thr Ser Thr 50 55 60 Ser Ser His Pro Gln Pro Pro Thr Met Thr Ala Ser Ser Ser Ser Ser 65 70 75 80 Ser His Gln Asn Asp Val Val Glu Ile Lys Asp Glu Asn Leu Ser Thr 85 90 95 Pro Asn Leu Asn Pro Ser Thr Ala Leu Asn Ser Lys Pro Ala Ser Ser 100 105 110 Phe Gly Leu Thr Pro Pro Pro Asn Ile Ala Val Asp Ser Glu Glu Tyr 115 120 125 Gln Ala Phe Leu Lys Ser Gln Leu His Leu Ala Cys Ala Ala Val Ala 130 135 140 Leu Thr Arg Gly Lys Ser Leu Asn Pro Gln Asp Ser Gly Ser Thr Ala 145 150 155 160 His Asp Lys Gly Ser Glu Thr Ala Ser Ala Ala Gln Ser Gly Ser His 165 170 175 Glu Met Ala Lys Ile Gln Asp Lys Asp Ala Gly Gly Pro Val Gly Ile 180 185 190 Pro Ser Leu Pro Pro Val Gln Lys Lys Pro Val Val Gln Val Arg Ser 195 200 205 Thr Thr Ser Gly Ser Ser Arg Glu Gln Ser Asp Asp Asp Glu Ala Glu 210 215 220 Gly Glu Ala Glu Thr Thr Gln Gly Met Asp Pro Ala Asp Ala Lys Arg 225 230 235 240 Val Arg Arg Met Leu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Arg 245 250 255 Arg Lys Gln Ala His Leu Thr Glu Leu Glu Thr Gln Val Ser Gln Leu 260 265 270 Arg Val Glu Asn Ser Ser Leu Leu Lys Arg Leu Thr Asp Ile Ser Gln 275 280 285 Lys Tyr Asn Glu Ala Ala Val Asp Asn Arg Val Leu Lys Ala Asp Val 290 295 300 Glu Thr Leu Arg Ala Lys Val Lys Met Ala Glu Glu Thr Val Lys Arg 305 310 315 320 Val Thr Gly Leu Asn Pro Leu Phe Gln Ala Met Ser Glu Ile Ser Ser 325 330 335 Met Val Met Pro Ser Tyr Ser Gly Ser Pro Ser Asp Thr Ser Ala Asp 340 345 350 Ala Ala Val Pro Val Gln Asp Asp Pro Lys His His Tyr Tyr Gln Gln 355 360 365 Pro Pro Asn Asn His Met Pro Thr Asn Asp Pro Arg Ile Gln Asn Gly 370 375 380 Met Val Asp Val Pro Pro Ile Glu Asn Val Gln Gln Asn Pro Ala Thr 385 390 395 400 Ala Ala Val Gly Gly Asn Lys Met Gly Arg Thr Ala Ser Met Gln Arg 405 410 415 Val Ala Ser Leu Glu His Leu Gln Lys Arg Ile Arg Gly Glu Ile Ser 420 425 430 Ser Cys Gly Thr Gln Gly Arg Gly Glu Gln 435 440

Claims

1. A method for preparing harvested leaves, comprising i) modifying a tobacco plant by reducing the activity and / or expression of at least one bZIP transcription factor to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6; and ii) obtaining harvested leaves from said tobacco plant, wherein said harvested leaves are cut harvested leaves.

2. A method for obtaining a tobacco plant extract, comprising obtaining a tobacco plant propagation material or a tobacco plant extract from a tobacco plant or a part of the tobacco plant, wherein the tobacco plant is prepared as follows: the tobacco plant is modified by reducing the activity and / or expression of at least one bZIP transcription factor to achieve a reduced conversion of nicotine to nornicotine and / or a reduced level of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO:

6.

3. The method of claim 1 or 2, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

4. The method of claim 3, wherein the gene silencing technology is RNAi.

5. The method of claim 3, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

6. A method for producing processed tobacco leaves, comprising using a tobacco plant prepared as follows: modifying the tobacco plant by reducing the activity and / or expression of at least one bZIP transcription factor to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, wherein the processed tobacco leaves are flue-cured tobacco leaves.

7. The method of claim 6, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

8. The method of claim 7, wherein the gene silencing technology is RNAi.

9. The method of claim 7, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

10. A cut processed tobacco leaf: a. A tobacco plant cell comprising a cell prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO: 2; an amino acid sequence of SEQ ID NO: 4; an amino acid sequence of SEQ ID NO: 5; and an amino acid sequence of SEQ ID NO: 6; b. obtained by processing a tobacco plant prepared as follows: modifying the tobacco plant by reducing the activity and / or expression of at least one bZIP transcription factor to achieve a reduced conversion of nicotine to nornicotine and / or a reduced level of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6; c. obtained by processing the cut harvested leaves obtained by the method of claim 1; or Any combination of Wherein the cut processed tobacco leaves are non-viable processed tobacco leaves.

11. The cut processed tobacco leaves of claim 10, wherein the tobacco plants or harvested leaves are processed by curing, fermentation, pasteurization, or a combination thereof.

12. The cut processed tobacco leaf of claim 10 or 11, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

13. The cut processed tobacco leaf of claim 12, wherein the gene silencing technology is RNAi.

14. The cut processed tobacco leaf of claim 12, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

15. A flue-cured tobacco material made from a tobacco plant, part thereof, extract thereof, or cell culture thereof prepared as follows: a tobacco plant is modified by reducing the activity and / or expression of at least one bZIP transcription factor to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO:

6.

16. The flue-cured tobacco material of claim 15, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

17. The flue-cured tobacco material of claim 16, wherein the gene silencing technology is RNAi.

18. The cured tobacco material of claim 16, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

19. A tobacco blend comprising the cured tobacco material of any one of claims 15-18.

20. A tobacco industry product comprising a tobacco plant extract obtained from a modified tobacco plant prepared by reducing the activity and / or expression of at least one bZIP transcription factor to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, wherein the tobacco industry product is a smoking article or a smokeless tobacco product.

21. A tobacco industry product comprising a tobacco plant extract obtained from a modified tobacco plant prepared as follows: reducing the activity and / or expression of at least one bZIP transcription factor to achieve reduced conversion of nicotine to nornicotine and / or reduced levels of at least one tobacco-specific nitrosamine (TSNA) precursor in the tobacco plant compared to an unmodified tobacco plant, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2; an amino acid sequence of SEQ ID NO: 4; an amino acid sequence of SEQ ID NO: 5; and an amino acid sequence of SEQ ID NO: 6, wherein the tobacco industry product is a non-combustible aerosol delivery system.

22. The tobacco industry product of claim 20 or 21, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

23. The tobacco industry product of claim 22, wherein the gene silencing technology is RNAi.

24. The tobacco industry product of claim 22, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

25. A tobacco industry product prepared from leaves harvested from a tobacco plant obtained by the method of any one of claims 6 to 9, wherein the tobacco industry product is a smoking article or a smokeless tobacco product.

26. A tobacco industry product prepared from leaves harvested from a tobacco plant obtained by the method of any one of claims 6 to 9, wherein the tobacco industry product is a non-combustible aerosol delivery system.

27. The tobacco industry product of claim 21 or 26, wherein the non-combustible aerosol delivery system is a tobacco heating device.

28. The tobacco industry product of claim 27, wherein the tobacco heating device is an aerosol generating device.

29. A combustible smoke-generating article comprising A tobacco plant or part thereof from the species Nicotiana tabacum or Nicotiana rustica, or an extract thereof, prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or A tobacco cell culture prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or The flue-cured tobacco material of any one of claims 15 to 18, or The tobacco blend of claim 19.

30. The combustible smoking article of claim 29, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

31. The combustible smoking article of claim 30, wherein the gene silencing technology is RNAi.

32. The combustible smoking article of claim 30, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

33. A non-flammable aerosol supply system comprising A tobacco plant or part thereof from the species Nicotiana tabacum or Nicotiana rustica, or an extract thereof, prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or A tobacco cell culture prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or The flue-cured tobacco material of any one of claims 15 to 18, or The tobacco blend of claim 19.

34. The non-flammable aerosol supply system of claim 33, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

35. The non-flammable aerosol delivery system of claim 34, wherein the gene silencing technology is RNAi.

36. The non-combustible aerosol delivery system of claim 34, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

37. A smokeless tobacco product comprising A tobacco plant or part thereof from the species Nicotiana tabacum or Nicotiana rustica, or an extract thereof, prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or A tobacco cell culture prepared by reducing the activity and / or expression of at least one basic region-leucine zipper (bZIP) transcription factor, wherein the at least one bZIP transcription factor comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 4; the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6, or The flue-cured tobacco material of any one of claims 15 to 18, or The tobacco blend of claim 19.

38. The smokeless tobacco product of claim 37, wherein reducing the activity and / or expression of at least one bZIP transcription factor comprises: Mutating a bZIP transcription factor; using a bZIP transcription factor inhibitor selected from antisense oligonucleotides, miRNA, siRNA, and a combination thereof; Or use gene silencing technology.

39. The smokeless tobacco product of claim 38, wherein the gene silencing technology is RNAi.

40. The smokeless tobacco product of claim 38, wherein mutating the bZIP transcription factor comprises mutating the tobacco plant genome to knock out at least one basic region-leucine zipper (bZIP) transcription factor.

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