Tobacco material derived from plant body of genus nicotiana, tobacco product, plant body of genus nicotiana, and method for imparting cooling sensation to tobacco product

Abstract

Provided is a tobacco product in which a tobacco material itself produces a cooling sensation. A tobacco material derived from a plant body of the genus Nicotiana according to the present disclosure is derived from a plant body of the genus Nicotiana into which a mutation causing suppression of the function of an endogenous JOX gene has been introduced. This tobacco material has a cold-sensitive receptor TRPM8 activity.

Description

Tobacco material derived from Nicotiana plants, tobacco products, Nicotiana plants, and method for imparting a cooling sensation to tobacco products

[0001] The present invention relates to tobacco materials derived from Nicotiana plants, tobacco products, Nicotiana plants, and methods for imparting a cooling sensation to tobacco products.

[0002] Conventionally, tobacco products have been designed to provide a desired taste or aroma (hereinafter sometimes referred to as "smoking flavor") by adding flavorings or the like to tobacco materials derived from Nicotiana plants. However, from the standpoint of production efficiency, quality stability, and the like, there is a demand for improving the smoking flavor of the tobacco leaves themselves, which are the raw material for tobacco products.

[0003] Jasmonic acid (JA) is a plant hormone that contributes to defense responses against insect pests and diseases. Various JA analogs are known. In plants, the active form is jasmonate-isoleucine (JA-Ile), which is formed by binding JA to the amino acid isoleucine. JA and JA-Ile are reversibly converted. JA and JA-Ile are converted to the inactive forms 12-hydroxy-jasmonic acid (12-OH-JA) and 12-hydroxy-jasmonate-isoleucine (12-OH-JA-Ile) by the metabolic enzymes JOX (JAO) and CYP94B1 / B3, respectively. It has been reported that suppression of JOX or CYP94B gene function increases the accumulation of JA analogs in Arabidopsis (Non-Patent Documents 1-3) and wild tobacco (Non-Patent Document 4).

[0004] As an effect of JA as a contributing factor to the flavor and aroma components of tobacco materials, Non-Patent Document 5 reports enhanced synthesis of cembratrienediol (CBT), one of the main components of leaf surface resin secreted from glandular trichomes, an organ present on the leaf surface. Non-Patent Document 5 discloses that exogenous application of methyl jasmonate, a JA analog, to tobacco increases CBT. Furthermore, Non-Patent Document 6 discloses that exogenous application of methyl jasmonate to leaves immediately before harvest increases the levels of several phenylalanine-derived aroma components, which are components of tobacco leaves, and improves the flavor and aroma of tobacco leaves.

[0005] China Patent Publication CN112094855A

[0006] Smirnova et al., Molecular Plant 10, 1159-1173, 2017Caarls et al., PNAS, vol.114, No.24, 6388-6393, 2017Heitz et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 9, pp. 6296-6306, 2012Tang et al., Plant Diversity 42, 111-119, 2020Sui et al., Molecules, 23, 2511, 2018Xu et al., The effects of exogenous methyl jasmonate on volatile compounds, key enzymes, and sensory quality in tobacco (a model plant), https: / / doi.org / 10.21203 / rs.3.rs-3902707 / v1

[0007] Although it has been reported that suppressing the function of JOX genes results in the accumulation of JAs in plants, it has not been reported that suppressing the function of JOX genes improves the flavor and aroma of tobacco materials, particularly the expression of a cooling sensation.

[0008] An object of one aspect of the present invention is to provide a tobacco product in which the tobacco material itself exerts a cooling sensation.

[0009] In order to solve the above-mentioned problems, the following means are provided: According to one aspect of the present invention, there is provided a tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the endogenous gene in its genome that specifically causes functional inhibition in at least one of the following: (a) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:3, (b) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6, and (c) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:9, and the tobacco material has cold receptor TRPM8 activity.

[0010] According to another aspect of the present invention, there is provided a tobacco material according to the above aspect, which comprises a TRPM8 agonist.

[0011] According to yet another aspect of the present invention, there is provided a tobacco material according to any of the above aspects, wherein the cold receptor TRPM8 activity of the tobacco material is at least twice as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant.

[0012] According to yet another aspect of the present invention, there is provided a tobacco material according to any of the above aspects, which is a dry material.

[0013] According to yet another aspect of the present invention, there is provided a tobacco material according to any of the above aspects, which is a powder of dry material.

[0014] According to yet another aspect of the present invention, there is provided a tobacco product comprising the tobacco material according to any of the above aspects.

[0015] According to yet another aspect of the present invention, there is provided a Nicotiana plant in which a mutation that specifically causes functional inhibition has been introduced into at least one of the following endogenous genes in the genome: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:9.

[0016] According to yet another aspect of the present invention, there is provided a Nicotiana plant according to the above aspect, wherein the Nicotiana plant is any one of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana rustica.

[0017] According to yet another aspect of the present invention, there is provided a Nicotiana plant according to any of the above aspects, into which a mutation that specifically causes the functional suppression has been introduced by mutagen treatment, genome editing, or gene knockout.

[0018] According to yet another aspect of the present invention, there is provided a Nicotiana plant according to any of the above aspects, wherein the mutation that specifically causes the functional suppression specifically causes an enhanced response to jasmonic acid.

[0019] According to yet another aspect of the present invention, there is provided a method for imparting a cooling sensation to a tobacco product, wherein the tobacco product comprises tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the genome of at least one of the following endogenous genes in its genome that specifically causes functional inhibition: (a) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the tobacco material is substantially free of cooling flavoring; and the tobacco material has cooling sensation receptor TRPM8 activity.

[0020] According to one aspect of the present invention, a tobacco product can be realized in which the tobacco material itself exerts a cooling sensation.

[0021] 1-5。 α-CBT analysis results for Evaluation Example 1-1. α-CBT analysis results for the first year of the field test for Evaluation Example 1-3. α-CBT analysis results for the second year of the field test for Evaluation Example 1-3. α-CBT analysis results for the first year of the field test for Evaluation Example 1-5. α-CBT analysis results for the second year of the field test for Evaluation Example 1-5. α-CBT analysis results for the first assay of components in cigarette smoke for Evaluation Example 1-5. α-CBT analysis results for the second assay of components in cigarette smoke for Evaluation Example 1-5. α-CBT analysis results for the first assay of components in JOX mutant leaves for Evaluation Example 1-5. α-CBT analysis results for the second ... This is a diagram showing the results of solavetivone analysis when ripe leaves were dried to a yellow color in Evaluation Example 2-1.

[0022] One aspect of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in the embodiments and examples are also included in the technical scope of the present invention.

[0023] The following embodiments and examples are more specific embodiments of any of the above aspects. The following items can be incorporated into each of the above aspects, either singly or in combination.

[0024] In addition, in this specification, "A to B" means A or more and B or less unless otherwise specified.

[0025] [Tobacco Material] A tobacco material according to one embodiment of the present invention is a material derived from a Nicotiana plant into which a mutation that causes functional suppression of the endogenous JOX gene has been introduced. Examples of parts of the plant include leaves, midribs separated from leaves, stem remains, and flowers. The tobacco material according to one embodiment of the present invention has cold receptor TRPM8 activity.

[0026] (JOX gene) The JOX (jasmonate-induced oxygenase) gene is a gene encoding JOX. The JOX gene may also be referred to as the JAO (jasmonic acid oxidase) gene. In this specification, the term "JOX gene" encompasses both the JOX gene and the JAO gene. JOX is an enzyme involved in the metabolism of jasmonic acid (JA).

[0027] The Nicotiana plant has a mutation in its genome that specifically causes functional inhibition in at least one of the following: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:9.

[0028] As used herein, the region of the endogenous gene that encodes a polypeptide is referred to as a coding region (CDS). Furthermore, as used herein, the terms "polypeptide" and "protein" have substantially the same meaning and can be used interchangeably.

[0029] As used herein, "sequence identity" refers to the percentage of a sequence that is identical to a reference sequence (e.g., a nucleotide sequence, an amino acid sequence, etc.), where the mismatched portions of the sequences are those containing substitutions, additions, deletions, or insertions.

[0030] JOX genes include the JOX-S1 gene, the JOX-S2 gene, and the JOX-T2 gene. SEQ ID NO: 1 is the genomic sequence of the JOX-S1 gene (sometimes referred to as "NtJOX-S1") encoded by the S genome of Nicotiana tabacum. SEQ ID NO: 2 is the nucleotide sequence of the CDS of the JOX-S1 gene. SEQ ID NO: 3 is the amino acid sequence of the JOX-S1 protein. SEQ ID NO: 4 is the genomic sequence of the JOX-S2 gene (sometimes referred to as "NtJOX-S2") encoded by the S genome of Nicotiana tabacum. SEQ ID NO: 5 is the nucleotide sequence of the CDS of the JOX-S2 gene. SEQ ID NO: 6 is the amino acid sequence of the JOX-S2 protein. SEQ ID NO: 7 is the genomic sequence of the JOX-T2 gene (sometimes referred to as "NtJOX-T2") encoded by the T genome of Nicotiana tabacum. SEQ ID NO: 8 is the nucleotide sequence of the CDS of the JOX-T2 gene. SEQ ID NO: 9 is the amino acid sequence of the JOX-T2 protein.

[0031] As described below, Nicotiana tabacum is an amphidiploid and has a genome derived from its parent plant, Nicotiana sylvestris (also referred to as the "S genome") and a genome derived from Nicotiana tomentosiformis (also referred to as the "T genome"). When the Nicotiana plant is Nicotiana tabacum, it has cold receptor TRPM8 activity by specifically suppressing the function of at least one JOX gene (i.e., at least one of the JOX-S1 gene, the JOX-S2 gene, and the JOX-T2 gene) present in either the S genome or the T genome.

[0032] A tobacco material according to one embodiment of the present invention may be a tobacco material derived from a Nicotiana plant in which the functions of the JOX-S1 gene and the JOX-S2 gene, the JOX-S1 gene and the JOX-T2 gene, or the JOX-S2 gene and the JOX-T2 gene are specifically suppressed. Furthermore, a tobacco material according to one embodiment of the present invention may be a tobacco material derived from a Nicotiana plant in which the functions of the JOX-S1 gene, the JOX-S2 gene, and the JOX-T2 gene are specifically suppressed.

[0033] The functional suppression and mutation of the JOX gene will be explained in detail in the section on Nicotiana plants below.

[0034] (Activation of the Cold Receptor TRPM8) The transient receptor potential cation channel melastatin 8 (TRPM8) is a receptor closely related to the sensation of coolness. TRPM8 is a type of TRP channel and has been confirmed to be expressed on human sensory neurons. Known methods for activating TRPM8 include applying a temperature stimulus below 28°C or administering a cooling substance (e.g., Menthol, WS-5, WS-3, etc.). Activation of TRPM8 induces the influx of cations into cells. This induction depolarizes neurons, transmitting cold sensation information.

[0035] A tobacco material according to one embodiment of the present invention has TRPM8 activity. Furthermore, the smoke from combustion of the tobacco material according to one embodiment of the present invention also has TRPM8 activity, and tobacco products containing the tobacco material can provide a cooling sensation to users. The tobacco material according to one embodiment of the present invention may contain a TRPM8 agonist. Examples of such TRPM8 agonists include substances newly generated by functional suppression of an endogenous JOX gene, substances whose expression or activation is increased or whose accumulation is increased compared to a wild-type Nicotiana plant due to functional suppression of an endogenous JOX gene, and the like.

[0036] Whether or not a tobacco material has TRPM8 activity can be evaluated, for example, by the following procedure.

[0037] (When evaluating the TRPM8 activity of tobacco material) Tobacco material is ground, and a solvent (e.g., hexane, ethanol, ethyl acetate, acetonitrile, etc.) is added to the resulting ground sample, followed by shaking and centrifugation to obtain a supernatant containing the components of the tobacco material; The supernatant is evaporated to obtain a dry product; DMSO is added to the dry product to dissolve it, and then the dry product is diluted with HBSS solution to obtain a measurement composition; The measurement composition is contacted with TRPM8-expressing cells (e.g., HEK293T cells transformed to express TRPM8), and the fluorescence intensity is measured by fluorescent calcium imaging; The maximum fluorescence intensity obtained after contact with the measurement composition is obtained as the activity of the TRPM8-expressing cells due to the tobacco material; The above maximum fluorescence intensity is compared with a control maximum fluorescence intensity when the measurement composition is contacted with cells that do not express TRPM8 (e.g., non-transformed HEK293T cells), and if the above maximum fluorescence intensity is higher than the control maximum fluorescence intensity, the tobacco material is evaluated to have TRPM8 activity.

[0038] (When evaluating TRPM8 activity in combustion smoke from tobacco material) A cigarette containing tobacco material is burned under ISO 3308:2012 conditions, and the mainstream smoke produced by burning the cigarette is collected with a glass fiber filter; The components in the mainstream smoke collected with the glass fiber filter are extracted by shaking with a solvent (e.g., ethanol, methanol, ethyl acetate), and filtered with a PVDF (polyvinylidene fluoride) filter to obtain an extract; The filtered extract is diluted with HBSS solution to obtain a measurement composition; The measurement composition is contacted with TRPM8-expressing cells (e.g., HEK293T cells transformed to express TRPM8), and the fluorescence intensity is measured by fluorescent calcium imaging; The maximum fluorescence intensity obtained after contact with the measurement composition is taken as the activity of the TRPM8-expressing cells due to the tobacco material; The maximum fluorescence intensity is compared with the control maximum fluorescence intensity obtained when the measurement composition is contacted with cells that do not express TRPM8 (e.g., non-transformed HEK293T cells), and if the maximum fluorescence intensity is higher than the control maximum fluorescence intensity, the tobacco material is evaluated to have TRPM8 activity.

[0039] The tobacco material according to one embodiment of the present invention preferably has a TRPM8 activity that is at least twice as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant, more preferably at least five times as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant, and even more preferably at least ten times as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant.

[0040] A tobacco material according to one embodiment of the present invention is leaf tobacco harvested from the above-mentioned Nicotiana plant. More preferably, the tobacco material according to one embodiment of the present invention is dried leaf tobacco harvested from the above-mentioned Nicotiana plant. As used herein, "leaf tobacco" is synonymous with "leaf" and refers to "fresh leaf" that has not undergone the drying process described below. Furthermore, the tobacco material according to one embodiment of the present invention may be obtained by processing dried leaf using any method. Examples of such processing methods include drying, aging, extraction, harmonizing, flavoring, high-temperature and high-pressure treatment, distillation, crushing, and shredding the dried leaf. The obtained tobacco material may be in any form, such as cut filler, powder, sheet, granules, or extract. These forms of the tobacco material are preferred from the perspective of applying the dried material to tobacco products.

[0041] Cured leaves are obtained by drying tobacco leaves. Any drying method can be used, including, but not limited to, yellow drying, air drying, hot air drying, and hot air drying.

[0042] [Method for producing dried leaves] A method for producing dried leaves according to one embodiment of the present invention may include a drying step of drying tobacco leaves harvested from the Nicotiana plant. In order to further improve the cool feeling imparted to the tobacco material, the drying step is preferably yellow drying or air drying, with air drying being more preferred. In this specification, yellow drying is a heat-type drying method used for flue-cured tobacco, which is a drying method that produces a yellow color in the leaf tobacco. Air drying is a drying method that utilizes natural temperature, humidity, and ventilation conditions.

[0043] In order to further improve the cooling sensation that dried leaves provide, the tobacco leaves used in the drying step are preferably unripe or ripe, and more preferably unripe.

[0044] As used herein, immature leaf tobacco refers to leaf tobacco harvested several days after the stem is cut, and ripe leaf tobacco refers to leaf tobacco harvested at the optimum ripeness of the tobacco leaves.

[0045] In order to further enhance the cooling sensation expressed by the dried leaves, it is further preferable that the method for producing tobacco material according to one aspect of the present invention includes a drying step in which immature tobacco leaves harvested from the Nicotiana plant are air-dried.

[0046] Tobacco Products. A tobacco product according to one embodiment of the present invention comprises the tobacco material described above. The tobacco product may be in any form, including cut tobacco products, cigars, pipe tobacco products, cigarettes (cigarettes), electronic cigarettes, smokeless tobacco products (including snus and snuff), and waterpipe tobacco products. In particular, the tobacco product may be any of electronic cigarettes, cigarettes, and smokeless tobacco products. Examples of electronic cigarette products include non-combustion high-temperature heated tobacco products that use aerosol generated by heating a tobacco portion containing tobacco material as an aerosol source; non-combustion low-temperature heated tobacco products that have an atomization section separate from the tobacco portion that atomizes the aerosol source, and inhale the tobacco flavor entrained by the aerosol generated by heating the atomization section as it passes through the tobacco portion; and unheated tobacco products that inhale the flavor without heating the tobacco portion.

[0047] The tobacco product is preferably a cigarette or an electronic cigarette, as these products provide a cooling sensation that is easily perceived.

[0048] [Nicotama Plant] One embodiment of the present invention relates to a Nicotama plant into which a mutation that causes functional silencing of the JOX gene has been introduced. The Nicotama plant has a mutation that specifically causes functional silencing introduced into at least one of the following endogenous genes in its genome: (a) an endogenous gene containing, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; (b) an endogenous gene containing, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; or (c) an endogenous gene containing, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.

[0049] (Mutation) As used herein, "mutation" has the meaning commonly understood in the technical field to which the present application belongs, and refers to, for example, any change (e.g., substitution, deletion, insertion, addition, duplication, inversion, or translocation) of a base in a wild-type genome or an amino acid residue in a wild-type polypeptide. Therefore, "mutation of an endogenous gene" refers to a mutation of a gene that does not produce an original functional polypeptide (including a mutation that produces a polypeptide with reduced function or no function), a mutation of a gene that produces a polypeptide but reduces the amount produced, a mutation of a gene that produces a polypeptide but reduces the stability of the polypeptide, loss of a gene (a genomic DNA sequence including a coding region or an untranslated region), or a mutation that suppresses transcription from a gene (such as deletion of a transcriptional regulatory region or a transcription initiation region).

[0050] The mutation may occur in at least one of the promoter sequence (including a sequence located upstream (5') of the coding region), the terminator sequence (including a sequence located downstream (3') of the coding region), the 5' untranslated region, the 3' untranslated region, the conserved sequences at both ends of an intron (e.g., GT at the 5' end and AG at the 3' end), and the coding region. In particular, the mutation may occur within the coding region of the endogenous JOX gene. When a mutation occurs in at least one of the promoter sequence, the terminator sequence, and the coding region, the mutation may be any one of substitution, deletion, insertion, addition, duplication, inversion, and translocation. In particular, the function of an endogenous gene may be suppressed if the coding region contains a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid, which comprises an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of a JOX polypeptide. Here, the number of amino acids deleted, substituted or added in each amino acid sequence is, for example, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0051] Furthermore, in a Nicotiana plant according to one embodiment of the present invention, a mutation that causes functional suppression of the endogenous JOX gene may be introduced outside the coding region of the endogenous JOX gene. When the mutation is introduced outside the coding region of the endogenous JOX gene, the mutation may be an insertion of a polynucleotide that expresses an antisense RNA molecule, an RNAi molecule, or a co-suppressor molecule that promotes degradation of mRNA transcribed from the endogenous JOX gene.

[0052] When a substitution results in loss of function, the substitution may occur in at least one of the promoter sequence, terminator sequence, 5' untranslated region and 3' untranslated region, conserved sequences at both ends of an intron, and coding region.

[0053] For example, substitutions in nucleotide sequences important for regulating gene expression, such as those in the promoter sequence, 5' untranslated region, and 3' untranslated region of a gene, can result in a decrease in the transcriptional activity of the gene or a decrease in the stability of the transcript from the gene. Both of these decreases can result in a decrease in the translation product due to a decrease in the transcript from the gene. Substitutions in the above-mentioned conserved sequences of introns (splice mutations) can cause abnormal splicing of mRNA, resulting in abnormal mRNAs with unnecessary introns added or inserted. Abnormal mRNAs can produce abnormal translation products, for example, due to frameshifts, or translation cannot be terminated.

[0054] If the nucleotide substitution in the coding region is a missense mutation (resulting in a reduced abundance of the wild-type polypeptide), the substitution will result in an amino acid different from the original amino acid, and may result in a polypeptide with reduced or no original function.

[0055] Furthermore, substitutions in the coding region can result in incomplete translation products or translation products that do not maintain their original function. Incomplete translation products arise due to the conversion of a codon encoding an amino acid to a stop codon (nonsense mutation). Incomplete translation products lack one or more consecutive amino acid residues, including the C-terminal amino acid residue, compared to the original translation product. The nonsense mutation occurs in any codon upstream of the original stop codon, preferably one or more codons upstream from the original stop codon. Therefore, translation products from genes containing nonsense mutations are incomplete. Translation products that lack their original function are generated by amino acid substitutions. In this case, the amount of transcript may be equivalent to that of a wild-type Nicotiana plant (hereinafter sometimes abbreviated as "wild-type plant"). The translation product may exhibit a change in three-dimensional structure or a reduction in its function as a functional domain. One preferred embodiment of the mutation of the present invention is an amino acid substitution that results in a translation product that lacks its original function. The amino acid substitution is preferably a non-conservative substitution, which has a high possibility of changing the function of the translation product. Non-conservative substitutions include substitutions of amino acids with different charge or hydrophobicity (e.g., substitutions of a basic amino acid with an acidic amino acid, a basic or acidic amino acid with a neutral amino acid, a neutral amino acid with a basic or acidic amino acid, or a polar amino acid with a non-polar amino acid), and substitutions of amino acids with side chains with different bulkiness (steric size).

[0056] Another example of a phenomenon caused by a nonsense mutation is that when a nonsense mutation is present in the protein-coding region of the JOX gene, nonsense-mediated mRNA decay can occur (Brogna and Wen (2009) Nat. Structural Mol. Biol. 16: 107-113). Because nonsense-mediated mRNA decay causes the degradation of transcripts, nonsense mutations can result in a decrease in the amount of transcripts. For nonsense-mediated mRNA decay to occur, it is preferable that the JOX gene contains at least one exon containing a nonsense mutation. A preferred embodiment of a nonsense mutation that causes nonsense-mediated mRNA decay is that at least one nonsense mutation is present in the first or second exon of the JOX gene.

[0057] Mutations other than substitutions (such as deletions and insertions) occurring in the promoter sequence, 5' untranslated region, and / or 3' untranslated region can result in a decrease in the amount of transcripts and polypeptides due to decreased transcriptional activity or stability, similar to substitutions. Mutations other than substitutions into conserved intron sequences can also result in the translation of polypeptides with amino acid sequences different from the original, similar to substitutions. Mutations other than substitutions into coding regions can also result in the translation of polypeptides with amino acid sequences different from the original due to deletions or insertions of amino acid residues (caused by deletions or insertions of multiples of three consecutive bases) or frameshifts. Furthermore, large deletions including the entire gene or insertions of large fragments into the gene can result in the loss of expression of the gene itself.

[0058] The mutation that specifically causes the functional suppression may be introduced by mutagen treatment, genome editing, or gene knockout. In particular, the mutation may be introduced by mutagen treatment. In a Nicotiana plant according to one embodiment of the present invention, the mutation that specifically causes the functional suppression specifically enhances the response to jasmonic acid.

[0059] The mutagen treatment of the gene can be carried out by artificially applying the mutagen to a Nicotiana plant (and, if necessary, in combination with suppression of gene repair function). Examples of mutagens that can be used include chemical agents such as ethylmethanesulfonate (EMS), sodium azide, ethidium bromide, and nitrous acid, but are not limited to these as long as they cause mutations in the genomic DNA of Nicotiana plants. Examples of mutagens include gamma rays, heavy ion beams, X-rays, neutron rays, and UV rays, but are not limited to these as long as they are radiation that causes mutations in the genomic DNA of Nicotiana plants. EMS is preferred as a mutagen. These techniques are preferred because they do not require the addition of exogenous factors to the target plant. The gene recombination can be carried out by homologously recombining part or all of the target gene with a recombinant sequence according to known genetic engineering techniques. Genome editing of the gene can be performed by known techniques (e.g., zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the CRISPR / Cas9 system). The gene knockout can be performed by inserting a known transposon (mobile genetic element) or T-DNA.

[0060] Nucleotide substitutions in the JOX gene by EMS treatment can result in, for example, (I) frameshift mutations, (II) truncation mutations (in which the N-terminal amino acid residue is essentially deleted), (III) splice mutations, or (IV) nonsense mutations, because EMS treatment tends to induce specific nucleotide changes in DNA (C→T substitutions and G→A substitutions).

[0061] In the CRISPR / Cas9 system, if guide RNA and Cas9 protein are present in target cells, and in the case of TALEN and ZFN, if fusion protein (DNA binding domain and nuclease fused) is present in target cells, genome editing can be performed.Therefore, the above-mentioned guide RNA and Cas9 protein, as well as the above-mentioned fusion protein, can be directly introduced into target cells.Methods for directly introducing them into target cells include PEG method, electroporation method, and particle bombardment method.In addition, a vector into which a construct (including a polynucleotide encoding guide RNA and Cas9 protein, and any promoter and / or terminator) is inserted can be introduced into target cells and tissues via Agrobacterium or the like.

[0062] In the CRISPR / Cas9 system, a complementary sequence of a nucleotide sequence immediately upstream of XGG on the genome forms a base pair with a part of the guide RNA, and the double-stranded genomic DNA is cleaved by Cas9 within the nucleotide sequence.

[0063] In TALEN, each of the pair of DNA-binding domains of the dimer-forming artificial nuclease binds to a nucleotide sequence present on both ends of the FokI cleavage domain via a 5- to 20-base spacer. The nucleotide sequences are present on one strand and the other strand of double-stranded genomic DNA, and therefore one of the pair of DNA-binding domains binds to one strand and the other to the other strand. The DNA-binding domain is composed of repeating units (modules) of 33 to 34 amino acid residues, with the number of modules corresponding to the number of bases to be bound.

[0064] In ZFNs, similar to TALENs, a pair of DNA-binding domains of a dimer-forming artificial nuclease bind to nucleotide sequences present on both ends of a FokI cleavage domain via a 5- to 20-base spacer. The DNA-binding domains are composed of multiple zinc finger modules.

[0065] As described above, the mutation introduced into a Nicotiana plant according to one embodiment of the present invention has been described as an artificially induced mutation, but is not limited thereto. For example, a mutation or disruption of the JOX gene may occur through spontaneous mutation. Spontaneous mutation of a gene generally occurs due to replication errors and genetic damage. Such damage can be caused by exposure to known naturally occurring mutagens (e.g., radiation or ultraviolet light).

[0066] Gene mutation or disruption can be determined by detecting the presence or absence of a mutation in the gene. Methods for detecting a mutation in a gene include: (1) a method in which a DNA sequence containing the mutation is amplified by PCR or the like, and then the DNA base sequence is directly decoded using a commercially available sequencer, (2) a method in which sequence differences are detected by differences in electrophoretic distance using SSCP (Single Strand Conformation Polymorphism), (3) a method in which SNP (Single Nucleotide Polymorphism) is detected using Cycle PCR, (4) a method in which the presence or absence of a mutation is detected by cleaving mismatch sites using T7 Endonuclease I or the like, (5) a CAPS (Cleaved Amplified Polymorphic Sequence) method in which the presence or absence of a mutation can be determined based on the presence or absence of cleavage by restriction enzyme treatment, and (6) a dCAPS (Derived Amplified Polymorphic Sequence) method in which the presence or absence of a mutation can be determined based on the presence or absence of cleavage by restriction enzyme treatment using a primer set that intentionally contains mismatches. (7) A method for determining the presence or absence of a mutation by detecting whether or not a probe that specifically hybridizes to a mutant sequence has hybridized (PCR using a TaqMan probe); (8) A method for performing single-base extension using a primer adjacent to the mutation and detecting the presence or absence of a mutation based on the mass difference of the incorporated base (MassARRAY analysis); (9) In the case of deletions or insertions, a method for detecting mutations based on differences in electrophoretic mobility is available. However, any method that can determine the presence or absence of a mutation is sufficient. Alternatively, gene mutation or disruption can be determined by comparing the size and expression level of the protein resulting from the gene modification with those of the wild-type protein. Specifically, such a comparison can be performed, for example, by Western blotting.

[0067] Suppression of gene expression includes suppression of transcription from the gene to mRNA, suppression of translation from the gene to a polypeptide via mRNA (e.g., degradation of the mRNA), and suppression of the function of the translated polypeptide. mRNA degradation can result from the nonsense-mediated mRNA decay. Suppression of transcription can be achieved by inhibiting transcription factors that promote transcription from the gene and by inhibiting access of transcription initiation factors to the gene. Suppression of translation can be achieved using antisense RNA molecules, RNAi molecules, or co-suppression molecules. Suppression of polypeptide function can be achieved by molecules that inhibit the function of a functional polypeptide by binding to it (e.g., decoy nucleic acids, ribozymes, antibodies, and inhibitory peptides).

[0068] The vector used for transforming Nicotiana plants for the purpose of suppressing gene expression or introducing mutations into genes is not particularly limited, as long as it is capable of expressing a polynucleotide inserted therein in plant cells. Suitable vectors include, for example, pBI-, pPZP-, and pSMA-based vectors, which can introduce a polynucleotide of interest into plant cells via Agrobacterium. Binary vector-based plasmids (e.g., pBIG, pBIN19, pBI101, pBI121, and pPZP202) are particularly preferred.

[0069] When gene expression is suppressed by RNAi, a trigger sequence used to suppress the expression of a target gene by RNAi is inserted as a mutation into the vector. The trigger sequence is, for example, a polynucleotide (sense RNA portion) represented by a base sequence of at least 21 to 30 consecutive bases (e.g., 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more bases) that is part of a polynucleotide (which may have 0.1 to 1% substitutions) encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 2, and a polynucleotide (antisense RNA portion) represented by a base sequence complementary to the polynucleotide. The above-mentioned base sequence of "at least 21 to 30 consecutive bases" more specifically means a base sequence of 21 or more consecutive bases, 23 or more bases, 25 or more bases, 30 or more bases, 35 or more bases, 40 or more bases, 45 or more bases, 50 or more bases, 60 or more bases, 70 or more bases, 80 or more bases, 90 or more bases, or 100 or more bases.

[0070] The suppression (of transcription, translation, or polypeptide function) can be achieved, for example, by directly introducing a molecule for achieving the suppression into a plant, or by introducing a nucleic acid molecule encoding the molecule into a plant (transformation of the plant). Here, as a result of the transformation of the plant, the nucleic acid molecule is integrated into one or more arbitrary regions in the genome of the plant. In the case of Nicotiana tabacum, which is an amphidiploid plant of the genus Nicotiana, the nucleic acid molecule does not need to be integrated into both the S genome and the T genome as a result of the transformation of the plant, as long as the suppression is achieved.

[0071] (Function Suppression) In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene that contains, as its coding region, a polynucleotide consisting of the nucleotide sequence of a JOX gene. As used herein, the term "endogenous gene function suppression" refers to a state in which a gene on the genome does not perform its original function. Therefore, "endogenous gene function suppression" encompasses "mutation of the endogenous gene," "disruption of the endogenous gene," and "suppression of expression of the endogenous gene" by a gene other than the endogenous gene (including a foreign gene). Furthermore, specifically causing function suppression refers to suppressing only the function of the target gene without suppressing the functions of other genes. For example, it is desirable to avoid simultaneous suppression of the function of multiple genes under the control of the same transcription factor by suppressing the function of the JOX gene, which could lead to metabolic abnormalities.

[0072] In one embodiment of the Nicotiana plant of the present invention, the functional suppression may be a reduction in the abundance of the original functional polypeptide translated from the coding region of the endogenous JOX gene compared to a wild-type plant.

[0073] "Decreased abundance" of a polypeptide means that the abundance of the polypeptide is 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the abundance of the wild-type polypeptide. The abundance of the polypeptide relative to the abundance of the wild-type polypeptide can be appropriately selected from the above-mentioned values ​​so as to bring about the functional inhibition in Nicotiana plants.

[0074] Preferably, the reduction in the abundance of the polypeptide in a Nicotiana plant according to one embodiment of the present invention is genetically stably inherited in cultured cells, calli, protoplasts, seeds, and progeny obtained from the Nicotiana plant. Thus, a Nicotiana plant according to one embodiment of the present invention may be an individual developed from cultured cells, calli, protoplasts, seeds, and progeny produced through artificial manipulation, and these materials for obtaining the individual are included in the scope of the present invention.

[0075] The functional inhibition may be a reduction in translation of the original functional polypeptide compared to a wild-type plant. The translation of the polypeptide occurs due to a reduction in mRNA (due to the abundance of mRNA, such as instability of the mRNA itself, promotion of mRNA degradation, or suppression of mRNA transcription) or a reduction in the amount of translation from mRNA (due to a deficiency, inhibition of recruitment, or functional deficiency of translation components (tRNA and ribosomes)).

[0076] The functional inhibition may be a reduction in the abundance of mRNA transcribed from the JOX gene compared to a wild-type plant. The reduction in the amount of mRNA transcription occurs, for example, by suppressing transcription from an endogenous gene to mRNA. Transcription inhibition can be achieved by, for example, inhibiting access of a transcription initiation factor to the endogenous gene as a result of introducing a mutation into the endogenous gene.

[0077] The functional inhibition may be the promotion of degradation of mRNA transcribed from the endogenous gene. mRNA degradation can be caused by the production of abnormal mRNA (causing nonsense-mediated mRNA decay), the presence of exogenous factors that degrade mRNA, the activation of endogenous components that degrade mRNA, or the presence of a degradation-promoting sequence in the mRNA. Accelerated degradation of mRNA transcribed from the endogenous gene results in a decrease in the amount of mRNA in the Nicotiana plant. That is, in the Nicotiana plant, the functional inhibition may be a decrease in the amount of mRNA transcribed from the endogenous gene compared to a wild-type plant. Here, "a decrease in the amount of mRNA transcribed from an endogenous gene" refers to the presence of 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the transcript, based on the amount of the transcript of the endogenous gene in a wild-type plant.

[0078] In the Nicotiana plant, the mutation may be insertion of a polynucleotide expressing a factor that promotes degradation of mRNA transcribed from the endogenous gene outside the region where the endogenous gene is located. The factor may be an antisense RNA molecule, an RNAi molecule, or a co-suppression molecule.

[0079] In one embodiment of the present invention, a Nicotiana plant has suppressed function of an endogenous gene that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of a nucleotide sequence encoding an endogenous JOX gene and that contains as its coding region a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid.

[0080] Stringent conditions refer to conditions under which a double-stranded polynucleotide specific to the so-called nucleotide sequence is formed, but the formation of a non-specific double-stranded polynucleotide is significantly suppressed. In other words, they can be said to be conditions under which hybridization occurs between highly homologous nucleic acids, for example, at a temperature 15°C, preferably 10°C, and more preferably 5°C lower than the melting temperature (Tm value) of a double-stranded polynucleotide that is perfectly matched to a probe. For example, hybridization conditions can be exemplified by conditions under which hybridization occurs in a general hybridization buffer at 68°C for 20 hours. One example is 0.25M Na ２ HPO ４ The hybridization is carried out for 16 to 24 hours in a buffer solution consisting of 1x Denhardt's solution, pH 7.2, 7% SDS, 1 mM EDTA, and 1x Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C. ２ HPO ４, pH 7.2, 1% SDS, 1 mM EDTA, followed by two 15-minute washes at 60-68°C, preferably 65°C, and more preferably 68°C. Another example is prehybridization overnight at 42°C in a hybridization solution containing 25% formamide, or, for more stringent conditions, 50% formamide, 4x SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, and 20 μg / ml denatured salmon sperm DNA, followed by addition of a labeled probe and incubation at 42°C overnight for hybridization. The washing solution and temperature conditions for the subsequent washes can be approximately "1x SSC, 0.1% SDS, 37°C," or, more stringent conditions, approximately "0.5x SSC, 0.1% SDS, 42°C," or, even more stringent conditions, approximately "0.2x SSC, 0.1% SDS, 65°C." As such, the more stringent the hybridization washing conditions, the more likely it is that DNA with high homology to the probe sequence will be isolated. However, the above combinations of SSC, SDS, and temperature conditions are merely examples, and one skilled in the art can achieve similar stringency by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.). For example, those skilled in the art can easily obtain such genes by referring to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive, Plainview, NY (1989)).

[0081] The Nicotiana plant according to one embodiment of the present invention is not particularly limited as long as it is a plant belonging to the Nicotiana genus, and examples thereof include Nicotiana acaulis, Nicotiana acuminata, Nicotiana acuminata var. multzjlora, Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana Nicotiana bigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana ingrown ingulba), Nicotiana kawakamii, Nicotiana naitianaknightiana, Nicotiana langsdorfi, Nicotiana linearis, Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp. hesperis, Nicotiana otophora otophora), Nicotiana paniculata, Nicotiana pauczjlora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rosulata subsp. Ingulba, Nicotiana rotundifolia, Nicotiana rustica Nicotiana rustica (Mulberry tobacco), Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifoliasolanifolia, Nicotiana spegauinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa, Nicotiana tomentosiformis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotiana wigandioides wigandioides), and hybrids of Nicotiana plants. Among these, Nicotiana tabacum and Nicotiana rustica, which are used as raw materials for tobacco leaf production, are particularly preferred. Nicotiana sylvestris can also be preferably used. The Nicotiana plant according to one embodiment of the present invention may particularly belong to Nicotiana tabacum, Nicotiana sylvestris, or Nicotiana rustica.

[0082] Individuals resulting from the mutation or disruption of the above genes are referred to herein as mutants (also simply referred to as mutants) of Nicotiana plants. Among Nicotiana plants, Nicotiana tabacum is an amphidiploid and has both a genome derived from its parent plant, Nicotiana sylvestris (also referred to as the "S genome" or "S subgenome") and a genome derived from Nicotiana tomentosiformis (also referred to as the "T genome" or "T subgenome"). In Nicotiana tabacum, genes with the same name are almost always present in both the S genome and the T genome. In the case of Nicotiana tabacum, the mutant may have the above mutation in either the S genome or the T genome. The mutant may also have the above mutation in both the S genome and the T genome. The mutation for eliminating a function may be a single mutation or multiple mutations in a single gene, and the type of mutation is not important. In the case of Nicotiana tabacum, any or all of the four alleles, two of which are present in each of the S genome and the T genome, may have mutations, and if mutations are present in multiple alleles, these mutations may be the same or different.

[0083] A Nicotiana plant according to one embodiment of the present invention is preferably an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 62%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, and further comprising, as a coding region, a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid, wherein a mutation that specifically causes functional inhibition has been introduced into the endogenous gene in the genome; and / or an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 62%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, and further comprising, as a coding region, a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid, wherein a mutation that specifically causes functional suppression has been introduced into the endogenous gene in a genome; and / or An endogenous gene contains, as its coding region, a polynucleotide encoding a polypeptide having 62%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 9, and further contains, as its coding region, a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid. A mutation that specifically causes functional suppression has been introduced into the endogenous gene in a genome that contains, as its coding region, a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid.

[0084] A Nicotiana plant according to one embodiment of the present invention may also include breeding progeny obtained by crossing. Mutant breeding has been conducted for many plant species, including rice, wheat, barley, and soybean. For example, mutants isolated from a mutant population treated with a mutagen contain numerous mutations in addition to the target gene. Therefore, backcrossing is generally performed to remove excess mutations. By crossing with a cultivar with superior traits, the traits possessed by the mutant can be introduced into the cultivar, resulting in a cultivar with higher added value. Because the traits possessed by mutants are derived from mutations, backcrossing requires the selection of individuals with mutations. In this case, the fewer mutations that result in the target trait (in this case, cold receptor TRPM8 activity), the fewer mutations need to be considered, thereby reducing the effort required for backcrossing. Efficient backcrossing requires a method for easily detecting the presence or absence of mutations and whether the mutations are homozygous or heterozygous. This method can be performed using the mutation detection method described below. In addition, by performing Marker Assisted Selection (MAS) using background markers that show polymorphism between mutants and cultivars, lines with a high reversion rate to cultivars can be efficiently obtained with a small number of crosses. SNPs and Simple Sequence Repeats (SSRs), which are well known in tobacco, can be used as polymorphic markers. If necessary, new polymorphic markers can be obtained and used by decoding the genome sequence of the tobacco used to identify differences in base sequence and number of repeat sequences.

[0085] [Method for producing a Nicotiana plant] A method for producing a Nicotiana plant according to one embodiment of the present invention comprises the step of introducing a mutation that specifically causes functional suppression of at least one of the endogenous genes (a) to (c) into the endogenous gene in the genome of a Nicotiana plant. Details of the mutation introduced into Nicotiana plants and the like are described in the section [Nicotiana plants].

[0086] The introducing step may include introducing the mutation into the coding region of at least one of the endogenous genes (a) to (c). Further, the introducing step may be performed by mutagen treatment, genome editing, or gene knockout. In particular, the introducing step may be mutagen treatment.

[0087] In the above production method, an individual exhibiting a desired phenotype may be further selected from a mutant population of plants having mutations. As an example of selecting an individual, a procedure for selecting a desired individual from a mutant population (panel) obtained by treatment with a mutagen will be described.

[0088] Loss-of-function tobacco mutants with homozygous mutations in genes present in the S genome and the T genome can be obtained, for example, by the following method. The following describes an example of a method for obtaining loss-of-function tobacco mutants with homozygous mutations in both one gene on the S genome and one gene on the T genome. As described above, tobacco plants are treated with a mutagen to create a panel of tobacco mutants with mutations throughout the entire tobacco genome, and genomic DNA is extracted. Target genes (polynucleotides) are amplified from the panel's genomic DNA using gene-specific primers for the S genome and the T genome, and the nucleotide sequence of the product is determined. Lines with homozygous mutations are selected by first obtaining lines (M2) with homozygous mutations in both the S genome and the T genome, and then crossing them to produce F1. Furthermore, the resulting self-bred progeny (F2) are cultivated, and from these lines with homozygous mutations in both the S and T genomes are obtained (with a probability of 1 / 16 due to bifactorial recessiveness).

[0089] Furthermore, loss-of-function tobacco mutants with homozygous mutations in two genes on the S genome and one gene on the T genome can be obtained, for example, by the following method. A line with homozygous mutations in both the S and T genomes obtained as described above is crossed with a line with a homozygous mutation in the other allele on the S genome to produce an F1. The resulting self-pollinated progeny (F2) are then cultivated, and a line with homozygous mutations in both genes on the S genome and one gene on the T genome is obtained from among them (the probability of obtaining this is 1 / 64 due to triple-factor recessiveness).

[0090] Selection of individuals exhibiting a desired phenotype may be carried out by measuring the activity of the cold receptor TRPM8.

[0091] Therefore, the method for producing a Nicotiana plant according to one embodiment of the present invention may further include one or more of the following steps: preparing a panel of tobacco mutants in which mutations have been introduced throughout the tobacco genome, extracting genomic DNA from lines included in the panel, determining the base sequence of the JOX gene in the genomic DNA, selecting lines from the panel that contain homozygous mutations, and confirming the activity of the cold receptor TRPM8 in the lines.

[0092] The strain can be crossed with a non-mutagenized strain at any time before the step of confirming the cold receptor TRPM8 activity is performed. Crossing allows for the elimination of mutations that may exist in genes other than the JOX gene. In certain embodiments, the strain carrying a mutation in the JOX gene can be backcrossed multiple times with a non-mutagenized strain (the original strain used to generate the panel).

[0093] Genomic DNA from tobacco mutants can be extracted by known methods, or a commercially available extraction kit can be used. The genomic DNA may be crudely purified or may be a purified product that has undergone several purification steps.

[0094] Polynucleotide amplification can be carried out by, for example, PCR, but may also be carried out by other known gene amplification methods, such as LCR (ligase chain reaction) or LAMP (loop-mediated isothermal amplification).

[0095] Primer sequences for amplifying each polynucleotide can be designed, for example, from the nucleotide sequence. For example, regions specific to each JOX gene (JOX-S1 gene, JOX-S2 gene, or JOX-T2 gene) are identified based on the results of homology analysis with the nucleotide sequence of each JOX gene. The nucleotide sequence of SEQ ID NO: 1 is the genomic sequence of the JOX-S1 gene, the nucleotide sequence of SEQ ID NO: 4 is the genomic sequence of the JOX-S2 gene, and SEQ ID NO: 7 is the genomic sequence of the JOX-T2 gene. By designing primers for these regions, the desired JOX gene can be specifically amplified from extracted genomic DNA. The site to be designed can be selected from regions specific to each gene, preferably an intron, a 5' untranslated region, or a 3' untranslated region.

[0096] When multiple genes similar to the JOX-S1 gene, JOX-S2 gene, or JOX-T2 gene exist on the S genome or T genome, primers may be designed based on regions specific to each gene, or primers may be designed based on specific regions common to multiple genes present on each genome, such as S type or T type.

[0097] The length of the primer is preferably 15 to 30 bases, particularly preferably 17 to 25 bases. The primer sequence may be designed based on the sequence of a region specific to the base sequence or a region common to both base sequences. Furthermore, as long as it can function as a primer for amplifying a sequence of a predetermined number of bases including a mutation site, the sequence may contain one or more substitutions, deletions, and / or additions. Furthermore, the primer may be labeled with a fluorescent substance, a radioactive substance, or the like, as needed.

[0098] The length of each polynucleotide to be amplified is not particularly limited as long as it is a length that allows use of various detection methods described below, but is, for example, 20 to 5,000 bases, more preferably 50 to 2,000 bases, even more preferably 100 to 700 bases, and still more preferably 100 to 500 bases.

[0099] [Method for imparting a cooling sensation to a tobacco product] Another aspect of the present invention includes a method for imparting a cooling sensation to a tobacco product containing the tobacco material derived from a Nicotiana plant. Because the tobacco material has cold receptor TRPM8 activity and exhibits a cooling sensation, the tobacco product is substantially free of a cooling flavoring that imparts a cooling sensation. Examples of cooling flavorings include menthol, WS-5 (ethyl-2-(p-menthane-3-carboxamide) acetate), and WS-3 (N-ethyl-p-menthane-3-carboxamide).

[0100] Another aspect of the present invention provides a method for determining whether a Nicotiana plant has cold receptor TRPM8 activity, comprising the steps of: obtaining a sample by harvesting a part of the Nicotiana plant; detecting a mutation in the sample that specifically causes functional suppression of the endogenous JOX gene in the genome; and determining that the Nicotiana plant in which the mutation is detected is a Nicotiana plant with cold receptor TRPM8 activity.

[0101] In the method for determining a Nicotiana plant having cold receptor TRPM8 activity, the functional inhibition confers cold receptor TRPM8 activity to tobacco leaves or cured leaves of the Nicotiana plant. In other words, the determination method is used in a method for producing a Nicotiana plant having cold receptor TRPM8 activity.

[0102] One aspect of the present invention provides a method for breeding Nicotiana plants having cold receptor TRPM8 activity, comprising the step of crossing Nicotiana plants having cold receptor TRPM8 activity determined by the above-described method for determining Nicotiana plants having cold receptor TRPM8 activity.

[0103] Mutant breeding has been performed on many plant species. For example, mutants isolated from a mutant population treated with a mutagen have numerous mutations in addition to the target gene. Therefore, backcrossing is generally performed to remove excess mutations. In this crossing, a cultivated variety possessing superior traits is crossed with the mutant, allowing the desired traits possessed by the mutant to be introduced into an existing cultivated variety. The resulting breeding progeny can be a variety that adds high added value to an existing cultivated variety. In this case, the fewer the number of target mutations that confer cold receptor TRPM8 activity, the fewer mutations that need to be focused on, reducing the effort required for backcrossing.

[0104] [Summary] By summarizing the above embodiments, the present invention can be summarized as follows.

[0105] (A1) A tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the endogenous gene in its genome that specifically causes functional inhibition in at least one of the following: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9, and the tobacco material has cold receptor TRPM8 activity.

[0106] (A2) A tobacco material derived from a Nicotiana plant, the Nicotiana plant comprising: (a) an endogenous gene having, as a coding region, a polynucleotide encoding a polypeptide having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of catalyzing the hydroxylation of jasmonic acid; (b) an endogenous gene having, as a coding region, a polynucleotide encoding a polypeptide having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, and having the activity of catalyzing the hydroxylation of jasmonic acid; and (c) A tobacco material having cold receptor TRPM8 activity, wherein at least one endogenous gene has, as its coding region, a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid, and wherein a mutation that specifically causes functional inhibition has been introduced into the endogenous gene in the genome, the endogenous gene having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9.

[0107] (A3) A tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the endogenous gene in its genome that specifically causes functional inhibition in at least one of the following: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, and the tobacco material has cold receptor TRPM8 activity.

[0108] (A4) The tobacco material according to any one of (A1) to (A3), which contains a TRPM8 agonist. (A5) The tobacco material according to any one of (A1) to (A4), wherein the cold receptor TRPM8 activity of the tobacco material is at least twice as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant.

[0109] (A6) The tobacco material according to any one of (A1) to (A5), which is a dry material. (A7) The tobacco material according to any one of (A1) to (A6), which is a powder of a dry material.

[0110] (A8) The tobacco material according to (A6) or (A7), wherein the dried material is dried leaves of tobacco leaf harvested from the Nicotiana plant. (A9) The tobacco material according to any one of (A6) to (A8), wherein the dried material is dried leaves obtained by air-drying tobacco leaf harvested from the Nicotiana plant.

[0111] (A10) The tobacco material according to any one of (A1) to (A9), wherein the Nicotiana plant belongs to Nicotiana tabacum, Nicotiana sylvestris, or Nicotiana rustica. (A11) The tobacco material according to any one of (A1) to (A10), wherein the Nicotiana plant belongs to Nicotiana tabacum.

[0112] (A12) The tobacco material according to any one of (A1) to (A11), in which a mutation that specifically causes functional inhibition has been introduced into both the endogenous gene according to (a) and the endogenous gene according to (c). (A13) The tobacco material according to any one of (A1) to (A11), in which a mutation that specifically causes functional inhibition has been introduced into both the endogenous gene according to (b) and the endogenous gene according to (c). (A14) The tobacco material according to any one of (A1) to (A11), in which a mutation that specifically causes functional inhibition has been introduced into all of the endogenous gene according to (a), the endogenous gene according to (b), and the endogenous gene according to (c).

[0113] (B1) A tobacco product comprising the tobacco material according to any one of (A1) to (A14).

[0114] (C1) A Nicotiana plant in which a mutation that specifically causes functional inhibition has been introduced into at least one of the endogenous genes in the genome: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9.

[0115] (C2) (a) an endogenous gene having, as a coding region, a polynucleotide encoding a polypeptide having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and having the activity of catalyzing the hydroxylation of jasmonic acid; (b) an endogenous gene having, as a coding region, a polynucleotide encoding a polypeptide having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, and having the activity of catalyzing the hydroxylation of jasmonic acid; and (c) A Nicotiana tabacum plant, in which a mutation that specifically causes functional inhibition has been introduced into at least one of its endogenous genes in the genome, the endogenous genes having as their coding region a polynucleotide that has 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 9, and that encodes a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid.

[0116] (C3) A Nicotiana plant in which a mutation that specifically causes functional inhibition has been introduced into at least one of the endogenous genes in the genome: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0117] (C4) The Nicotiana plant according to any one of (C1) to (C3), wherein the Nicotiana plant is any one of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana rustica. (C5) The Nicotiana plant according to any one of (C1) to (C4), wherein the Nicotiana plant belongs to Nicotiana tabacum.

[0118] (C6) A Nicotiana plant according to any one of (C1) to (C5), in which a mutation that specifically causes functional suppression has been introduced into both the endogenous gene according to (a) and the endogenous gene according to (c). (C7) A Nicotiana plant according to any one of (C1) to (C5), in which a mutation that specifically causes functional suppression has been introduced into both the endogenous gene according to (b) and the endogenous gene according to (c). (C8) A Nicotiana plant according to any one of (C1) to (C5), in which a mutation that specifically causes functional suppression has been introduced into all of the endogenous gene according to (a), the endogenous gene according to (b), and the endogenous gene according to (c).

[0119] (C9) The Nicotiana plant according to any one of (C1) to (C8), wherein the mutation that specifically causes the functional suppression has been introduced by mutagen treatment, genome editing, or gene knockout. (C10) The Nicotiana plant according to any one of (C1) to (C9), wherein the mutation that specifically causes the functional suppression specifically causes an enhanced response to jasmonic acid.

[0120] (D1) A method for imparting a refreshing or cool sensation to a tobacco product, wherein the tobacco product comprises tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the genome of at least one of the following endogenous genes: (a) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:9, wherein the tobacco material is substantially free of cooling flavoring and has cooling receptor TRPM8 activity.

[0121] (D2) A method for imparting a refreshing or cool sensation to a tobacco product, the method comprising: introducing a mutation that specifically causes functional inhibition of at least one of the following endogenous genes into a Nicotiana plant: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:9, to produce a mutant of the Nicotiana plant; and incorporating tobacco material derived from the mutant into a tobacco product, wherein the tobacco material is substantially free of cooling flavorings and has cooling receptor TRPM8 activity.

[0122] (D3) The at least one endogenous gene is: (a) an endogenous gene having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and comprising, as a coding region, a polynucleotide encoding a polypeptide having activity to catalyze the hydroxylation of jasmonic acid; (b) an endogenous gene having, as a coding region, a polynucleotide encoding a polypeptide having 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, and having the activity of catalyzing the hydroxylation of jasmonic acid; and (c) The method according to (D1) or (D2), wherein the endogenous gene has at least 60% or more, preferably 62% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, or even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 9, and contains as a coding region a polynucleotide encoding a polypeptide having the activity of catalyzing the hydroxylation of jasmonic acid.

[0123] (D4) The method according to (D1) or (D2), wherein the at least one endogenous gene is at least one of: (a) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3; (b) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0124] (D5) The method according to any one of (D1) to (D4), wherein the tobacco material is a dry material. (D6) The method according to any one of (D1) to (D5), wherein the tobacco material is a powder of a dry material.

[0125] (D7) The method according to (D5) or (D6), wherein the dried material is dried leaves of tobacco leaves harvested from the Nicotiana plant. (D8) The method according to any one of (D5) to (D7), wherein the dried material is dried leaves obtained by air-drying tobacco leaves harvested from the Nicotiana plant.

[0126] (D9) The method according to any one of (D1) to (D8), wherein the Nicotiana plant belongs to Nicotiana tabacum, Nicotiana sylvestris, or Nicotiana rustica. (D10) The method according to any one of (D1) to (D9), wherein the Nicotiana plant belongs to Nicotiana tabacum.

[0127] (D11) The method according to any one of (D1) to (D10), wherein the at least one endogenous gene is both the endogenous gene according to (a) and the endogenous gene according to (c). (D12) The method according to any one of (D1) to (D10), wherein the at least one endogenous gene is both the endogenous gene according to (b) and the endogenous gene according to (c). (D13) The method according to any one of (D1) to (D10), wherein the at least one endogenous gene is all of the endogenous gene according to (a), the endogenous gene according to (b), and the endogenous gene according to (c).

[0128] Example 1: Generation of Tobacco JOX Mutants. The genome sequence of the tobacco (Nicotiana tabacum) cultivar "Tsukuba No. 1" was analyzed to identify NtJOX-S1 (genome sequence: SEQ ID NO: 1, CDS sequence: SEQ ID NO: 2, deduced amino acid sequence: SEQ ID NO: 3) and NtJOX-S2 (genome sequence: SEQ ID NO: 4, CDS sequence: SEQ ID NO: 5, deduced amino acid sequence: SEQ ID NO: 6), which are thought to be derived from the S subgenome, and NtJOX-T2 (genome sequence: SEQ ID NO: 7, CDS sequence: SEQ ID NO: 8, deduced amino acid sequence: SEQ ID NO: 9), which are thought to be derived from the T subgenome. A mutant population of the tobacco cultivar "Tsukuba No. 1" induced by EMS treatment was analyzed. Three lines of tobacco mutants harboring mutations in NtJOX-S1, NtJOX-S2, or NtJOX-T2 were identified for each gene.

[0129] More specifically, for NtJOX-S1, we identified the NtJOX-S1-1 line, which had a nonsense mutation in exon 2 due to a substitution of C at position 2427 in the genome sequence shown in SEQ ID NO: 1 with T. We also identified the NtJOX-S1-2 line, which had a nonsense mutation in exon 2 due to a substitution of G at position 2378 in the genome sequence shown in SEQ ID NO: 1 with A. We also identified the NtJOX-S1-3 line, which had a nonsense mutation in exon 2 due to a substitution of C at position 2337 in the genome sequence shown in SEQ ID NO: 1 with T. For NtJOX-S2, we identified the NtJOX-S2-1 line, which had a nonsense mutation in exon 1 due to a substitution of G at position 1258 in the genome sequence shown in SEQ ID NO: 4 with A. We also identified the NtJOX-S2-2 line, which had a nonsense mutation in exon 2 due to a substitution of C at position 2335 in the genome sequence shown in SEQ ID NO: 4 with T. Similarly to NtJOX-S2-1, we also found the NtJOX-S2-3 line, which had a nonsense mutation in exon 1 due to a substitution of G at position 1258 in the genome sequence shown in SEQ ID NO: 4 with A. Regarding NtJOX2-T, we also found the NtJOX-T2-1 line, which had a nonsense mutation in exon 1 due to a substitution of C at position 1196 in the genome sequence shown in SEQ ID NO: 7 with T. We also found the NtJOX-T2-2 line, which had a splicing mutation on the 5' side of the second intron due to a substitution of G at position 2677 in the genome sequence shown in SEQ ID NO: 7 with A. We also found the NtJOX-T2-3 line, which had a nonsense mutation in exon 1 due to a substitution of C at position 1322 in the genome sequence shown in SEQ ID NO: 7 with T.

[0130] DNA was extracted from each individual in the M2 generation of the lineages identified above. PCR was performed using this DNA as a template with the primers shown in Table 1. The sequence of the amplified product was determined by sequence analysis, and individuals homozygous for the mutation were selected. PCR was performed using KOD One (registered trademark) PCR Master Mix (TOYOBO Corporation).

[0131]

[0132] The NtJOX-S1-1 line was crossed with the NtJOX-T2-1 line to obtain the F1 generation (NtJOX1-F1-1 line). The NtJOX-S1-2 line was crossed with the NtJOX-T2-2 line to obtain the F1 generation (NtJOX1-F1-2 line). The NtJOX-S2-1 line was crossed with the NtJOX-T2-1 line to obtain the F1 generation (NtJOX2-F1-1 line). The NtJOX-S2-2 line was crossed with the NtJOX-T2-2 line to obtain the F1 generation (NtJOX2-F1-2 line). Each F1 generation was cultivated and self-fertilized to obtain the F2 generation (NtJOX1-F2-1 line, NtJOX1-F2-2 line, NtJOX2-F2-1 line, NtJOX2-F2-2 line).

[0133] From the F2 generation, individuals homozygous for the mutation were selected by sequence analysis using iSeq 100 (Illumina). Sequence analysis and library preparation for sequence analysis (2-step tailed PCR method) were performed according to the procedures described below.

[0134] DNA was extracted from each individual in the F2 generation. Using this DNA as a template, PCR was performed using the primers shown in Table 1 to amplify the sequence surrounding the mutation, yielding an amplified product. To prepare a library for sequence analysis, PCR was performed to add an adapter sequence for analysis (5' end - P7 sequence, or P5 sequence - barcode sequence for each individual - sequence primer binding sequence - overhang sequence - 3' end) to the amplified product for sequence analysis using iSeq 100 (Illumina). The library was then subjected to sequence analysis using iSeq 100 (Illumina), the sequence information was determined, and individuals homozygous for the mutation were selected.

[0135] As a result, we found two homozygous individuals, joxs1joxt2-1 and joxs1joxt2-2, carrying mutations in both the NtJOX-S1 and NtJOX-T2 genes. We also found two homozygous individuals, joxs2joxt2-1 and joxs2joxt2-2, carrying mutations in both the NtJOX-S2 and NtJOX-T2 genes. These individuals were selfed to obtain F3 seeds.

[0136] Evaluation Example 1-1: Measurement of the number of glandular trichomes in tobacco JOX mutants. Four JOX mutant lines (F3 generation of joxs1joxt2-1, joxs1joxt2-2, joxs2joxt2-1, and joxs2joxt2-2) obtained in Example 1 and a control, Tsukuba No. 1, were grown in a greenhouse. Thirteen weeks after sowing, each line was observed under a stereomicroscope, and the number of glandular trichomes present within a 1-cm margin of the leaf (the fifth expanded leaf counting from the top) was counted at four locations per leaf, and the average was calculated. The results revealed that the number of glandular trichomes secreting leaf surface resin components was significantly increased (at the 1% level by Tukey-Kramer test) in all four JOX mutant lines (Figure 1).

[0137] Evaluation Example 1-2: Measurement of the amount of leaf surface resin components in tobacco JOX mutants Steam distillation is a commonly used method for collecting aroma components contained in plants. By steam distilling tobacco leaves, essential oils containing many leaf surface resin components can be obtained. The amount of essential oil containing leaf surface resin components is one indicator of the amount of flavor and aroma components in tobacco leaves. Therefore, the amount of leaf surface resin components in each tobacco JOX mutant obtained in Example 1 was measured by steam distillation.

[0138] The plant materials used were the JOX mutant joxs1joxt2-1 line, conventionally cultivated in a field, and Tsukuba 1 as a control. Essential oils were obtained by steam distillation using 5 kg of mid-to-upper leaves (fresh leaves) from joxs1joxt2-1 and Tsukuba 1 at the harvesting stage. Specifically, 35 L of tap water was placed in a distillation pot and brought to a boil at 100°C. After boiling, the boiler was stopped, and a basket containing 5 kg of fresh leaves was placed in the pot. The basket was secured in a position that prevented it from being submerged in water. The lid of the distillation pot was closed, the boiler was restarted, and distillation continued for 2 hours. The chiller was set to 20°C. The steam-distilled water obtained after 2 hours of distillation was collected and 20% (w / w) sodium chloride was added. 1 L of ethyl acetate was added to the treated steam-distilled water, and the mixture was then transferred to a separatory funnel. The ethyl acetate layer was collected and concentrated to dryness using a rotary evaporator. The yield of essential oil was approximately 30 mg / kg for joxs1joxt2-1 and approximately 12 mg / kg for Tsukuba 1, confirming a more than two-fold increase in the JOX mutant.

[0139] In addition, steam distillation was performed on 300 g of dried leaves of JOXS1JOXT2-1 and Tsukuba No. 1, which had been harvested and dried to a yellow color after optimal harvesting, to obtain essential oils. Specifically, 4 L of tap water was placed in a stockpot and brought to a boil at 100°C using an induction heater. After boiling, the heating was stopped and 300 g of harvested leaves were added to the stockpot. The leaves were secured in a position so they were not submerged in water. The lid of the stockpot was then closed, the boiler was restarted, and distillation was continued for 2 hours. A tap was connected to the condenser, and tap water was passed through it to act as a chiller. The steam-distilled water obtained after 100 minutes of distillation was collected and 20% (w / w) sodium chloride was added. 1 L of ethyl acetate was added to the steam-distilled water after the treatment, and the mixture was subjected to liquid-liquid transfer using a separatory funnel. The ethyl acetate layer was collected and concentrated using a rotary evaporator to obtain a dry solid. The amount of essential oil obtained was approximately 670 mg / kg for joxs1joxt2-1 and approximately 380 mg / kg for Tsukuba 1. This indicates that the amount of essential oil increased approximately two-fold in the JOX mutants, even in dry leaves.

[0140] Furthermore, we attempted to obtain leaf surface resin components using the following method, which is different from steam distillation. For JOXS1JOXT2-1 and Tsukuba No. 1, 20 g of dried yellow leaves were immersed in 400 ml of ethanol or hexane for 5 minutes at room temperature. The solution was filtered and then evaporated to dryness. The leaf surface resin components obtained using ethanol solvent were approximately 576 mg for JOXS1JOXT2-1 and 367 mg for Tsukuba No. 1; using hexane solvent, the leaf surface resin components were approximately 180 mg for JOXS1JOXT2-1 and 158 mg for Tsukuba No. 1. These results confirm that leaf surface resin components are increased in JOXS1JOXT2-1.

[0141] Evaluation Example 1-3: Analysis of tobacco JOX mutant components α-cembratrienediol (hereinafter also referred to as α-CBT), a representative component of leaf surface resin, was analyzed by GC-MS. The JOX mutant joxs1joxt2-1 line obtained in Example 1, which had been cultivated in a field by conventional methods, and Tsukuba No. 1 were used as controls. Field cultivation was carried out twice, in the first and second years.

[0142] The middle and upper leaves were harvested at the appropriate time and dried to turn yellow. Approximately four dried yellow leaves per plant were collected from three plants, for a total of approximately 12 leaves per replicate. For joxs1joxt2-1, three replicates were used for field-grown samples in the first year, and five replicates were used for field-grown samples in the second year. For Tsukuba 1, three replicates were used for each year. The deboned laminae were crushed and analyzed for α-CBT.

[0143] In the first-year field-grown samples, joxs1joxt2-1 showed a significant increase (at the 1% level by t-test) compared to the control, Tsukuba No. 1 (Fig. 2). It also showed higher values ​​than American yellow dry leaf (FCV / US) and Brazilian yellow dry leaf (FCV / BR), which were also analyzed in the same way.

[0144] Additionally, for second-year field-grown samples, the effects of drying method and harvest time on the amount of leaf resin components were examined. For joxs1joxt2-1, two drying methods were used: one sample was yellow-dried, and the other was air-dried. Furthermore, for joxs1joxt2-1, mid- to upper-middle leaves were used, and two harvest times were used: immature leaves harvested a few days after cutting, and ripe leaves harvested at the appropriate time. In other words, for joxs1joxt2-1, two drying methods and two harvest times were combined, for a total of three levels. The three levels of joxs1joxt2-1 tested are as follows:・joxs1joxt2-1_yellow-dried_ripe: A sample of ripe leaves of joxs1joxt2-1, dried to a yellow color. ・joxs1joxt2-1_air-dried_immature: A sample of immature leaves of joxs1joxt2-1, dried to an air color. ・joxs1joxt2-1_air-dried_ripe: A sample of ripe leaves of joxs1joxt2-1, dried to an air color.

[0145] No replicates were used for each level. After deboning, the lamina was crushed and analyzed for α-CBT. Compared with the control leaves of Tsukuba 1 and the yellow-dried leaves of joxs1joxt2-1, the air-dried leaves of jox1sjox2t-1 showed a significant increase in α-CBT in both immature and mature leaves. Compared with the yellow-dried leaves of Tsukuba 1, the air-dried leaves of joxs1joxt2-1 showed approximately 7-fold higher α-CBT in immature leaves and approximately 4-fold higher α-CBT in mature leaves (Figure 3).

[0146] [Evaluation Example 1-4] Sensory evaluation of tobacco JOX mutants Sensory evaluation was carried out using yellow dried leaves of joxs1joxt2-1 and Tsukuba 1, which were produced in field cultivation in the first year.

[0147] A finely pulverized sample was prepared from yellow dried leaves and spread on an unflavored cigarette. Specifically, the yellow dried leaves of JOXS1JOXT2-1 were pulverized. Propylene glycol was added to the resulting pulverized sample in an amount three times the weight of the pulverized sample, and mixed to prepare a finely powdered sample. The finely powdered sample was spread at a concentration of 3% by weight on 0.7 g of shredded tobacco from an unflavored cigarette, to prepare a tobacco product sample (i.e., a cigarette spread with JOXS1JOXT2-1).

[0148] Similarly, yellow dried leaves of Tsukuba No. 1 were finely pulverized. Propylene glycol was added to the resulting finely pulverized sample in an amount three times the weight of the finely pulverized sample, and mixed to prepare a finely powdered sample. The finely powdered sample was spread at a concentration of 3% by weight on 0.7 g of unflavored cigarette shredded tobacco to prepare a tobacco product sample (i.e., a cigarette with Tsukuba No. 1 spread on it).

[0149] Five trained panelists (AE) evaluated the degree of favorable change in smoking taste of cigarettes after spreading various samples on a three-point scale (0: not good, 1: good, 2: very good) compared to unflavored cigarettes without any samples spread on them, and also described the smoking taste characteristics.

[0150] As a result, all panelists rated cigarettes coated with Tsukuba No. 1 (i.e., a wild-type strain without a functionally suppressive mutation in the JOX gene) as "0." On the other hand, for cigarettes coated with joxs1joxt2-1, three panelists rated them as "1" and two as "2," indicating that cigarettes coated with joxs1joxt2-1 had a pleasant aroma and flavor. Furthermore, four out of five panelists described the smoking taste of cigarettes coated with joxs1joxt2-1 as being "cool" or "refreshing."

[0151] Finely ground samples were prepared from "yellow dried Tsukuba No. 1 leaves," "dried leaves obtained by yellowing mature leaves of joxs1joxt2-1," "dried leaves obtained by air-drying immature jox1sjox2t-1 leaves," and "dried leaves obtained by air-drying mature jox1sjox2t-1 leaves," all of which were cultivated in the second year of field cultivation. These samples were then spread on unflavored cigarettes. Seven trained panelists (AG) subjectively evaluated the intensity of "cooling sensation" on a 6-point scale (0: no cooling sensation, 1-5: higher numbers indicate stronger cooling sensation). The results showed that none of the panelists perceived a "cooling sensation" or "cooling sensation" in cigarettes spread with Tsukuba No. 1, whereas they perceived a "cooling sensation" or "cooling sensation" in cigarettes spread with any of the JOX mutant samples. Furthermore, the "cooling sensation" or "cool feeling" in cigarettes with the JOX mutant spread on them was stronger in air-dried leaves than in yellow-dried leaves, and stronger in immature leaves than in ripe leaves (Table 2).

[0152]

[0153] Furthermore, the leaf surface resin components of the dried leaves extracted with ethanol and hexane in Evaluation Example 1-2 were spread on unflavored cigarettes. Regardless of the leaf surface resin components of the dried leaves extracted with either solvent, none of the five evaluation panel members recognized a "refreshing" or "cooling sensation" for Tsukuba No. 1, but a "refreshing" or "cooling sensation" for the joxs1joxt2-1 mutant.

[0154] Evaluation Example 1-5: Cold Receptor TRPM8 Assay in Tobacco JOX Mutants Transient receptor potential cation channel melastatin 8 (TRPM8) is one of the TRP channels confirmed to be strongly expressed in human sensory neurons and is known to be involved in the perception of cold. TRPM8 is activated by temperature stimulation below 28°C or by the administration of cold-sensing substances such as menthol or icillin. Activated TRPM8 induces an influx of cations into cells, depolarizing the neurons and thereby transmitting cold-sensing signals. Taking advantage of this property, TRPM8 is also being used to identify novel cold-sensing substances. The following procedure was used to investigate whether components contained in JOX mutants activate the human cold receptor (TRPM8).

[0155] First, we constructed stable TRPM8-expressing cells. Specifically, a TRPM8 expression vector was constructed by inserting a human TRPM8 expression gene (NCBI Reference Sequence: NM_024080.5) into the multiple cloning site of the pF5A CMV-neo Flexi vector (Promega). The TRPM8 expression vector was then transfected into the human embryonic stem cell line HEK293T (ECACC) using Lipofectamine 2000 (Thermo Fisher Scientific). The resulting transformed cells were cultured at 37°C under 5% CO2 for 3 days, then the medium was replaced with selective medium containing 200 μg / mL Geneticin (Thermo Fisher Scientific) and further cultured for 10 days to obtain stable TRPM8-expressing cells.

[0156] The TRPM8-stably expressing cells were seeded at 30,000 cells / well in a 96-well black plate (Corning). After 24 hours of incubation at 37°C under 5% CO2, the calcium-sensitive dye Fluo-8 AM (AAT Bioquest) was transfected into the TRPM8-stably expressing cells. The change in fluorescence intensity at Ex / Em = 480 nm / 520 nm was then measured using an FDSS / μCELL (Hamamatsu Photonics). The test substance was appropriately diluted with HBSS (Thermo Fisher Scientific) and added to the cell supernatant 30 seconds after the start of measurement. The maximum fluorescence intensity after test substance addition was used to indicate the activity of the TRPM8-expressing cells.

[0157] (Assay of components in cigarette smoke containing a finely ground JOX mutant sample) Test samples included unflavored cigarettes (control cigarettes) and control cigarettes coated with a finely ground sample of dried yellow leaves of the JOX mutant JOX variant (joxs1joxt2-1) cultivated in the field for the first year (Evaluation Example 1-3) (JOX cigarettes). The cigarettes were burned under ISO 3308:2012 conditions using a linear smoking device (Cerulean SM410), and the combustion smoke of 10 cigarettes each was collected using a Cambridge filter. The Cambridge filter was added with 10 mL of ethanol, shaken for 30 minutes, and filtered through a 0.45 μm PVDF filter. The filtered ethanol eluate was diluted with HBSS solution to a final concentration of 0.3%.

[0158] The TRPM8 activity of each sample was calculated as follows: First, the maximum fluorescence intensity of each TRPM8(+) and TRPM8(-) sample was normalized by dividing it by the maximum fluorescence intensity of the negative control (a solution without captured smoke). The normalized TRPM8(+) value was then divided by the normalized TRPM8(-) value, and the resulting value, minus 1 (TRPM8(+) / TRPM8(-)-1), was used as the TRPM8 activity intensity.

[0159] The assay was performed twice, with each test performed according to the above procedure counting as one repetition. The results of the first repetition (Test #1) are shown in Figure 4, and the results of the second repetition (Test #2) are shown in Figure 5. In Figures 4 and 5, the control cigarettes are labeled "Control," and the JOX cigarettes are labeled "Cig-JOX." In Test #1, the TRPM8 activity of the JOX cigarettes was 2.01-fold higher than that of the control cigarettes. In Test #2, the TRPM8 activity of the JOX cigarettes relative to the control cigarettes was greater than that of Test #1.

[0160] (Assay of JOX mutant leaf components) Test samples included the second-year field-grown samples from Evaluation Example 1-3: joxs1joxt2-1_yellow-dried_ripe, joxs1joxt2-1_air-dried_immature, and Tsukuba No. 1 as a control. For each sample, the deboned lamina was crushed to prepare a crushed sample. 1 g of the crushed sample was transferred to a 15 mL centrifuge tube and 10 mL of hexane was added. After shaking at room temperature for 1 hour, the tube was centrifuged and the supernatant was transferred to a recovery flask. The supernatant was evaporated (minimum pressure 200 mbar, water bath 35°C) to obtain a dry product. 10 mg of the dry product was dissolved in 200 μL of DMSO and diluted with HBSS solution to a final concentration of 50 μg / mL. TRPM8 activity was calculated as in the smoke component assay.

[0161] The assay was performed in duplicate, with each test performed according to the above procedure counting as one replicate. The results of the first replicate (Test #1) are shown in Figure 6, and the results of the second replicate (Test #2) are shown in Figure 7. In both Test #1 and Test #2, the joxs1joxt2-1_yellow-dried-ripe sample and the joxs1joxt2-1_air-dried-immature sample exhibited higher TRPM8 activity than the control, Tsukuba No. 1.

[0162] [Evaluation Example 1-6] Search for JOX genes in Nicotiana plants To search for JOX genes in Nicotiana plants, blastn (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)) was performed on NCBI. The CDS sequences of NtJOX-S1, Nt-JOX-S2, and NtJOX-T2 were used as query sequences. The Nucleotide collection (nr / nt) database was used, and Nicotiana (taxid: 4085) was selected as the target organism.

[0163] The nucleotide sequences of the putative JOX genes obtained as a result of blastn are listed in Table 3 (query: NtJOX-S1), Table 4 (query: NtJOX-S2), and Table 5 (query: NtJOX-T2).

[0164] Tables 3 to 5 also show the predicted amino acid sequences encoded by the nucleotide sequences of each gene, as well as the accession of the predicted amino acid sequences, the sequence identity (Per. Identity) to each query sequence (predicted amino acid sequence), the expected value (E value), and the query sequence coverage (Query Cover) (calculated using genetic information processing software GENETYX (Japan Server Co., Ltd.)).

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[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Phylogenetic analysis using amino acid sequences confirmed that the sequences shown in Tables 3 to 5 are JOX genes.

[0172] The sequences used in the phylogenetic analysis are as follows: ・The deduced amino acid sequences of NtJOX-S1, NtJOX-S2, and NtJOX-T2 ・The deduced amino acid sequences of N. tabacum, N. sylvestris, N. tomentosiformis, and N. attenuata, which are included in Tables 3 to 5 ・The deduced amino acid sequences of AtJOX1, 2, 3, and 4 in Arabidopsis thaliana (Caarls et al., 2017; Smirnova et al., 2017)

[0173] The Arabidopsis AT1G05010.1 sequence was used as an outgroup. The AT1G05010.1 sequence belongs to the same 2-oxoglutarate (2OG) Fe(II)-dependent oxygenase family as AtJOX1,2,3,4, but is classified into a different clade from JOX (Caarls et al., 2017).

[0174] The phylogenetic tree is shown in Figure 8. The sequences used in the phylogenetic analysis were aligned using MAFFT (a multiple sequence alignment program (cbrc.jp), Ver. 7, Katoh et al., 2019). The phylogenetic tree was also constructed using Molecular Evolutionary Genetics Analysis (MEGA) (Ver. 11.0.13, https: / / www.megasoftware.net / , Kumar et al., 2018) with maximum likelihood (Bootstrap: 1000). The deduced amino acid sequence of N. attenuata is shown to correspond to NaJOX-like-1, 2, 3, and 4 in Tang et al., 2020.

[0175] Phylogenetic analysis showed that the nucleotide and amino acid sequences of the genes of N. tabacum, N. sylvestris, N. tomentosiformis, and N. attenuata included in Tables 3 to 5 were predicted to be JOX genes and the amino acid sequences encoded thereby.

[0176] From the above, it was found that the JOX genes in the genus Nicotiana share a sequence identity (Per. Identity) of 62% or more in the amino acid sequence with NtJOX-S1, NtJOX-S2, and NtJOX-T2.

[0177] Example 2: Creation of a JOX triple mutant The JOX mutant joxs1joxt2-1 line and the JOX mutant joxs2joxt2-1 line obtained in Example 1 were crossed to obtain an F1 generation. The F1 generation was then cultivated and self-fertilized to obtain an F2 generation. Individuals of the F2 generation that were homozygous for the mutation were identified by Sanger sequencing. Specifically, DNA was extracted from each individual of the F2 generation, and the primers shown in Table 1 were used to generate the Tks Gflex TM The sequence surrounding the mutation was amplified by PCR using DNA Polymerase (Takara Bio Inc.), and the sequence of the amplified product was determined by Sanger sequencing using the primers used in PCR.

[0178] As a result, we identified an individual homozygous for mutations in all three genes, NtJOX-S1, NtJOX-S2, and NtJOX-T2 (joxs1joxs2joxt2, hereafter referred to as the JOX triple mutant). The JOX triple mutant individuals were selfed to obtain the F3 generation. The F3 generation of the JOX triple mutant and Tsukuba 1, a control, were cultivated in a conventional field to obtain field-grown samples.

[0179] Evaluation Example 2-1: Analysis of Components of the Tobacco JOX Triple Mutant Solavetivone is a phytoalexin (antibacterial substance) found in tobacco. It is known to increase in Nicotiana plants in response to environmental stresses, such as pests and diseases. Therefore, the solavetivone content in JOX mutant plants can be used as an evaluation index for the enhancement of jasmonic acid responses due to mutations that suppress JOX gene function. Solavetivone itself is also known as a flavor component in Nicotiana plants and their dried tobacco materials. Therefore, tobacco materials containing solavetivone are useful for improving the flavor and taste of tobacco products. Therefore, we analyzed solavetivone and α-CBT in the JOX triple mutant obtained in Example 2. α-CBT is the same component analyzed in Evaluation Example 1-3.

[0180] Dried leaf samples were prepared for the field-grown samples of Tsukuba No. 1 and JOXS1 JOXS2 JOXT2 lines obtained in Example 2, with reference to the dried leaf sample preparation method in Evaluation Examples 1-3. As with Evaluation Examples 1-3, several levels were set for the preparation of dried leaf samples depending on the drying method and harvesting period. Two drying methods were set: samples that underwent yellow drying, and samples that underwent air drying. Two harvesting periods were set: immature leaves from the middle and upper stems harvested on the third day after stemming, and ripe leaves from the middle and upper stems harvested at the appropriate time. These levels were combined, and a total of two levels were subjected to analysis. The two levels tested were as follows: - Yellow-dried samples of ripe leaves from JOXS1 JOXS2 JOXT2 and the control Tsukuba No. 1 - Air-dried samples of immature leaves from JOXS1 JOXS2 JOXT2 and the control Tsukuba No.

[0181] No replication was performed for each level. The deboned lamina was crushed and analyzed for solavetivone and α-CBT by GC-MS in the same manner as in Evaluation Example 1-3.

[0182] The results of GC-MS analysis are shown in Figures 9–12. In Figures 9–12, joxs1joxs2joxt2 are abbreviated as "joxs1s2t2." When immature leaves were air-dried, the α-CBT content in joxs1joxs2joxt2 was 4.1 times higher than that in the control, Tsukuba 1 (Figure 9). When mature leaves were yellow-dried, the α-CBT content in joxs1joxs2joxt2 was 2.0 times higher than that in the control, Tsukuba 1 (Figure 10). Furthermore, while solavetivone was below the detection limit in the control, Tsukuba 1, at all levels, solavetivone was detected in joxs1joxs2joxt2, demonstrating increased solavetivone content in joxs1joxs2joxt2 (Figures 11 and 12). These findings suggest that the JOX triple mutant not only enhances the jasmonic acid response but also increases the content of flavor components that may contribute to improved aroma and flavor.

[0183] [Evaluation Example 2-2] Sensory evaluation of the JOX triple mutant As in Evaluation Example 2-1, immature leaves of joxs1joxs2joxt2 were air-dried to prepare dried leaf samples. Sensory evaluation was carried out using these dried leaf samples.

[0184] First, dried leaf samples of JOXS1, JOXS2, and JOXT2 were finely pulverized. Propylene glycol was added to the resulting finely pulverized sample in an amount three times the weight of the finely pulverized sample, and mixed to prepare a finely powdered sample (hereinafter also referred to as a JOX tobacco material sample). The finely powdered sample was then spread at a rate of 3% by weight onto 0.7 g of unflavored cigarette shredded tobacco to prepare a tobacco product sample (hereinafter also referred to as a JOX spread sample).

[0185] As a control, an unflavored cigarette (hereinafter also referred to as an unspread sample) was used, in which the JOX tobacco material sample was not spread.

[0186] Thirty-four trained panelists compared the "strength of the cooling or refreshing sensation" of the JOX-spread samples with that of the unspread samples on a five-point scale (0: no cooling sensation, 1-4: the stronger the cooling sensation, the higher the score), and also described the smoking characteristics.

[0187] For the JOX-spread sample, 3 panelists gave it a score of "0," 15 gave it a score of "1," 8 gave it a score of "2," 6 gave it a score of "3," and 2 gave it a score of "4." This indicates that cigarettes with joxs1joxs2joxt2 spread on them have a "refreshing" or "cool" sensation. The average score for "strength of the refreshing / cooling sensation" was 1.7 points (Table 6). Furthermore, 18 out of 34 panelists described the smoking taste of the JOX-spread sample as being "refreshing" or "cool."

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[0189] [Example 3] Example using JOX mutants [Evaluation Example 3] Sensory evaluation of JOX mutants at different chopped blend ratios From the above-mentioned evaluation examples 1-4, the ``immature leaf / air-dried leaf sample of the joxs1joxt2-1 line'' was selected as a sample that expressed a ``refreshing sensation'' or ``cool sensation'' and used for sensory evaluation.

[0190] First, the above-mentioned dried leaf sample was shredded to prepare a tobacco material in the form of cut filler (hereinafter also referred to as "JOX tobacco shreds").

[0191] Standard shredded tobacco and JOX shredded tobacco were mixed to prepare five levels of blended materials using the following procedure: Before preparing the blended materials, the JOX shredded tobacco and standard shredded tobacco were left to stand in open conditions at 60% RH and 25°C for 24 hours to condition the moisture content to 10%.

[0192] Level 1 (hereinafter also referred to as "0% blend") used 20 g of standard shredded tobacco. The blend ratio of JOX shredded tobacco in Level 1 was 0 wt% on a wet weight basis. Level 2 (hereinafter also referred to as "5% blend") was prepared by mixing 1 g of JOX shredded tobacco with 19 g of standard shredded tobacco to create a blended material. The blend ratio of JOX shredded tobacco in Level 2 was 5 wt% on a wet weight basis. Level 3 (hereinafter also referred to as "10% blend") was prepared by mixing 2 g of JOX shredded tobacco with 18 g of standard shredded tobacco to create a blended material. The blend ratio of JOX shredded tobacco in Level 3 was 10 wt% on a wet weight basis. Level 4 (hereinafter also referred to as "15% blend") was prepared by mixing 3 g of JOX shredded tobacco with 17 g of standard shredded tobacco to create a blended material. The blend ratio of JOX shredded tobacco in Level 4 was 15% by weight on a wet weight basis. For Level 5 (hereinafter also referred to as "20% blend"), 4g of JOX shredded tobacco was mixed with 16g of standard shredded tobacco to create a blend material. The blend ratio of JOX shredded tobacco in Level 5 was 20% by weight on a wet weight basis.

[0193] 0.7 g of each mixed material was dispensed and filled into the tobacco segment of a combustible smoking article comprising a tobacco segment and a filter segment to prepare five levels of cigarette samples.

[0194] Five trained panelists rated the "strength of the refreshing / cooling sensation" for each cigarette level on a six-point scale (0 point: no refreshing sensation, 1 point to 5 point: if a refreshing sensation was felt, the stronger the score, the higher the score).

[0195] As a result, the average scores for the "intensity of cool and refreshing sensation" evaluated by five panelists for each cigarette level were 0 (0% blend), 0.6 (5% blend), 1.3 (10% blend), 1.9 (15% blend), and 1.5 (20% blend) (Table 7). This suggests that tobacco products using dried leaves of the JOX mutant joxs1joxt2-1 line as cut filler tobacco material also exhibit a "cool and refreshing sensation."

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[0197] The present invention can be used in tobacco products.

Claims

1. A tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the genome of at least one of the following endogenous genes in its genome that specifically causes functional inhibition: (a) an endogenous gene comprising, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:3; (b) an endogenous gene comprising, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6; and (c) an endogenous gene comprising, as its coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:9; and the tobacco material has cold receptor TRPM8 activity.

2. The tobacco material according to claim 1, comprising a TRPM8 agonist.

3. The tobacco material according to claim 1 or 2, wherein the cold receptor TRPM8 activity of the tobacco material is at least twice as high as the cold receptor TRPM8 activity of tobacco material derived from a wild-type Nicotiana plant.

4. The tobacco material according to any one of claims 1 to 3, which is a dry material.

5. The tobacco material according to any one of claims 1 to 3, which is a powder of dry material.

6. A tobacco product comprising the tobacco material according to any one of claims 1 to 5.

7. A Nicotiana plant in which a mutation that specifically causes functional inhibition has been introduced into at least one of the following endogenous genes in the genome: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

9.

8. The Nicotiana plant according to claim 7, wherein the Nicotiana plant is any one of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana rustica.

9. A Nicotiana plant according to claim 7 or 8, wherein the mutation that specifically causes the functional suppression has been introduced by mutagen treatment, genome editing, or gene knockout.

10. A Nicotiana plant according to any one of claims 7 to 9, wherein the mutation that specifically causes the functional suppression specifically causes an enhanced response to jasmonic acid.

11. A method for imparting a cooling sensation to a tobacco product, wherein the tobacco product comprises tobacco material derived from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into the endogenous gene in its genome that specifically causes functional inhibition of at least one of the following: (a) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3; (b) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and (c) an endogenous gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 60% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the tobacco material is substantially free of cooling flavorings; and the tobacco material has cooling sensation receptor TRPM8 activity.

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