Dried material of plant of genus nicotiana, tobacco product, plant of family solanaceae, and production method therefor

Abstract

Provided is a tobacco product having improved fragrance inhaling taste. A dried material of a plant of the genus Nicotiana according to the present disclosure is a dried material of a plant of the genus Nicotiana into which a mutation causing the suppression of the function of an endogenous caffeic acid-O-methyltransferase gene has been introduced. This dried material has a coniferin content higher than that of a dried material of a wild-type plant of the genus Nicotiana.

Description

Dried material of Nicotiana plant, tobacco product, Solanaceae plant and method for producing the same

[0001] The present invention relates to a dried material of a Nicotiana plant, a tobacco product, a Solanaceae plant, and a method for producing the same.

[0002] The flavor and aroma of tobacco products is a complex quality that appeals to human sensory organs and is thought to be based on the balance of various components contained in the raw material, tobacco leaves. Numerous flavor-contributing components have been investigated to improve the flavor and aroma of tobacco. For example, Patent Document 1 describes that the flavor and aroma were improved by adding coniferin to shredded tobacco.

[0003] Japanese Patent No. 3001531

[0004] Coniferin is one of the metabolites produced by the phenylpropanoid metabolic pathway, but there are no known examples of increasing coniferin by modifying the functions of various genes involved in the phenylpropanoid metabolic pathway. Mutants that increase the coniferin content in tobacco leaves and cured leaves of Nicotiana plants without adding coniferin have not been developed.

[0005] An object of one aspect of the present invention is to provide a tobacco product with an improved aroma and flavor.

[0006] In order to solve the above-mentioned problems, the following means are provided: One aspect of the present invention provides a dried material of a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into at least one of the following: (a) an endogenous caffeic acid-O-methyltransferase gene, the coding region of which is a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene, the coding region of which is a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, such that the mutation causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene; and the dried material has a higher coniferin content than dried material of a wild-type Nicotiana plant.

[0007] According to another aspect of the present invention, there is provided a dried material of a Nicotiana plant according to the above aspect, wherein the dried material is dried tobacco leaves harvested from the Nicotiana plant.

[0008] According to yet another aspect of the present invention, there is provided a dried material of a Nicotiana plant according to any of the above aspects, wherein the coniferin content is at least twice the coniferin content of cured tobacco leaves harvested from the same amount of the wild-type Nicotiana plant.

[0009] According to yet another aspect of the present invention, there is provided a dried material of a Nicotiana plant according to any of the above aspects, the plant belonging to Nicotiana tabacum or Nicotiana rustica.

[0010] According to yet another aspect of the present invention, there is provided a tobacco product comprising a dry material according to any of the above aspects.

[0011] According to yet another aspect of the present invention, there is provided a Solanaceae plant having a mutation introduced into its genome that causes functional suppression of an endogenous caffeic acid-O-methyltransferase gene, the Solanaceae plant having resistance to disease caused by Ralstonia solanacearum.

[0012] According to yet another aspect of the present invention, there is provided the Solanaceae plant according to the above aspect, wherein the Solanaceae plant is a Nicotiana plant or a tomato plant.

[0013] According to yet another aspect of the present invention, there is provided a Solanaceae plant according to the above aspect, wherein the Nicotiana plant belongs to Nicotiana tabacum or Nicotiana rustica.

[0014] According to yet another aspect of the present invention, there is provided a Solanaceae plant according to any of the above aspects, wherein the endogenous caffeic acid-O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0015] According to yet another aspect of the present invention, there is provided a method for producing a Solanaceae plant that is resistant to disease caused by Ralstonia solanacearum, the method comprising the step of introducing into the Solanaceae plant a mutation that specifically causes functional suppression of an endogenous caffeic acid-O-methyltransferase gene.

[0016] According to one aspect of the present invention, a tobacco product with improved flavor and taste can be realized.

[0017] Fig. 1 shows the results of a disease index survey of tobacco COMT mutants in a Ralstonia solanaceum assay. Fig. 2 shows the results of a disease index survey of tobacco COMT mutants in a Ralstonia solanaceum assay. Fig. 3 shows the results of a disease index survey of tobacco COMT mutants in a Ralstonia solanaceum assay.

[0018] The present invention will be described in detail below. However, the following description is for the purpose of explaining the present invention and is not intended to limit the present invention. The embodiments described below are more specific embodiments of any of the above aspects. The following items can be incorporated into each of the above aspects alone or in combination.

[0019] [Definition] "Ralstonia solanacearum" is a pathogenic fungus that causes soil-borne plant diseases of Solanaceae plants. For example, plant diseases caused by Ralstonia solanacearum include bacterial wilt of Solanaceae plants and tobacco bacterial wilt (Granville wilt) of Nicotiana plants.

[0020] "Disease resistance" refers to the property of a crop that makes it less susceptible to disease and less susceptible to damage from the disease. A resistant variety refers to a plant variety that has the above property. In this specification, "disease resistance" is interpreted to include disease resistance, and any of the following properties may be interpreted as resistance: not being infected with the target disease and not showing symptoms, not showing symptoms even after infection, slow onset of symptoms even after infection and onset, or only mild symptoms even after infection and onset. In this specification, when simply referring to "disease resistance," it is intended to mean resistance to plant diseases caused by Ralstonia solanacearum.

[0021] "Solanaceae plants" are a family of plants in the order Solanales, and Nicotiana plants belong to the Solanaceae family. In addition to recreational crops such as Nicotiana, other species include the Capsicum genus, which is often used as a spice or food crop; the Solanum genus, which includes the tomato (Solanum lycopersicum); and the Petunia and Physalis genus, which are often used as ornamental plants. These species contain many useful plants with high commercial potential. As used herein, the term "plant" encompasses the entire plant (e.g., adult, seedling, and seed) and plant tissues (e.g., leaves, stems, flowers, roots, reproductive organs, embryos, and parts thereof). The definition herein also includes the current generation into which a mutation has been introduced and the progeny obtained by crossbreeding such plants.

[0022] "Tobacco" refers to plants of the genus Nicotiana, which mostly belong to the Solanaceae family, and the main commercially cultivated species are Nicotiana tabacum and Nicotiana rustica. In this specification, "nicotinoid plants" simply refers to plants of the genus Nicotiana, which belong to the Solanaceae family, and a typical example is Nicotiana tabacum.

[0023] "Caffeate 3-O-methyltransferase (COMT)" is an enzyme responsible for the next conversion in the phenylpropanoid metabolic pathway (Maury et al. (1999) Plant Physiology, 121: 215-223, Hemm et al. (2004) Plant J 38: 765-778, Chun et al. (2019) Plant Signaling & Behavior, 14:8).

[0024] (i) Conversion of caffeate to ferrate (ii) Conversion of caffeoyl aldehyde to coniferaldehyde (iii) Conversion of 5-hydroxyferuloyl CoA to sinapoyl CoA (iv) Conversion of 5-hydroxyferulic acid to sinapic acid (v) Conversion of 5-hydroxyconiferaldehyde to sinapaldehyde (vi) Conversion of 5-hydroxyconiferyl alcohol to sinapyl alcohol The caffeate-O-methyltransferase gene of a Solanaceae plant is not particularly limited as long as it is an enzyme capable of carrying out any of the conversions (i) to (vi). In particular, in Nicotiana plants, the COMT1 gene is intended as the COMT gene.

[0025] After extensive research, the inventors discovered that COMT gene mutants in Nicotiana tabacum plants increase coniferin content in tobacco and dried leaves. While various genes involved in the phenylpropanoid metabolic pathway have been reported (e.g., Hemm et al. (2004) Plant J 38: 765-778; Chun et al. (2019) Plant Signaling & Behavior, 14:8), no studies have demonstrated that altering the function of genes in this pathway to increase coniferin content. In Arabidopsis thaliana, mutants of BGLU45 and BGLU46, which are glucosidases specifically expressed in stems and degrade coniferin, have been shown to increase coniferin content in stems and roots (Chapelle et al. 2012 Plant Physiology 160: 1204-1217). However, coniferin content in leaves has not been reported. Furthermore, coniferin is not produced through the conversion of (i) to (vi) in the phenylpropanoid metabolic pathway, in which COMT is directly involved. As a result of intensive research by the present inventors, it was found that suppression of the function of the COMT gene increases the coniferin content in tobacco leaves and dried leaves compared to the wild type.

[0026] Furthermore, as a result of extensive research, the inventors discovered that a COMT gene mutant in a Solanaceae plant confers resistance to Ralstonia solanacearum. In particular, they discovered that a mutant of the COMT1 gene (hereinafter simply referred to as a "COMT mutant"), a COMT gene in Nicotiana plants, confers resistance to Ralstonia solanacearum through the suppression of a single gene function. COMT mutants in Nicotiana plants have not been reported to date. Furthermore, Nicotiana plants with resistance to Ralstonia solanacearum have been achieved through the mutation of multiple microgenes, and resistance to Ralstonia solanacearum through the suppression of a single gene function has not been reported to date. While disease resistance has been investigated in plants with suppressed COMT gene function (reviewed in Miedes et al. (2014) Frontiers in Plant Science 5: 358), no examples of resistance to Ralstonia solanacearum have been reported.

[0027] [1. Dried Material] A dried material according to one embodiment of the present invention is a dried portion of a plant harvested from a Nicotiana plant into which a mutation causing functional suppression of the endogenous caffeic acid-O-methyltransferase gene has been introduced. Examples of plant portions include tobacco leaves, midribs separated from tobacco leaves, stem remains, and flowers. In particular, a dried material according to one embodiment of the present invention is cured tobacco leaves harvested from a Nicotiana plant into which a mutation causing functional suppression of the endogenous caffeic acid-O-methyltransferase gene has been introduced. The following description focuses on the case where the dried material is a cured leaf. The cured leaf has a higher coniferin content than cured leaves derived from wild-type Nicotiana plants. Details of the Nicotiana plant according to one embodiment of the present invention are described in detail in Section [2. Solanaceae Plants, Such as Nicotiana Plants]. The use of such cured leaves can improve the aroma and flavor of tobacco products.

[0028] The endogenous caffeic acid-O-methyltransferase gene is at least one of the following endogenous caffeic acid-O-methyltransferase genes (a) and (b): (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0029] The cured leaves according to one embodiment of the present invention may have a coniferin content that is at least twice that of cured tobacco leaves harvested from the same amount of a wild-type Nicotiana plant. Furthermore, the cured leaves according to this embodiment may have a coniferin content that is at least 2.1 times, or even 2.3 times, that of cured tobacco leaves harvested from the same amount of a wild-type Nicotiana plant. Hereinafter, the expressions "increased coniferin content" or "high coniferin content" refer to a high coniferin content compared to a wild-type Nicotiana plant. Furthermore, the term "coniferin content" simply refers to the coniferin content of the tobacco leaves or cured leaves. The coniferin content may be measured by any method. For example, it may be measured by conventionally known component analysis using GC-MS and LC-MS / MS.

[0030] In one embodiment of the present invention, the coniferin content per gram of dried leaves (hereinafter also referred to as μg / g, ppm, etc.) may be 25 μg or more, preferably 28 μg or more, and more preferably 30 μg or more. When the coniferin content in the dried leaves exceeds this lower limit, dried leaves with excellent flavor and taste can be obtained. Furthermore, the coniferin content per gram of dried material may be 100 μg or less, preferably 90 μg or less, and more preferably 80 μg or less, but is not limited thereto.

[0031] Cured leaves are obtained by drying tobacco leaves. Any drying method can be used, including, but not limited to, yellow drying, natural drying, hot air drying, and hot air drying.

[0032] (Tobacco Material) One aspect of the present invention provides a tobacco material obtained from the above-described dried leaves. The tobacco material may be obtained by processing the dried leaves in any manner. For example, the dried leaves may be processed by drying, aging, extraction, harmonizing, flavoring, high-temperature and high-pressure treatment, distillation, pulverization, and shredding. The obtained tobacco material may be in any form, including, for example, cut filler, powder, sheet, granules, and extract. These forms of the tobacco material are preferred from the viewpoint of applying the tobacco material to tobacco products.

[0033] One aspect of the present invention provides a tobacco product containing the above-described dried material or tobacco material. 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, but are not limited to, 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 portion separate from the tobacco portion that atomizes the aerosol source, heat the atomization portion, and inhale the tobacco flavor entrained by the generated aerosol as it passes through the tobacco portion; and unheated tobacco products that inhale the flavor without heating the tobacco portion.

[0034] The coniferin content of the above-mentioned leaf tobacco may be at least twice the coniferin content of leaf tobacco harvested from the same amount of a wild-type Nicotiana plant. Furthermore, the coniferin content of the leaf tobacco according to this embodiment may be at least 2.2 times, or at least 2.6 times, or even more than the coniferin content of dried leaf tobacco harvested from the same amount of a wild-type Nicotiana plant.

[0035] For details, see the above-mentioned items regarding Nicotiana plants and methods for producing the same.

[0036] 2. Solanaceae Plants, Such as Nicotiana Plants One aspect of the present invention provides a Nicotiana plant into which a mutation that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene has been introduced (hereinafter, sometimes abbreviated as a "high-coniferin Nicotiana plant"). The coniferin content of the leaves of the Nicotiana plant is higher than that of leaves harvested from the same amount of a wild-type Nicotiana plant. According to this configuration, by suppressing the function of a single gene, a plant can be obtained whose tobacco leaves and cured leaves have a high coniferin content.

[0037] Another aspect of the present invention is a Solanaceae plant that has resistance to disease caused by Ralstonia solanacearum and that has a mutation introduced into it that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene (hereinafter, sometimes abbreviated as a "disease-resistant Solanaceae plant"). According to this configuration, a plant that has acquired resistance to Ralstonia solanacearum can be achieved by suppressing the function of a single gene.

[0038] Hereinafter, the "high coniferine-containing Nicotiana plant" and the "disease-resistant Solanaceae plant" may be collectively referred to as "COMT gene mutant plant."

[0039] (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).

[0040] The mutation may be present 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 be present in the coding region of an endogenous caffeic acid-O-methyltransferase gene. When a mutation is present 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 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of a COMT polypeptide, and the polynucleotide encoding a polypeptide having COMT activity is included. 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.

[0041] Furthermore, a COMT gene mutant plant may have a mutation introduced outside the coding region of the endogenous caffeic acid-O-methyltransferase gene that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene. When the mutation is introduced outside the coding region of the endogenous caffeic acid-O-methyltransferase 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 caffeic acid-O-methyltransferase gene.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 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. Preferably, the amino acid substitution is a non-conservative substitution, which has a high potential to alter 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), as well as substitutions of amino acids with side chains with different bulk (steric size).

[0046] 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 COMT gene, nonsense-mediated mRNA decay can occur (Brogna and Wen (2009) Nat. Structural Mol. Biol. 16: 107-113). Because nonsense-mediated mRNA decay causes transcript degradation, nonsense mutations can result in a decrease in transcript abundance. For nonsense-mediated mRNA decay to occur, it is preferable that the COMT gene contains at least one exon containing a nonsense mutation. In particular, it is more preferable that the exon containing the nonsense mutation is not the most downstream (3') exon constituting the COMT gene. The COMT gene in wild-type Solanaceae plants consists of four exons and three introns. Therefore, a preferred embodiment of a nonsense mutation that causes nonsense-mediated mRNA decay is one in the first to third exons of the COMT gene.

[0047] 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.

[0048] The mutation may be introduced by mutagen treatment, genome editing or gene knockout. In particular, the mutation may be introduced by mutagen treatment.

[0049] The mutagen treatment of the gene can be carried out by artificially applying the mutagen to a Solanaceae plant (and, if necessary, in combination with suppression of gene repair function). Mutagens can be, for example, 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 Solanaceae plants. Mutagens can also be, for example, gamma rays, heavy ion beams, X-rays, neutron rays, or UV rays, but are not limited to these as long as they are radiation that causes mutations in the genomic DNA of Solanaceae 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.

[0050] Nucleotide substitutions in the COMT 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] As described above, the COMT gene mutant plant in which the mutation is artificially induced is described, but is not limited thereto. For example, in the COMT gene mutant plant, the mutation or disruption of the gene may be caused by spontaneous mutation. Spontaneous mutation of the gene is generally caused by replication errors and genetic damage. The cause of the damage may be exposure to known naturally occurring mutagens (e.g., radiation, ultraviolet rays, etc.).

[0056] The various mutations described above can be easily introduced into Solanaceae plants by those skilled in the art. That is, based on this sequence information, regions in the genomes of various Solanaceae plants encompassed by the concept of the present invention into which mutations should be introduced can be appropriately determined.

[0057] 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.

[0058] 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).

[0059] The vector used for transforming a Solanaceae plant for the purpose of suppressing gene expression or introducing a mutation into a gene 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.

[0060] 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.

[0061] 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, a Solanaceae plant that is an amphidiploid, 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.

[0062] (Function Suppression) In one embodiment of the present invention, a COMT gene mutant plant has suppressed function of an endogenous gene containing a polynucleotide consisting of the nucleotide sequence of the COMT gene as its coding region. 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 suppressing function" refers to suppressing only the function of the target gene without suppressing the functions of genes other than the target gene. 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 caffeic acid-O-methyltransferase gene, which could lead to metabolic abnormalities.

[0063] In a COMT gene mutant plant according to one embodiment 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 caffeic acid-O-methyltransferase gene compared to a wild-type plant.

[0064] "Decreased abundance" of a polypeptide refers to an abundance of the polypeptide that 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 values ​​so as to confer disease resistance in a Solanaceae plant.

[0065] Preferably, the decrease in the abundance of the polypeptide in a COMT gene mutant plant according to one embodiment of the present invention is genetically stably inherited in cultured cells, calli, protoplasts, seeds, and progeny obtained from the COMT gene mutant plant. Therefore, a COMT gene mutant 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.

[0066] 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)).

[0067] The functional inhibition may be a reduction in the abundance of mRNA transcribed from the endogenous caffeic acid-O-methyltransferase gene compared to a wild-type plant. The reduction in the amount of mRNA transcription occurs, for example, by suppressing transcription from the endogenous gene to mRNA. Transcription suppression can be achieved by, for example, inhibiting access of transcription initiation factors to the endogenous gene as a result of introducing a mutation into the endogenous gene.

[0068] 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 COMT gene mutant plant. That is, in the COMT gene mutant 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.

[0069] In the COMT gene mutant 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.

[0070] In one embodiment of the present invention, a COMT gene mutant plant has a 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 COMT gene and contains a polynucleotide as a coding region that encodes a polypeptide having COMT activity.

[0071] 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)).

[0072] (Nicotiana Plants) A plant with a mutant COMT gene according to one embodiment of the present invention may be a Nicotian plant. The Nicotiana plant is not particularly limited as long as it is a plant belonging to the genus Nicotiana, 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 (Nicotianakawakamii), Nicotiana knightiana, Nicotiana langsdorfi, Nicotiana linearis, Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp. hesperis Hesperis, Nicotiana 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 rustica (Mulberry tobacco), Nicotiana setchellii, Nicotiana simulanssimulans, Nicotiana solanifolia, Nicotiana spegauinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa, Nicotiana tomentosiformis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana berntina Examples of such plants include Nicotiana velutina, Nicotiana 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. A COMT gene mutant plant according to one embodiment of the present invention may particularly belong to Nicotiana tabacum or Nicotiana rustica.

[0073] Individuals resulting from the mutation or disruption of the above genes are referred to herein as mutants of Nicotiana plants (also simply referred to as mutants). 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") and a genome derived from Nicotiana tomentosiformis (also referred to as the "T genome"). 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 one type of mutation or multiple mutations in one 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.

[0074] The functions of the COMT genes in both the S genome and the T genome may be specifically suppressed, or the function of the COMT gene in either the S genome or the T genome may be specifically suppressed. Specific suppression of the function of either the S genome or the T genome refers to suppressing the function of only the COMT gene in one of the S genome and the T genome without suppressing the function of the COMT gene in the other genome. To specifically suppress the function of only one of the COMT genes in the S genome or the T genome, it is preferable to introduce a change in the nucleotide sequence into only that one COMT gene.

[0075] In one embodiment of the present invention, a COMT gene mutant plant has suppressed function of an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2. SEQ ID NO: 1 represents the amino acid sequence of endogenous caffeate-O-methyltransferase (also referred to herein as "NtCOMT-S") encoded by the S genome of Nicotiana tabacum, and SEQ ID NO: 2 represents the amino acid sequence of endogenous caffeate-O-methyltransferase (also referred to herein as "NtCOMT-T") encoded by the T genome of Nicotiana tabacum.

[0076] SEQ ID NO: 3 shows the CDS sequence of the NtCOMT-S gene (encoding the amino acid shown in SEQ ID NO: 1). SEQ ID NO: 5 is the genomic DNA sequence of the NtCOMT-S gene, which contains the nucleotide sequence shown in SEQ ID NO: 3 in its coding region. SEQ ID NO: 4 shows the CDS sequence of the NtCOMT-T gene (encoding the amino acid shown in SEQ ID NO: 2). SEQ ID NO: 6 is the genomic DNA sequence of the NtCOMT-T gene, which contains the nucleotide sequence shown in SEQ ID NO: 4 in its coding region.

[0077] An endogenous caffeate-O-methyltransferase gene in a Nicotiana plant with a high coniferin content according to one embodiment of the present invention may be (a) an endogenous caffeate-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, an endogenous caffeate-O-methyltransferase gene in a Nicotiana plant according to one embodiment of the present invention may be (b) an endogenous caffeate-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. This embodiment may satisfy one or more or all of the following conditions: The endogenous gene (a) contains a mutation that causes functional suppression of the endogenous gene (a); The endogenous gene (a) does not contain a mutation that causes functional suppression of the endogenous gene (b); and The endogenous gene (b) contains a mutation that causes functional suppression of the endogenous gene (b). The endogenous gene of (b) does not contain a mutation that causes functional suppression of the endogenous gene of (a).

[0078] In a preferred embodiment, the high-coniferine Nicotiana plant has suppressed function of an endogenous gene comprising, as a coding region, a polynucleotide that has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and encodes a polypeptide having COMT activity. When the high-coniferine Nicotiana plant has two endogenous genes, the coding region of one of the endogenous genes encodes a polypeptide having the sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0079] An endogenous caffeate-O-methyltransferase gene of a disease-resistant Solanaceae plant according to one embodiment of the present invention may be (a) an endogenous caffeate-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, an endogenous caffeate-O-methyltransferase gene of a Nicotiana plant according to one embodiment of the present invention may be (b) an endogenous caffeate-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. This embodiment may satisfy one or more or all of the following conditions: The endogenous gene (a) contains a mutation that causes functional suppression of the endogenous gene (a); The endogenous gene (a) does not contain a mutation that causes functional suppression of the endogenous gene (b); and The endogenous gene (b) contains a mutation that causes functional suppression of the endogenous gene (b). The endogenous gene of (b) does not contain a mutation that causes functional suppression of the endogenous gene of (a).

[0080] In a preferred embodiment, the disease-resistant Solanaceae plant has suppressed function of an endogenous gene comprising, as a coding region, a polynucleotide that has 90% or more (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and encodes a polypeptide having COMT activity. When the disease-resistant Solanaceae plant has two endogenous genes, the coding region of one of the endogenous genes encodes a polypeptide having the sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0081] As used herein, the term "sequence identity (of an amino acid sequence)" refers to the percentage of identity between a reference (amino acid) sequence and a reference (amino acid) sequence, where the non-identical portions of the sequences are those in which substitutions, additions, deletions, or insertions (of amino acid residues) exist.

[0082] Here, the phrase "a polypeptide having 80% or more (or 90% or more) sequence identity to the amino acid sequence shown in ..." used to identify a polypeptide using an amino acid sequence listed in the Sequence Listing may refer to a polypeptide that is normally present in a plant of the Solanaceae family. As used herein, the terms "polypeptide" and "protein" have substantially the same meaning and may be used interchangeably.

[0083] Therefore, the specific polypeptides whose abundance is reduced in disease-resistant Solanaceae plants according to one embodiment of the present invention may be polypeptides that have a sequence identity of 80% or more with the respective amino acid sequences shown in the sequence listing, and it is preferable that the sequence identity be a higher percentage (e.g., 90% or more).

[0084] Furthermore, the above-mentioned specific polypeptides whose abundance is reduced in Nicotiana plants with high coniferin content according to one embodiment of the present invention may be polypeptides having a sequence identity of 90% or more with the respective amino acid sequences shown in the sequence listing, and it is preferable that the sequence identity is a higher percentage (e.g., 95% or more).

[0085] A COMT gene mutant plant according to one embodiment of the present invention has an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1 or 2, and the function of an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having COMT activity is suppressed. 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.

[0086] In a COMT gene mutant plant according to one embodiment of the present invention, the function of an endogenous gene comprising a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 or 4 as a coding region is suppressed.

[0087] In one embodiment of the present invention, a COMT gene mutant 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 the nucleotide sequence set forth in SEQ ID NO: 3 or 4 and contains, as a coding region, a polynucleotide that encodes a polypeptide having COMT activity.

[0088] A Nicotiana plant having a high coniferin content in one embodiment of the present invention has a sequence identity of 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and has suppressed function of an endogenous gene that includes, as a coding region, a polynucleotide encoding a polypeptide having COMT activity.

[0089] A disease-resistant Solanaceae plant according to one embodiment of the present invention has a sequence identity of 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and has suppressed function of an endogenous gene that includes, as a coding region, a polynucleotide that encodes a polypeptide having COMT activity.

[0090] Polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 1 also encompass degenerate sequences of the nucleotide sequence shown in SEQ ID NO: 3 due to the degeneracy of the genetic code. Polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 2 also encompass degenerate sequences of the nucleotide sequence shown in SEQ ID NO: 4 due to the degeneracy of the genetic code.

[0091] The disease-resistant Solanaceae plant according to one embodiment of the present invention may further have an excellent flavor and taste when used in tobacco products. This may be achieved by increasing or decreasing metabolites due to functional inhibition of COMT activity. For example, the improved flavor and taste may be achieved by increasing coniferin in tobacco leaves. The increased coniferin content in tobacco leaves may be, for example, at least twice the coniferin content of tobacco leaves harvested from the same amount of wild-type Nicotiana plant.

[0092] (Tomato Plant) A disease-resistant Solanaceae plant according to one embodiment of the present invention may be a tomato plant (Solanum lycopersicum). A mutation that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene may be introduced into the tomato plant outside the coding region of the endogenous caffeic acid-O-methyltransferase gene. In this case, functional suppression may be a decrease in the abundance of mRNA transcribed from the endogenous caffeic acid-O-methyltransferase gene compared to a wild-type plant. More preferably, functional suppression may be promotion of degradation of mRNA transcribed from the endogenous gene.

[0093] The mutation may be the insertion of a polynucleotide that expresses an antisense RNA molecule, an RNAi molecule, or a co-suppressor molecule that promotes the degradation of mRNA transcribed from the endogenous caffeic acid-O-methyltransferase gene.

[0094] [3. Method for producing a COMT gene mutant plant] One aspect of the present invention provides a method for producing a Nicotiana plant with high coniferin content, comprising the step of introducing a mutation that specifically causes functional suppression of the endogenous COMT gene into the endogenous gene in the genome of the Nicotiana plant. Also provided is a method for producing a Solanaceae plant that is resistant to disease caused by Ralstonia solanacearum, comprising the step of introducing a mutation that specifically causes functional suppression of the endogenous COMT gene into the endogenous gene in the genome of the Solanaceae plant. Details of the mutation introduced into Solanaceae plants such as Nicotiana plants are described in Section [2. Solanaceae plants such as Nicotiana plants].

[0095] The introducing step may include introducing the mutation into the coding region of the endogenous caffeic acid-O-methyltransferase gene. Further, the introducing step may be performed by mutagen treatment, genome editing, or gene knockout. In particular, the introducing step may be mutagen treatment.

[0096] The introducing step may also include introducing a mutation that specifically suppresses the function of the endogenous caffeate-O-methyltransferase gene into the genome of the Solanaceae plant outside the coding region of the endogenous caffeate-O-methyltransferase gene. The suppression of function is a decrease in the abundance of mRNA transcribed from the endogenous caffeate-O-methyltransferase gene compared to a wild-type plant. More preferably, the suppression of function may be promotion of degradation of mRNA transcribed from the endogenous gene. Furthermore, the method may include inserting 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 caffeate-O-methyltransferase gene.

[0097] 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.

[0098] One example of a method for producing COMT gene mutant plants is to treat Solanaceae plants with a mutagen such as EMS, as described above, to generate mutations throughout the genome of the Solanaceae plants, thereby creating a mutant population (panel) of Solanaceae plants, and then extracting genomic DNA. Using gene-specific primers, nucleic acids encoding the COMT gene are amplified from each panel of genomic DNA or from a pool of these genomic DNAs, and the nucleotide sequence of the product is determined. Lines carrying homozygous mutations are then selected. The nucleotide sequence of the gene may be amplified by PCR. This allows for the identification of candidate lines that may suppress gene function on the COMT gene. The transcription level of the gene transcript may then be confirmed by reverse transcription PCR. This allows for the identification of mutants with reduced transcription levels of the normal COMT gene. In the case of Nicotiana plants, lines carrying homozygous mutations in the S genome and T genome are isolated, and these lines are then crossed to produce F1 hybrids. Furthermore, the self-fertilized progeny (F2) are cultivated, and from among them, a line containing homozygous mutations in both the S genome and the T genome is obtained (due to bifactorial recessiveness, this is obtained with a probability of 1 / 16).

[0099] Selection of individuals exhibiting a desired phenotype can be performed based on mutations in the nucleotide sequence and the transcription amount of the transcript as described above, but can also be performed based on coniferin content. For example, individuals whose coniferin content in tobacco leaves or cured leaves is increased compared to that of wild-type tobacco leaves or cured leaves can be selected. In addition to the selection criteria based on coniferin content, resistance to disease caused by Ralstonia solanaceum can also be used as a selection criterion.

[0100] Therefore, the method of one embodiment may further include one or more steps: creating a population (panel) of Solanaceae plants in which mutations have been introduced throughout the genome of the Solanaceae plant, extracting genomic DNA from lines included in the panel, determining the base sequence of the COMT gene in the genomic DNA, selecting lines containing homozygous mutations from the panel, and confirming disease resistance or metabolites in the lines.

[0101] The line can be crossed with a line that has not been subjected to the mutation treatment at any time before the step of confirming disease resistance or metabolites is performed. Crossing allows for the elimination of mutations that may exist in genes other than the COMT gene. In certain embodiments, the line that has a mutation in the COMT gene can be backcrossed multiple times with a line that has not been subjected to the mutation treatment (the original line used to create the panel).

[0102] Genomic DNA can be extracted from COMT mutant plants by known methods, or a commercially available extraction kit can be used. The genomic DNA may be crudely purified or may be purified through several purification steps.

[0103] Polynucleotide amplification can be carried out by, for example, PCR, but other known gene amplification methods such as LCR (ligase chain reaction) or LAMP (loop-mediated isothermal amplification) may also be used.

[0104] Primer sequences for amplifying each polynucleotide can be designed, for example, from the nucleotide sequence. For example, in the case of Nicotiana tabacum plants, S-type-specific and T-type-specific regions are first identified based on the results of a homology analysis between the nucleotide sequences of SEQ ID NO: 5 (the genomic sequence of the S-type COMT gene) and SEQ ID NO: 6 (the genomic sequence of the T-type COMT gene). By designing primers in these regions, the S-type and T-type COMT genes can be specifically amplified, respectively, from extracted genomic DNA (including S-type and T-type). The site to be designed can be selected from S-type- or T-type-specific regions, but is preferably an intron, 5' untranslated region, or 3' untranslated region. 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 a region specific to the nucleotide sequence or a region common to both nucleotide 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 or a radioactive substance, if necessary.

[0105] 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.

[0106] (Method for producing a disease-resistant Nicotiana plant) In one embodiment of the method of the present invention, the method for producing a disease-resistant Solanaceae plant may be a method for producing a disease-resistant Nicotiana plant. In particular, the Nicotiana plant may belong to Nicotiana tabacum or Nicotiana rustica. Details of Nicotiana plants are described in Section 2. Solanaceae plants such as Nicotiana plants.

[0107] Functionally deficient mutants of Nicotiana tabacum plants with mutations in two alleles (a total of four alleles, including alleles in both the T and S genomes, in the case of Nicotiana tabacum) can be obtained, for example, by the following method. As described above, Nicotiana tabacum plants are treated with a mutagen to create a mutant population (panel) with mutations throughout the genome, and genomic DNA is extracted. Using gene-specific primers, target genes (polynucleotides) are amplified from the genomic DNA of the panel, the nucleotide sequences of the products are determined, and lines with homozygous mutations are selected. For example, in the case of Nicotiana tabacum, lines (M2) with homozygous mutations in both the S and T genomes are first obtained, and then crossed to create F1. Furthermore, the resulting self-pollinated progeny (F2) are cultivated, and lines with homozygous mutations in both the S and T genomes are obtained from among them. To obtain functionally deficient mutants of Nicotiana tabacum plants with mutations in only one of the S and T genomes, it is sufficient to confirm that the genes in the non-target genome in the obtained M2 are not mutated.

[0108] (Method for Producing a Disease-Resistant Tomato Plant) In one embodiment of the present invention, the method for producing a disease-resistant Solanaceae plant may be a method for producing a disease-resistant tomato plant. The tomato plant can be produced by the same method as the method for producing a Solanaceae plant described above. In particular, the introducing step preferably includes introducing a mutation that specifically suppresses the function of the endogenous caffeate-O-methyltransferase gene into the genome of the tomato plant, outside the coding region of the endogenous caffeate-O-methyltransferase gene. The suppression of function may be a reduction in the abundance of mRNA transcribed from the endogenous caffeate-O-methyltransferase gene compared to a wild-type plant. In particular, the method may include inserting 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 caffeate-O-methyltransferase gene.

[0109] [4. Breeding Progeny] One embodiment of the present invention may be the progeny of the above-described COMT gene mutant plant or the COMT gene mutant plant obtained by the above-described production method, or a breeding progeny obtained by crossing the COMT gene mutant plant. 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. In this case, by crossing with a cultivar with superior traits, the traits of the mutant can be introduced into the cultivar, resulting in a cultivar with higher added value. Because the traits of mutants are derived from mutations, backcrossing requires the selection of individuals with mutations. In this case, the fewer mutations that result in the desired traits (coniferin content and / or disease resistance in this specification), the fewer mutations that need to be focused on, thereby reducing the effort required for backcrossing. Efficient backcrossing requires a simple method for 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. In the case of Nicotiana plants, known SNPs and simple sequence repeats (SSRs) 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.

[0110] For example, in the case of Nicotiana tabacum, a plant of the Solanaceae family, suppression of the function of two COMT genes (NtCOMT-T and NtCOMT-S) results in high coniferin content and / or disease resistance. This makes it possible to breed using a bifactorial recessive inheritance pattern, targeting mutations in the NtCOMT-T gene and the NtCOMT-S gene, thereby reducing the amount of effort required for breeding.

[0111] Furthermore, the above-mentioned COMT gene mutant plant can be favorably used from the viewpoint of breeding because it can confer a high coniferin content and / or disease resistance due to the mutation of a single gene.

[0112] 5. Other Embodiments One embodiment of the present invention provides a method for determining whether a Nicotiana plant has an increased coniferin content, 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 COMT gene in the genome; and determining that the Nicotiana plant in which the mutation is detected is a Nicotiana plant with an increased coniferin content.

[0113] In the method for determining Nicotiana plants with increased coniferin content, the inhibition of the function results in an increase in the coniferin content in tobacco leaves or dried leaves of the Nicotiana plants. That is, the determination method is used in a method for producing Nicotiana plants with high coniferin content.

[0114] Another aspect of the present invention provides a method for determining whether a Solanaceae plant has disease resistance, comprising the steps of: obtaining a sample by harvesting a part of the Solanaceae plant; detecting a mutation in the genome contained in the sample that specifically causes functional suppression of the endogenous COMT gene; and determining that the Solanaceae plant in which the mutation is detected is a disease-resistant Solanaceae plant.

[0115] In the method for determining a disease-resistant Solanaceae plant, the suppression of the function results in disease resistance in the Solanaceae plant. In other words, the determination method is used in a method for producing a disease-resistant Solanaceae plant, etc.

[0116] One aspect of the present invention provides a method for breeding Nicotiana plants with high coniferin content, comprising the step of crossbreeding Nicotiana plants with high coniferin content determined by the method for determining Nicotiana plants with increased coniferin content described above.

[0117] Another aspect of the present invention provides a method for breeding a disease-resistant Solanaceae plant, which comprises the step of crossbreeding disease-resistant Solanaceae plants determined by the method for determining disease-resistant Solanaceae plants described above.

[0118] Mutant breeding has been conducted for many plant species. 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. In this crossing, the desired traits of the mutant can be introduced into an existing cultivated variety by crossing the mutant with a cultivated variety possessing superior traits. The resulting breeding progeny can be a variety that adds significant value to an existing cultivated variety. In this case, the fewer the number of target mutations that confer disease resistance, the fewer mutations that need to be focused on, reducing the effort required for backcrossing. The Solanaceae plant described above can be advantageously used from the perspective of breeding because disease resistance can be conferred by mutation of a single gene.

[0119] The present invention also includes, in one aspect, a dried material comprising a part of a Nicotiana plant harvested from the disease-resistant Nicotiana plant or its progeny or breeding progeny, cured tobacco leaves, and tobacco materials and tobacco products containing the dried material. Details of the dried material, tobacco materials, and tobacco products are as described in [1. Dried Material].

[0120] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0121] (Summary) By summarizing the above embodiments, the present invention can be summarized as follows.

[0122] (A1) A dried material from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into at least one of the following: (a) an endogenous caffeic acid-O-methyltransferase gene, the endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, the mutation causing functional inhibition of the endogenous caffeic acid-O-methyltransferase gene; and the dried material has a higher coniferin content than a dried material from a wild-type Nicotiana plant.

[0123] (A2) A dried material of a Nicotiana plant, comprising: (a) an endogenous caffeic acid-O-methyltransferase gene having 90% or more, preferably 91%, more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and including, as a coding region, a polynucleotide encoding a polypeptide having caffeic acid-O-methyltransferase (COMT) activity; and (b) A dried material from a Nicotiana plant, wherein the dried material has a higher coniferin content than that of a wild-type Nicotiana plant, and wherein a mutation that causes functional inhibition of the endogenous caffeic acid-O-methyltransferase gene has been introduced into at least one of the endogenous caffeic acid-O-methyltransferase genes, the endogenous caffeic acid-O-methyltransferase genes having, as a coding region, 90% or more, preferably 91%, more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and the endogenous caffeic acid-O-methyltransferase genes contain, as a coding region, a polynucleotide that encodes a polypeptide having caffeic acid-O-methyltransferase (COMT) activity.

[0124] (A3) A dried material from a Nicotiana plant, wherein the Nicotiana plant has a mutation introduced into at least one of the following: (a) an endogenous caffeic acid-O-methyltransferase gene, the gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene, the gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, the mutation causing functional inhibition of the endogenous caffeic acid-O-methyltransferase gene; and the dried material has a higher coniferin content than a dried material from a wild-type Nicotiana plant.

[0125] (A4) The dried material of a Nicotiana plant according to any one of (A1) to (A3), wherein the dried material is dried leaves of tobacco leaf harvested from a Nicotiana plant. (A5) The dried material of a Nicotiana plant according to any one of (A1) to (A4), wherein the coniferin content is at least twice that of dried leaves of tobacco leaf harvested from an equivalent amount of the wild-type Nicotiana plant.

[0126] (A6) The dried material of a Nicotiana plant according to any one of (A1) to (A5), wherein the coniferin content per 1 g of the dried material is 25 μg or more, preferably 28 μg or more, and more preferably 30 μg or more. (A7) The dried material of a Nicotiana plant according to any one of (A1) to (A5), wherein the coniferin content per 1 g of the dried material is 25 to 100 μg, preferably 28 to 100 μg, and more preferably 30 to 100 μg.

[0127] (A8) A dried material from a plant of the genus Nicotiana according to any one of (A1) to (A7), which belongs to Nicotiana tabacum or Nicotiana rustica. (A9) A dried material from a plant of the genus Nicotiana according to any one of (A1) to (A8), which belongs to Nicotiana tabacum.

[0128] (A10) A dried material of a Nicotiana plant according to any one of (A1) to (A9), wherein a mutation that causes functional inhibition of the endogenous caffeic acid-O-methyltransferase gene has been introduced only into the endogenous caffeic acid-O-methyltransferase gene according to (a). (A11) A dried material of a Nicotiana plant according to any one of (A1) to (A9), wherein a mutation that causes functional inhibition of the endogenous caffeic acid-O-methyltransferase gene has been introduced only into the endogenous caffeic acid-O-methyltransferase gene according to (b).

[0129] (A12) A dried material from a Nicotiana plant according to any one of (A1) to (A9), wherein a mutation that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene has been introduced into both the endogenous caffeic acid-O-methyltransferase gene described in (a) and the endogenous caffeic acid-O-methyltransferase gene described in (b). (A13) A dried material from a Nicotiana plant according to any one of (A1) to (A12), wherein the functional suppression is a decrease in the amount of the polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene compared to the amount of the polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene in a wild-type tobacco plant.

[0130] (B1) A tobacco product comprising a drying material according to any one of (A1) to (A13).

[0131] (C1) A Solanaceae plant having resistance to disease caused by Ralstonia solanacearum, in which a mutation that causes functional suppression of an endogenous caffeic acid-O-methyltransferase gene has been introduced into the genome. (C2) The Solanaceae plant according to (C1), which is a Nicotiana tabacum plant or a tomato plant. (C3) The Solanaceae plant according to (C1) or (C2), which is a Nicotiana tabacum plant.

[0132] (C4) The Solanaceae plant according to (C3), wherein the Nicotiana plant belongs to Nicotiana tabacum or Nicotiana rustica. (C5) The Solanaceae plant according to (C3) or (C4), wherein the Nicotiana plant belongs to Nicotiana tabacum.

[0133] (C6) A Solanaceae plant according to any one of (C1) to (C5), wherein the endogenous caffeic acid-O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0134] (C7) The endogenous caffeic acid-O-methyltransferase gene is: (a) an endogenous caffeic acid-O-methyltransferase gene having 80% or more, preferably 90% or more, more preferably 91%, even more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and including, as a coding region, a polynucleotide encoding a polypeptide having caffeic acid-O-methyltransferase (COMT) activity; (b) A Solanaceae plant according to any one of (C1) to (C5), which is at least one endogenous caffeic acid-O-methyltransferase gene having 80% or more, preferably 90% or more, more preferably 91%, even more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and comprising, as a coding region, a polynucleotide encoding a polypeptide having caffeic acid-O-methyltransferase (COMT) activity.

[0135] (C8) The Solanaceae plant according to any one of (C1) to (C5), wherein the endogenous caffeic acid-O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0136] (C9) The Solanaceae plant according to any one of (C1) to (C8), wherein the endogenous caffeic acid-O-methyltransferase gene is the endogenous caffeic acid-O-methyltransferase gene according to (a). (C10) The Solanaceae plant according to any one of (C1) to (C8), wherein the endogenous caffeic acid-O-methyltransferase gene is the endogenous caffeic acid-O-methyltransferase gene according to (b).

[0137] (C11) The Solanaceae plant according to any one of (C1) to (C8), wherein the endogenous caffeic acid-O-methyltransferase gene is both the endogenous caffeic acid-O-methyltransferase gene described in (a) and the endogenous caffeic acid-O-methyltransferase gene described in (b). (C12) The Solanaceae plant according to any one of (C1) to (C11), wherein the functional inhibition is a decrease in the amount of the polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene compared to the amount of the polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene in a wild-type tobacco plant.

[0138] (D1) A method for producing a Solanaceae plant that is resistant to disease caused by Ralstonia solanacearum, comprising the step of introducing into the Solanaceae plant a mutation that specifically causes functional suppression of an endogenous caffeic acid-O-methyltransferase gene. (D2) The method according to (D1), wherein the Solanaceae plant is a Nicotiana tabacum plant or a tomato plant. (D3) The method according to (D1) or (D2), wherein the Solanaceae plant is a Nicotiana tabacum plant.

[0139] (D4) The method according to (D3), wherein the Nicotiana plant belongs to Nicotiana tabacum or Nicotiana rustica. (D5) The method according to (D3) or (D4), wherein the Nicotiana plant belongs to Nicotiana tabacum.

[0140] (D6) The method according to any one of (D1) to (D5), wherein the endogenous caffeic acid-O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0141] (D7) The endogenous caffeic acid-O-methyltransferase gene is: (a) an endogenous caffeic acid-O-methyltransferase gene having 80% or more, preferably 90% or more, more preferably 91%, even more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and including, as a coding region, a polynucleotide encoding a polypeptide having caffeic acid-O-methyltransferase (COMT) activity; (b) The method according to any one of (D1) to (D5), wherein the endogenous caffeic acid-O-methyltransferase gene has 80% or more, preferably 90% or more, more preferably 91%, even more preferably 92%, even more preferably 93%, even more preferably 94%, even more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, even more preferably 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and contains, as a coding region, a polynucleotide encoding a polypeptide having caffeic acid-O-methyltransferase (COMT) activity.

[0142] (D8) The method according to any one of (D1) to (D5), wherein the endogenous caffeic acid-O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0143] (D9) The method according to any one of (D1) to (D8), wherein the endogenous caffeic acid-O-methyltransferase gene is the endogenous caffeic acid-O-methyltransferase gene according to (a). (D10) The method according to any one of (D1) to (D8), wherein the endogenous caffeic acid-O-methyltransferase gene is the endogenous caffeic acid-O-methyltransferase gene according to (b).

[0144] (D11) The method according to any one of (D1) to (D8), wherein the endogenous caffeic acid-O-methyltransferase gene is both the endogenous caffeic acid-O-methyltransferase gene described in (a) and the endogenous caffeic acid-O-methyltransferase gene described in (b). (D12) The method according to any one of (D1) to (D11), wherein the functional inhibition is a decrease in the amount of a polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene compared to the amount of a polypeptide translated from the coding region of the endogenous caffeic acid-O-methyltransferase gene in a wild-type tobacco plant.

[0145] An embodiment of the present invention will now be described.

[0146] Example 1: Obtaining tobacco COMT mutants Using the COMT nucleotide sequence (accession number NM_001325449 in GenBank at the National Center for Biotechnology Information), the genome sequence of the tobacco (Nicotiana tabacum) cultivar "Tsukuba No. 1" was analyzed, and a COMT (NtCOMT-S, ID: nttv1s720m00946, SEQ ID NO: 5) thought to be derived from the S genome and a COMT (NtCOMT-T, ID: nttv1s753m02122, SEQ ID NO: 6) thought to be derived from the T genome were identified. The CDS sequences of NtCOMT-S (SEQ ID NO: 3) and NtCOMT-T (SEQ ID NO: 4) were subjected to a homology search against the NCBI database, and the genome sequences shown in SEQ ID NOs: 5 and 6 were confirmed to be sequences related to the tobacco (Nicotiana tabacum) COMT gene, respectively. In addition, the amino acid sequences of NtCOMT-S (SEQ ID NO: 1) and NtCOMT-T (SEQ ID NO: 2) were similarly searched, and it was confirmed that the amino acid sequences shown in SEQ ID NOs: 1 and 2 were the amino acid sequences of COMT, respectively.

[0147] Tobacco mutants harboring mutations in COMT-S or COMT-T were isolated. The nucleotide sequences of the NtCOMT-S and NtCOMT-T gene regions of 2,000 tobacco mutant lines were analyzed to identify the mutations. Specifically, 2,000 tobacco mutants were generated by EMS treatment of seeds from the tobacco cultivar Tsukuba No. 1. Self-pollinated progeny seeds (M2 seeds) obtained from each M1 generation of 2,000 mutant individuals were sown. DNA extracted from eight seedlings per line was bulked and analyzed for nucleotide sequence (Tajima et al. (2011) Ann. Phytopathol. Soc. Jpn. 77: 258). Three lines were identified with nonsense mutations, splice mutations, or loss of translation initiation site in the coding region of the NtCOMT-S gene (SEQ ID NO: 3) and two lines were identified with NtCOMT-T (SEQ ID NO: 4). Two mutants were selected for each gene. Specifically, for NtCOMT-S, we selected the following lines: NtCOMT-S-1 (SEQ ID NO: 7) (splice_donor_variant: intron splice site mutation), in which G at position 5' to 2420 in the genome sequence shown in SEQ ID NO: 5 was mutated to A; and NtCOMT-S-2 (SEQ ID NO: 8) (stop_gained: nonsense mutation), in which C at position 5' to 2022 in the genome sequence shown in SEQ ID NO: 5 was mutated to T. For NtCOMT-T, we selected the following lines: NtCOMT-T-1 (SEQ ID NO: 9) (splice_donor_variant: intron splice site mutation), in which G at position 5' to 2420 in the genome sequence shown in SEQ ID NO: 6 was mutated to A; and NtCOMT-T-2 (SEQ ID NO: 10) (start_lost: start codon lost mutation), in which G at position 5' to 2003 in the genome sequence shown in SEQ ID NO: 6 was mutated to A. Seeds of these lines were sown, and DNA was extracted at the seedling stage. PCR was performed using this DNA as a template and primers to select individuals homozygous for the mutation. KOD One (registered trademark) PCR Master Mix (TOYOBO) was used for PCR. As a result, two lines with mutations in the NtCOMT-S gene and two lines with mutations in the NtCOMT-T gene were obtained. The line names, the COMT base sequences of the lines, and the primer sequences used are shown in Table 1.

[0148]

[0149] In Table 1, the underlined portions of SEQ ID NOs: 11 to 18 are sequences required for sequencing using iSeq 100 (Illumina).

[0150] NtCOMT-S-1 and NtCOMT-T-1 were grown and crossed in a greenhouse to obtain the F1 generation (NtCOMT-F1-1). NtCOMT-S-2 and NtCOMT-T-2 were grown and crossed in a greenhouse to obtain the F1 generation (NtCOMT-F1-2). Each F1 generation was grown and selfed in a greenhouse to obtain two F2 generations derived from different mutant lines (NtCOMT-F2-1 and NtCOMT-F2-2). These two F2 generations were sown, DNA was extracted from the transplanted seedlings, and PCR was performed using the primers listed in Table 1 to amplify the surrounding sequence containing the mutation. PCR was then performed again to assign P7 and P5 sequences and individual barcode sequences to the amplified product for sequencing using iSeq 100 (Illumina), and sequence analysis was performed to confirm the genotype of each individual. As a result, we found two individuals, NtCOMT-sstt-A and NtCOMT-sstt-B, which are homozygous for mutations in both the NtCOMT-S and NtCOMT-T genes. We also found two individuals, NtCOMT-SSTT-A (a sister line of NtCOMT-sstt-A) and NtCOMT-SSTT-B (a sister line of NtCOMT-sstt-B), which do not have mutations in either gene. These individuals were self-pollinated to obtain F3 generation seeds.

[0151] Example 2: Measurement of coniferin content Seventeen F3 generations of NtCOMT-sstt-A and NtCOMT-sstt-B were grown in the field and stemmed at the flowering stage. After seven weeks, four true leaves were harvested from each individual. Furthermore, as a control, Tsukuba 1 was grown in the same manner, and four true leaves were harvested. For both the mutant and the control Tsukuba 1, leaves were harvested from 15 individuals in good condition, and three individuals (12 leaves in total) were mixed together to form one sample, for a total of five samples. The harvested leaves were dried until yellow (dried leaf samples). Three leaves were selected from each sample, the midribs removed, and then freeze-dried (mature leaf samples).

[0152] Of the five samples of dried leaves and five samples of mature leaves, three samples from each group were crushed and subjected to analysis of coniferin using a liquid chromatograph tandem mass spectrometer.

[0153] A 0.1 g sample of ground tobacco was placed in a 14 mL screw-cap glass centrifuge tube, and 5.0 mL of a methanol solution containing the internal standard phlorizin (2.0 μg / mL) was added. The tube was then sealed with a screw cap and subjected to shaking extraction at 250 rpm and 70°C for 1 hour using a medium-sized, thermostatically controlled shaker, Bioshaker® BR-43FH・MR (Taitec Co., Ltd., Saitama). The extracted solution was filtered through a 0.45 μm pore size PTFE filter and subjected to analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0154] The analytical conditions were as follows: - High-performance liquid chromatograph: 1260 infinity (Agilent Technologies, Tokyo) - Mass spectrometer: 6470 Triple Quad LC / MS (Agilent Technologies, Tokyo) - Column: InfinityLab Poroshell 120 CS-C18, inner diameter 2.1 mm, length 150 mm, particle size 2.7 μm (Agilent Technologies, Tokyo) - Column temperature: 35°C - Eluent A: 20 mM ammonium formate - ultrapure water - Eluent B: 20 ​​mM ammonium formate - acetonitrile:ultrapure water = 9:1 (v / v) - Elution conditions: 1) 0 to 2 minutes: Eluent A:Eluent B = 95:5, run for 2 minutes (eluent A 95% / eluent B 5%) 2) 2 to 10 minutes: 1) Apply a linear gradient of eluent A:eluent B from 95:5 to 55:45. 2) 10 to 20 minutes: Apply a linear gradient of eluent A:eluent B from 55:45 to 0:100. 3) 20 to 25 minutes: Flow eluent B for 5 minutes (eluent A 0% / eluent B 100%). Flow rate: 0.2 mL / min. Injection volume: 2.0 μL. Ion source parameters: Ionization method: electrospray ionization (ESI). Nebulizer gas: nitrogen. Nebulizer gas temperature: 350°C. Nebulizer gas flow rate: 5 L / min. Nebulizer pressure: 35 psi. Sheath gas: 350°C. Sheath gas flow rate: 11 L / min. Capillary voltage: 3500 V. Mass spectrometer parameters: Ion polarity: negative. Collision gas: nitrogen. Measurement mode: selected reaction monitoring (SRM).

[0155] The analytical results are shown in Tables 2 and 3. The tables show the average analytical values ​​of the three samples of each lineage divided by the average value of the control, Tsukuba No. 1 (n=3). Significant differences from the analytical results of Tsukuba No. 1 are also shown (** indicates a significant difference from Tsukuba No. 1 at the p<0.01 level, * indicates a significant difference at the p<0.05 level).

[0156] Table 2 shows the average coniferin content in the dried leaf samples (compared to Tsukuba No. 1).

[0157]

[0158] Table 3 shows the average coniferin content in mature leaves.

[0159]

[0160] In both the NtCOMT-sstt-A and NtCOMT-sstt-B COMT mutants, coniferin levels were significantly higher than in the control in both mature and dry leaves: coniferin levels were significantly increased by 2.6-4.3 times in mature leaves and by 2.3-3.0 times in dry leaves compared to the control.

[0161] Next, quantitative analysis of coniferin was performed using SRM transitions. The quantitative conditions, including the molecular weights of the coniferin and the internal standard phlorizin, as well as the molecular weights of the precursor ions and product ions detected by SRM, are shown in Table 4. The cell accelerating voltage was 3 V.

[0162]

[0163] The quantitative value of coniferin was calculated using the calibration curve method from the ratio of the area of ​​the coniferin product ion to that of the internal standard, phlorizin. Coniferin standard solutions containing 2 μg / mL of phlorizin were analyzed at seven concentrations (0.05, 0.1, 0.2, 0.5, 1, 2, and 5 μg / mL) in the range of 0.05–5 μg / mL. A linear calibration curve was obtained, with a correlation coefficient of 0.9997 for the area ratio of the coniferin and phlorizin product ion signals. Table 5 shows the coniferin content (ppm) per 1 g of dried leaf sample.

[0164]

[0165] Table 6 shows the average coniferin content (ppm) per 1 g of dried leaf sample.

[0166]

[0167] The coniferin content in the control, Tsukuba No. 1, was 16.2 ppm (average), while in the COMT mutants it was 44.6 to 66.0 ppm (average). In other words, the coniferin content in the dried leaves of the COMT mutants was significantly higher than that of the control.

[0168] Tobacco materials made with fine powder of dried COMT mutant leaves and tobacco materials made with fine powder of the control, Tsukuba No. 1, were tested and evaluated for their smoking taste. The tobacco materials were prepared by premixing solvent and fine powder at a ratio of 9:1. Each tobacco material was sprayed onto the heated area of ​​a non-combustion heated smoking article, heated to temperatures between 250 and 340°C, and then subjected to a sensory evaluation. Five well-trained panelists participated in the sensory evaluation, and the smoking taste was evaluated by consensus. Compared to the control, Tsukuba No. 1, the sample made with the dried COMT mutant leaves exhibited improved aroma and flavor. In particular, the COMT mutant sample exhibited improved aroma and flavor, including a resinous aroma and a satisfying draw. These findings demonstrate that the dried COMT mutant leaves offer improved aroma and flavor.

[0169] Example 3: Ralstonia solanacearum Assay of Tobacco COMT Mutants The F3 generation of the COMT mutant NtCOMT-sstt-A and its sister line NtCOMT-SSTT-A, and the COMT mutant NtCOMT-sstt-B and its sister line NtCOMT-SSTT-B, were grown in a field infected with damping-off disease. The field was divided into three sections, and 17 individuals of each line were grown in each section. Ten individuals were surveyed per section, for a total of 30 individuals, and the disease index was investigated every week after the initial confirmation of the disease. The damping-off disease index was evaluated on a 6-point scale from 0 to 5 (0: no disease, 1: yellowing and wilting from the ground to the second leaf, 2: yellowing and wilting from the ground to the fourth or fifth leaf, 3: yellowing and wilting of approximately half of the leaves, 4: symptoms of damping-off disease on all leaves, 5: death).

[0170] (Results) The results of the disease index survey after flowering are shown in Figures 1 and 2. Figure 1 shows the results for the COMT mutant NtCOMT-sstt-A and its sister line NtCOMT-SSTT-A. Figure 2 shows the results for the COMT mutant NtCOMT-sstt-B and its sister line NtCOMT-SSTT-B.

[0171] The graphs in the figures show the average values ​​of n = 29 or 30, and statistical analysis was performed using the Mann-Whitney U test (***: p<0.001, **: p<0.01, *: p<0.05). Both COMT mutants showed significantly lower disease indices than the control.

[0172] The COMT mutant NtCOMT-sstt-A had a disease index 40 days after flowering that was more than 1 lower than that of the control (NtCOMT-SSTT-A). Similarly, the COMT mutant NtCOMT-sstt-B also had a disease index 40 days after flowering that was more than 1 lower than that of the control (NtCOMT-SSTT-B). These results suggest that the COMT mutants NtCOMT-sstt-A and NtCOMT-sstt-B have a high potential for significantly reducing yield loss due to the progression of disease caused by Ralstonia solanaceum.

[0173] Example 4: Ralstonia solanacearum assay of tobacco COMT single mutants NtCOMT-S-1 and NtCOMT-T-1 were grown and crossed in a greenhouse to obtain the F1 generation (NtCOMT-F1-1). The F1 generation was grown in a greenhouse and self-pollinated to obtain the F2 generation (NtCOMT-F2-1). The F2 generation was sown, DNA was extracted from the transplanted seedlings, and PCR was performed using the primers listed in Table 1 to amplify the surrounding sequence containing the mutation. PCR was performed again to provide the amplified product with P7 and P5 sequences and individual barcode sequences for sequencing using iSeq 100 (Illumina), and sequence analysis was performed to confirm the genotype of each individual. As a result, we found two individuals, NtCOMT-sstt-A, which have homozygous mutations in both the NtCOMT-S and NtCOMT-T genes; and NtCOMT-ssTT-A and NtCOMT-SStt-A, which have homozygous mutations in either the NtCOMT-S or NtCOMT-T gene. We also found an individual, NtCOMT-SSTT-A (a sister line of NtCOMT-sstt-A), which does not have mutations in either the NtCOMT-S or NtCOMT-T gene. These individuals were self-pollinated to obtain F3 generation seeds.

[0174] The COMT double mutant NtCOMT-sstt-A, its sister line NtCOMT-SSTT-A, and the COMT single mutants NtCOMT-SStt-A and NtCOMT-ssTT-A were investigated for their susceptibility to Ralstonia solanacearum using the same procedures as described in Example 3. That is, the F3 generation of NtCOMT-sstt-A, its sister line NtCOMT-SSTT-A, and NtCOMT-SStt-A and NtCOMT-ssTT-A were cultivated in a field contaminated with damping-off. The field was divided into three plots, and 17 individuals of each line were cultivated in each plot. 10 individuals from each plot were investigated. A total of 30 plants for NtCOMT-sstt-A, NtCOMT-SSTT-A, and NtCOMT-SStt-A, and a total of 28 plants for NtCOMT-ssTT-A, were investigated for the disease index every week after the first occurrence of disease. The damping-off index was evaluated on a 6-point scale from 0 to 5 (0: no disease, 1: yellowing and wilting from the ground to the second leaf, 2: yellowing and wilting from the ground to the fourth or fifth leaf, 3: yellowing and wilting of about half the leaves, 4: damping-off symptoms on all leaves, 5: death).

[0175] (Results) The results of the disease index survey after flowering are shown in Figure 3. Figure 3 shows the results for the COMT double mutant NtCOMT-sstt-A, its sister line NtCOMT-SSTT-A, and the COMT single mutants NtCOMT-SStt-A and NtCOMT-ssTT-A.

[0176] The graphs shown in the figures show the average values ​​for n = 28 or 30, and statistical analysis was performed using a Dunn's test. In Figure 3, each line is labeled "a," "b," or "c." When the same symbol is assigned between lines that have been flowering for the same number of days, it indicates that there is no statistically significant difference between the lines. When different symbols are assigned between lines that have been flowering for the same number of days, it indicates that there is a statistically significant difference between the lines. Specifically, when the p-value is calculated based on the disease index of each line that has been flowering for the same number of days, and the significance level is set at p = 0.05, the same symbol is assigned if there is no statistically significant difference between the lines, and different symbols are assigned if there is a statistically significant difference between the lines. Using the results at 29 days after flowering as an example, NtCOMT-SSTT-A is labeled "a" and NtCOMT-ssTT-A is labeled "b," indicating a significant difference in the disease tendency between the two lines. On the other hand, NtCOMT-sstt-A and NtCOMT-SStt-A were each assigned the symbol "c," indicating that there was no significant difference in the disease tendency between the two lines. In other words, the results at 29 days after flowering can be interpreted as follows: - Compared to NtCOMT-SSTT-A, NtCOMT-ssTT-A had a significantly lower disease index. - Compared to NtCOMT-ssTT-A, NtCOMT-sstt-A and NtCOMT-SStt-A had significantly lower disease indexes. - No significant difference was observed in the disease index between the NtCOMT-sstt-A and NtCOMT-SStt-A lines.

[0177] The results in Figure 3 reveal the following: All COMT mutants (i.e., NtCOMT-sstt-A, NtCOMT-SStt-A, and NtCOMT-ssTT-A) showed significantly lower disease indices compared to the control, NtCOMT-SSTT-A. In particular, the disease indices for NtCOMT-sstt-A and NtCOMT-SStt-A were more than 1 lower than the control (NtCOMT-SSTT-A) 36 days after flowering. This suggests that in addition to the COMT double mutant, the COMT single mutant is likely to be able to significantly reduce yield loss due to the progression of disease caused by Ralstonia solanaceum.

[0178] The present invention can be used in tobacco products.

Claims

1. A dried material from a Nicotiana plant, wherein a mutation that causes functional inhibition of the endogenous caffeic acid-O-methyltransferase gene has been introduced into at least one of the following: (a) an endogenous caffeic acid-O-methyltransferase gene, the endogenous caffeic acid-O-methyltransferase gene having, as its coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid-O-methyltransferase gene having, as its coding region, a polynucleotide that encodes a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; and the dried material has a higher coniferin content than dried material from a wild-type Nicotiana plant.

2. The dried material of Nicotiana plants according to claim 1, wherein the dried material is cured tobacco leaves harvested from Nicotiana plants.

3. A dried material of a Nicotiana plant according to claim 1 or 2, wherein the coniferin content is at least twice the coniferin content of dried tobacco leaves harvested from the same amount of the wild-type Nicotiana plant.

4. A dried material of a plant of the genus Nicotiana according to any one of claims 1 to 3, which belongs to Nicotiana tabacum or Nicotiana rustica.

5. A tobacco product comprising a drying material according to any one of claims 1 to 4.

6. A Solanaceae plant that has resistance to disease caused by Ralstonia solanacearum, in which a mutation that causes functional suppression of the endogenous caffeic acid-O-methyltransferase gene has been introduced into the genome.

7. The Solanaceae plant according to claim 6, wherein the Solanaceae plant is a Nicotiana plant or a Tomato plant.

8. The Solanaceae plant according to claim 7, wherein the Nicotiana plant belongs to Nicotiana tabacum or Nicotiana rustica.

9. The Solanaceae plant according to any one of claims 6 to 8, wherein the endogenous caffeic acid O-methyltransferase gene is at least one of: (a) an endogenous caffeic acid O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) an endogenous caffeic acid O-methyltransferase gene comprising, as a coding region, a polynucleotide that encodes a polypeptide having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

2.

10. A method for producing a Solanaceae plant that is resistant to disease caused by Ralstonia solanacearum, comprising the step of introducing into the Solanaceae plant a mutation that specifically causes functional suppression of an endogenous caffeic acid-O-methyltransferase gene.

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