WO2008117887A1 - Plant of genus nicotiana with reduced content of carbonyls in combustion smoke and method for producing the same - Google Patents

Abstract

It is intended to provide a plant of the genus Nicotiana with a reduced content of carbonyls in plant combustion smoke. A plant of the genus Nicotiana in which the expression of a gene encoding any one of the proteins shown in the following (a) to (c) has been suppressed is provided: (a) a protein having an amino acid sequence represented by SEQ ID NO: 6; (b) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 and capable of controlling the content of carbonyls in plant combustion smoke; and (c) a protein having an amino acid sequence with a homology of 85% or more to the amino acid sequence represented by SEQ ID NO: 6 and capable of controlling the content of carbonyls in plant combustion smoke.

Description

 Tobacco plant with reduced carbonyl content in combustion smoke and method for producing the same

Technical field

 The present invention relates to a tobacco genus plant having a reduced carbonyl content in combustion smoke and a method for producing the same. Light

Background art

 For tobacco products, it is written to reduce carbonyls in the combustion smoke.

It is desired. In order to reduce the amount of carbonyls contained in the smoke of tobacco products, it has been common to use a filter with activated carbon added as an adsorbent. The use of is being tried.

On the other hand, some parts of mainstream smoke, such as tar and nicotine, are directly related to smoking satisfaction and savory taste, and the amount of these ingredients remains relatively high. It is desirable to selectively remove only carbonyls in the component. However, the adsorbents used in the past adsorb not only carbonyls but also other components, which may adversely affect the taste of tobacco products. For example, Patent Documents 1 to 7 disclose filters to which amine substances are added for the purpose of adsorption of formaldehyde and the like. However, they can reduce carbonyls in the smoke component, but also remove tar and nicotine at the same time. In addition, there was a problem in odor, safety, and effects such as the appearance of a unique amine odor due to the decomposition of the added amine-based substance, and there was nothing practical for practical use in tobacco products. Further, Patent Document 7 discloses a cigarette wrapping paper to which an ammonia-containing compound is added as a combustion improving agent for the purpose of reducing carbonyls in combustion smoke. However, as described above, tar and nicotine are removed. So it's not practical. Patent document 8 also discloses the use of certain amino acids such as glycine to remove aldehydes in tobacco smoke. Glycine can reduce the level of formaldehyde in tobacco smoke, but it is not stable in the cigarette filter manufacturing process It has been known. In addition, it releases ammonia odor during storage, making it unsuitable for use in tobacco products.

 Several methods for selectively removing only carbonyls in the smoke component have been proposed. For example, reference 9 discloses a filter to which a high-mouth talcite compound that can selectively remove formaldehyde is added. In the description, the Ciacoal filter achieves a reduction in formaldehyde by indiscriminately reducing the total organic vapor (VOC), whereas the hydrotalcite compound addition filter does not significantly reduce the total organic vapor. It has been shown that formaldehyde reduction equivalent to that of Cyacoal filters is achieved.

 In Patent Document 10, it is considered that selectivity is enhanced by using crystalline zeolite having unique adsorption characteristics. However, it has been shown that a filter that incorporates it reduces nine components, such as reducing acrolein by 50% or more, but the effect on other components has not been clarified. Patent Document 11 discloses that, unlike activated carbon and chitosan, a filter to which a basic polypeptide is added reduces formaldehyde by about 15% compared to a commercial product without removing nicotine and tar. .

 As described above, it is considered that the use of several kinds of filters with specific adsorbents is effective as a method of specifically reducing the lower aldehydes contained in the smoke of tobacco products. However, such high functionality of the filter has limited practical use, such as increasing the ventilation resistance and making it difficult to obtain normal combustion smoke. Therefore, there has been a demand for a method for reducing the amount of carbon in the smoke of tobacco products by a method different from that for filters. One of the methods is the development of Tabacco plants with a low content of strong ruponils in combustion smoke. In addition, if such tobacco plant is used as a raw material for cigarettes and the like, the burden on development and production of a high-performance filter can be reduced, which can be expected to lead to a reduction in production cost.

 Patent Document 1 JP 5 9 _ 0 8 8 0 78

 Patent Document 2 Japanese Patent Application Laid-Open No. 5-9-1 5 1 8 8 2

 Patent Document 3 Japanese Patent Laid-Open No. Sho 6 0-0 0 5 4 6 6 9

Patent Document 4 Japanese Patent Application Laid-Open No. 09-166 Patent Document 5 Special Table 2 0 0 2— 5 2 8 1 0 5

 Patent Document 6 Special Table 2 0 0 2— 5 2 8 1 0 6

 Patent Literature 7 Special Table 2 0 0 3— 5 0 5 6 1 8

 Patent Document 8 US Patent No. 2 9 6 8 3 0 6 Specification

 Patent Document 9 International Publication No. 2 0 0 3/0 5 6 9 4 7 Pamphlet

 Patent Document 1 0 US Patent Application Publication No. 2 0 0 5 0 1 3 3 04 7 Patent Document 1 1 Japanese Patent Application Laid-Open No. 2 0 0 6-3 4 1 2 7

Disclosure of the invention

 The object of the present invention is to find a gene capable of controlling the content of carbonyls in the combustion smoke of tobacco genus plants, and to suppress the expression of the gene to thereby reduce the carbonyl content in the smoke. It is to create and provide plants.

 For the purpose of identifying genes that affect the quality of raw leaf tobacco, the present inventors focused on the fact that the degree of maturity of tobacco leaves greatly affects the quality of raw leaf tobacco, and the leaves of matured tobacco grown in soil cultivation Alternatively, we searched for genes whose expression level changes greatly in hydroponic tobacco leaves in which maturation was artificially induced by reducing the amount of nitrogen fertilization. As a result, we succeeded in identifying a gene that can control the maturity level of tobacco plants, and by suppressing the expression of this gene, we can promote the maturation of tapaco leaves and impart ease of drying. It has been found that the quality of raw leaf tobacco can be improved, and surprisingly, it has been found that by suppressing the expression of the gene, the content of carbonyls in the combustion smoke of dry leaf tobacco can be reduced. completed.

 The features of the present invention are summarized as follows.

 (1) A tobacco genus plant in which expression of a gene encoding any one of the proteins shown in the following (a) to (c) is suppressed.

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6

(b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the content of carbohydrates in plant combustion smoke (c) A protein comprising an amino acid sequence having an identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the carbonyl content in the combustion smoke of plants.

 (2) the gene is

 (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 5,

 (e) a polynucleotide encoding a protein having 85% or more identity to the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke;

 (f) Coding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke Polynucleotide to

 (g) It encodes a protein capable of hybridizing with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 5 under stringent conditions and capable of controlling the carbonyl content in plant combustion smoke. Polynucleotide

The tobacco genus plant according to (1).

 (3) The carbonyl compound is selected from the group consisting of formaldehyde, acetoaldehyde, acetone, acrolein, propion aldehyde, croton aldehyde, methyl ketylketone and ptyl aldehyde, (1) or (2) The tobacco genus plant of description.

 (4) The tobacco genus plant according to (3), wherein the carbole is formaldehyde or acrolein.

 (5) The tissue or cell of the tobacco genus plant according to any one of (1) to (4).

 (6) The tissue or cell according to (5), wherein the tissue is a leaf.

 (7) the nucleotide sequence shown in SEQ ID NO: 5, the nucleotide sequence shown in SEQ ID NO: 7, a nucleotide sequence having 90% or more identity to these sequences, and a partial sequence of 20 nucleotides or more of those nucleotide sequences A gene expression suppression vector comprising a base sequence selected from the group consisting of:

(8) An antisense method comprising a nucleotide sequence consisting of 20 or more consecutive nucleotide sequences The vector according to (7), comprising

 (9) The vector according to (7), comprising a sense strand of the base sequence and an antisense strand that pairs with the sense strand.

 (10) The vector according to (9), wherein the sense strand and the antisense strand are composed of 20 or more consecutive base groups of the base sequence.

 (1 1) The vector according to (9) or (1 0), which has a spacer between the sense strand and the antisense strand.

 (1 2) The vector according to any one of (7) to (11), which has a promoter operable in plant cells.

 (1 3) Tobacco cells or tissues are selected from RNA interference method, antisense method, gene disruption method, artificial mutation method, ribozyme method, co-suppression method and method of controlling transcription factor Including regenerating a plant by suppressing the expression of any gene of (a) to (g) defined in (1) and (2) using any one of the methods (1) to (1) (4) A method for producing the tobacco genus plant according to any one of the above.

 (14) The method according to (13), wherein the vector according to any one of (7) to (12) is introduced into a plant cell or tissue to regenerate a plant body.

 (15) The method according to (14), wherein a progeny is created using the plant body as a mother.

 (1 6) A progeny of the tobacco genus plant according to any one of (1) to (4), wherein the gene of any one of (a) to (g) defined in (1) or (2) Progeny characterized by suppressed expression.

 (1 7) A tobacco product produced from the tobacco genus plant according to any one of (1) to (4) or the progeny leaf according to (16).

 (1 8) In the tobacco genus plant, the smoke of dry leaf tobacco of the tobacco genus plant, comprising suppressing the expression of any of the genes defined in (a) to (g) defined in (1) or (2) To reduce the content of carbonyls therein.

 (1 9) Suppression of gene expression is selected from the group consisting of RNA interference method, antisense method, gene disruption method, artificial mutation method, ribozyme method, co-suppression method and method of controlling transcription factor The method according to (18), comprising the step of:

(20) A vector according to any one of (7) to (12) is introduced into a plant cell or tissue. The method described in (1 8) or (1 9).

 In the present specification, “a protein capable of controlling the carbonyl content in combustion smoke of plants” refers to the combustion smoke generated when burning tissues of tobacco plant such as dried leaves. It is a protein involved in biosynthesis or degradation of plant components that becomes a precursor of contained carbonyls or inhibits the formation of carbonyls by combustion reaction.

 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2007-083394, which is the basis of the priority of the present application.

Brief description of the drawings

 FIG. 1 shows the PCR amplification region of the # 1 049 1 gene.

 FIG. 2 shows the structure of the vector p S P 148. It was constructed by introducing 35 S promoter: spacer and NOS terminator between two DοχΙ of pDNR_l (CLONTEC H). 35 S promoter and NOS terminator were introduced from pB I 1 2 1 (C LONTECH). The spacer used was the M 1 u I fragment (1 525-20 73) of p S B 11 (A CCE S S I ON No. AB 027256).

 FIG. 3 shows the structure of the vector p S P 148— # 1 049 1 FR.

 FIG. 4 shows the structure of the vector p S P 107. The p ro K— l o x P fragment (C r e a t o r A c c e p t o r c t o r c o n s t r u c t i o n K i t, C 1 o n te c h) was introduced into p B I 1 2 1 (CLONTEC H) H i n d l I I—S se 838 7 I site.

 FIG. 5 shows the structure of the vector p S P 1 07— # 1 049 1.

 Figure 6 shows the results of measuring the expression of the # 1 04 1 9 gene in transgenic tobacco using real-time PCR.

 Panels A-D in FIG. 7 show the morphology of transgenic tobacco (T 1 generation, T 0 generation inbred progeny) and / or control tobacco (emergence stage).

Panel A in Figure 8 shows the dried leaves of Transgenic tobacco. Panel B in Figure 8 shows the dry leaves of the control tobacco. Panel A in Figure 9 shows the formaldehyde content in the smoke from the dry leaves of transgenic tobacco. Panel B in Fig. 9 shows the acrolein content in the smoke from the dry leaves of Transgenic tobacco.

 FIG. 10 shows the structure of the vector p S P 204.

 FIG. 11 shows the structure of the vector p S P 20 1.

 Figure 12 shows the map of the trigger sequence used in the expression suppression experiment.

 Figure 1 3 shows the vectors p S P 204—M_t r i g e r and p S P 204—3 ′

The structure of _t r i g e r is shown.

 FIG. 14 shows the result of measuring the expression of the # 1 04 19 gene in transgenic tobacco by real-time PCR. BEST MODE FOR CARRYING OUT THE INVENTION

 1. Tobacco plant of the present invention

 Tobacco plants with reduced carbonyl content in the combustion smoke of the present invention are:

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6,

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the content of carbonyls in plant combustion smoke; Or

 (c) A protein comprising an amino acid sequence having an identity of 85% or more with respect to the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the carbonyl content in plant combustion smoke

The expression of the gene encoding is suppressed, or

 (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 5,

 (e) a polynucleotide encoding a protein having 85% or more identity to the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke;

(f) Coding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke Polynucleotide to (g) It can hybridize under stringent conditions with a polynucleotide consisting of a base sequence complementary to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 and can control the content of carbonyls in the combustion smoke of plants. Polynucleotides encoding proteins.

It is characterized in that the expression of a gene containing is suppressed.

 The gene capable of controlling the carbonyl content in the combustion smoke of tobacco genus plants is SEQ ID NO:

This gene has the base sequence shown in FIG. 5 (full-length cDNA sequence) and encodes the amino acid sequence shown in SEQ ID NO: 6 (hereinafter sometimes referred to as # 1 049 1 gene).

 # 1 049 1 gene focuses on the fact that the degree of maturity of tobacco leaves greatly affects the quality of the raw tobacco, and using the DNA microarray of tobacco, we investigated the genes whose expression level changed significantly during the maturation process of tobacco leaves As a result, it was selected.

 The DNA of # 1 049 1 gene can be isolated by those skilled in the art by generally known methods. For example, hybridization technology (S outhern, EM., J Mo 1 B iol, 1 97 5, 98, 503.) is a polymerase chain reaction (PCR) technology (S aiki, RK. Eta 1. , Science, 1 98 5, 230, 1 350., Saiki, RK. Eta 1., Science 1 988, 239, 487.). Specifically, DNA consisting of the base sequence described in SEQ ID NO: 1 (probe sequence immobilized on tobacco DNA microarray) or a part thereof is used as a probe, and also described in SEQ ID NO: 1. Highly identical to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 from different varieties of tobacco genus plants or plants other than Tabacco plants, using oligonucleotides that specifically hybridize to DNA comprising the nucleotide sequence as primers ( Isolate DNA with 85% or greater). In this way, a DNA that hybridizes with a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and that can be isolated by a hybridization technique or a PCR technique is also applied to the present invention.

Hybridization conditions are not particularly limited as long as they can be highly pre-hybridized.For example, 0 · 25M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM E

60 ° C, preferably 65 ° C, in a buffer consisting of DTA, 1 X Denhardt's solution Luo preferably is 1 6-24 hours hybridizing under conditions of 68 ° C, further _{2 OmM Na 2 HP0 4, p} H 7. 2, 1% SDS, 1 m EDTA consists in buffer solution in 60 ° C The conditions are preferably such that washing is carried out twice for 15 minutes at 65 ° C, more preferably 68 ° C.

 In addition, the method for obtaining the full-length cDNA clone of the # 1 049 1 gene is not particularly limited, and it may be excised from a cDNA library of an organism having the gene with an appropriate restriction enzyme and purified. Genomic DNA is extracted from plant cells or tissues, for example, and a genomic library (plasmid, phage, cosmid, BAC, PAC, etc. can be used as vectors) is created from this library. It can be obtained by performing colony hybridization or plaque hybridization using a probe prepared based on the base sequence shown in SEQ ID NO: 5.

 In addition, once the base sequence of the gene of the present invention has been determined, thereafter, by chemical synthesis, or by PCR using the cDNA or genomic DNA of this gene as a cage, or the base sequence. It can be obtained by hybridizing with a DNA fragment having a DNA as a probe.

 In addition, about the base sequence of SEQ ID NO: 5, sequence comparison analysis by B LAST (P ro c. At l. Ac a d. Sei. USA, 1 990, 87, 2264

— 2268, Karlin, S. & A ltschul, S F., Proc. Nat 1. Ac ad. Sei. USA, 1 993, 90, 58 73-58 7 7) It was found to be identical to one of the cytochrome P450 genes induced by treatment. Among these cytochrome P450 genes, a gene encoding nicotine demethylase that converts nicotine to nornicotine was included, but an example of estimating the function of the nucleotide sequence of SEQ ID NO: 5 is shown. Neither is it disclosed, nor is its usage disclosed (Reference: International Publication No. 2005/0380 18, International Publication No. 2005/038033, International Publication No. 2005/1 1 1 2 1 7 No., US Patent Application Publication No. 2006/003 70

96, US Patent Application Publication No. 2006 004 1 949).

In addition, genes other than tobacco are disclosed in US Pat. No. 707 1 3 76. It was found that the CYP 98A3 gene encoding p-couma rate 3-hydroxylase (C 3 H) of Arabidopsis thaliana is approximately 70% identical in nucleotide sequence and 74% identical in amino acid sequence. However, the above description describes lignin, does not describe the relationship between lignin and carbohydrates in combustion smoke, and those skilled in the art also have no knowledge of lignin and carbonyls in combustion smoke. The relevance is unknown.

 Therefore, it is a novel finding that the # 1 049 1 gene can control the carbonyl content in combustion smoke.

 The protein encoded by the # 1 049 1 gene is a protein having the amino acid sequence shown in SEQ ID NO: 6, but is not limited to the protein in the present invention, and the content of carbonyls in the combustion smoke of tobacco plants As long as it is a protein that can control the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 6 has one or several amino acid deletions, substitutions or additions, or 85 It may be a protein containing an amino acid sequence having at least% identity.

 Here, the amino acid sequence in which one or several amino acids are deleted, substituted or added is specifically 1 to 10 amino acids, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 6. Is a sequence that has been deleted by mutation or substituted or added to another amino acid. The mutation may be an artificially introduced mutation or a naturally occurring mutation.

 The amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 6 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, 99%. The above identity is desirable. Sequence identity can be determined by F ASTA search or B LAST search.

The gene encoding the protein is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 5, but is not limited thereto, and is a polynucleotide capable of controlling the content of carbohydrates in the combustion smoke of tobacco genus plants. As long as the polynucleotide has 85% or more identity to the base sequence shown in SEQ ID NO: 5, or a base in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5. The polynucleotide may be a polynucleotide that hybridizes under stringent conditions with a polynucleotide that comprises a sequence or a polynucleotide that comprises a base sequence that is complementary to the polynucleotide that comprises the base sequence shown in SEQ ID NO: 5.

 Here, the base sequence in which one or several bases are deleted, substituted or added is specifically 1 to 10 base sequences shown in SEQ ID NO: 5, preferably:! To 5 base sequences. This refers to a sequence in which a base group has been deleted by mutation or substituted or added to another base. The mutation may be an artificially introduced mutation or a naturally occurring mutation.

 Further, the base sequence having 85% or more identity to the base sequence shown in SEQ ID NO: 5 is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more and 99% or more. Is desirable. Sequence identity can be determined by sequence comparison analysis tools such as FASTA and B LAST. In the present specification, the identity of a base sequence is calculated by performing BLAST analysis with default parameters.

 Moreover, the stringent conditions can be, for example, the hybridization conditions described above, in which so-called specific hybrids are formed and non-specific hybrids are not substantially formed.

 Suppression of expression of the gene encoding the protein includes suppression of transcription of the gene and suppression of translation into the protein, and includes not only complete termination of gene expression but also decrease of expression. By artificially or naturally mutating or destroying genes, or by using various genetic engineering methods such as RNA interference methods, antisense methods, ribozyme methods, co-suppression methods, methods of controlling transcription factors, etc. Cannot be expressed or suppressed.

 The tobacco genus plant of the present invention is characterized in that the content of carbonyls contained in combustion smoke generated when burning a plant, particularly dried leaves, is reduced.

 It is known that a variety of components exist in the smoke of dry leaf tobacco.

(Green CR. Etal., 1 996, Recent Ad v. Tobacco Sci., 22: 1 3 1—304). Therefore, it is considered that these components in the combustion smoke are related to each other and lead to smoking satisfaction and flavor. In other words, if the balance of the components in the combustion smoke is greatly changed, the satisfaction and flavor of smoking may be impaired. There is. Therefore, it is desirable to reduce specific components contained in combustion smoke by a highly selective method. In the present invention, by suppressing the expression of only 1 gene (# 1 049 1 gene), carbonyls contained in the smoke of dry leaf tobacco could be reduced. Specific examples of carbonyls contained in the smoke of dry leaf tobacco include formaldehyde, acetoaldehyde, acetone, acrolein, propionaldehyde, croton aldehyde, methyl ethyl ketone, and butyl aldehyde.

 In general, the components of cigarette combustion smoke are measured by collecting smoke that has been smoked and dried automatically with cigarettes wrapped under wrapping paper under ISO-compliant smoking conditions. On the other hand, a method using thermal decomposition has been reported as a method for collecting and measuring components in combustion smoke from a smaller amount of sample more easily (T rikai K. eta 1., 2 004, F 42: 1409-1 7.) (T rikai K. eta 1., 2005, Food Chem Toxicol .; 43: 559-68.). In the present invention, carbons in combustion smoke are measured by this pyrolysis method, but the present invention is not limited to this.

 In the present invention, tissues or cells of the tobacco genus plant of the present invention (for example, root, stem, leaf, flower, seed, embryo, ovule, ovary, shoot apex, cocoon, pollen, callus, protoplast, suspension) Cultured cells and the like).

2. Gene expression suppression vector

 The gene expression suppression vector of the invention is

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6,

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the content of carbonyls in the burning smoke of plants, Or

 (c) a protein comprising an amino acid sequence having an identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the content of carbonyls in plant combustion smoke;

A gene encoding, or

(d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 5, (e) a polynucleotide encoding a protein having 85% or more identity to the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke;

 (f) Coding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke Polynucleotide to

 (g) A protein that hybridizes with a polynucleotide consisting of a base sequence complementary to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 under stringent conditions and that can control the content of carbonyls in the combustion smoke of plants. Polynucleotide

A nucleic acid molecule for suppressing the expression of is inserted into an appropriate vector.

 The vector of the present invention includes, for example, the base sequence shown in SEQ ID NO: 5, the base sequence shown in SEQ ID NO: 7, a base sequence having 90% or more identity to these sequences, and 20 of these base sequences It includes a base sequence selected from the group consisting of partial sequences of bases or more. Such vectors include antisense vectors or RNA interference induction vectors.

 An antisense vector refers to a vector containing a nucleic acid molecule that suppresses the expression of the # 1 0 4 9 1 gene, its variant or homologue by an antisense method. A homologue is a gene corresponding to the # 1 0 4 9 1 gene, which has a different nucleotide sequence between tobacco varieties or in plants other than tobacco.

 The antisense method expresses an antisense RNA complementary to the mRNA transcribed from the target gene, binds the antisense RNA complementarily to the target gene, and prevents transcription of the target gene. As a method for suppressing the expression of the gene. Specifically, when the # 1 0 4 9 1 gene, its mutant or homologue, or a fragment thereof is inserted in the antisense direction downstream of a promoter that functions in plants, and introduced into a plant cell or tissue, the above Antisense RNA complementary to mRNA can be produced.

Nucleic acid molecules that suppress the expression of the # 1 0 4 9 1 gene, its mutants or homologues that are introduced into the antisense vector are, for example, the base sequence of the # 1 0 4 9 1 gene (sequence The full length antisense nucleic acid molecule of No. 5) may be 90% or more, more preferably 95% or more, more preferably 98% or more, 99% or more with respect to the base sequence shown in SEQ ID NO: 5. Antisense nucleic acid molecules having the same sequence may be used, and the base sequences of these nucleic acid molecules are 20 bases or more to less than full length, preferably 100 bases or more to less than full length, more preferably 500 bases or more to full length. It may be an antisense nucleic acid molecule having a base sequence consisting of less than. The length of the antisense DNA usually used is shorter than 5 kb, preferably shorter than 2.5 kb (reference documents: JP-A-60-232092, JP-A-2000-23685).

 The RNA interference-inducing vector is a vector containing a DNA that is a truncated form of dsRNA (double-stranded RNA, double-strandRNA) that triggers RNA interference. The ds RNA formed in the cell using this vector is cleaved into siRNA (sma 1 1 interfering RNA) of about 21-25 bases by double-stranded RNA-specific RNase (D icer). After that, a complex is formed as part of RISC (RNA-induced silencing complex), and the target mRNA is recognized and degraded by homology.

 For plant RNA interference, a vector expressed as a hairpin dsRNA is preferably used. This is done by placing a sequence that triggers RNA interference at both ends of the linker sequence (spacer) of tens to tens of bases or several ^ to several hundred bases so that it becomes IR (inverted repeat). This is a system that transcribes hairpin dsRNA using a promoter highly expressed in plants and produces siRNA in the cell. In addition to the hairpin type described above, siRNA expression systems include tandem types. In the tandem type, sense RNA and antisense RNA are transcribed from two promoters and hybridize in the cell to produce siRNA. For example, the DNA sequence that serves as a trapezoid for RNA interference is, for example, the nucleotide sequence of SEQ ID NO: 7 or 90% or more, more preferably

95% or more, more preferably 98% or more, 99% or more of a sequence having 20 or more bases, preferably 100 bases or more, more preferably 200 bases or more, and a complementary sequence thereof. Used (Reference: WO / 1 9 9

9/053050, WO 1 999/326 1 9 A, WO 200 1/75 1 64 A, Ch uang CF & Me yer ow itz EM: Proc Na tl A cad S ci USA 97: 4985, 2000).

 As an RNA interference induction vector used in the present invention, a sense strand of the base sequence shown in SEQ ID NO: 7 and an antisense strand complementary to the sense strand are sandwiched between a spacer (sequence forming a loop). It is preferable to include them in the same vector so as to be IR (inverted repeat).

 Here, as the vector, pBI, pPZP, and pSMA vectors that can introduce a target gene into a plant via agrobacterium are preferably used. In particular, binary vector systems (pBI 1 2 1, pBI 1 0 1, pBI 22 1, pBI 2 1 1 3, pBI 1 0 1.2, etc.) or intermediate vector systems (p LGV23Neo, pNCAT) Etc.) is preferred. A binary vector contains a right border (RB) and a left border (LB) of the T1 DNA region, and can contain elements such as a promoter and a plant selection marker along with the target gene between both borders. scherichiacoli) and a vector vector that can replicate in agrobacterium. When agrobacterium carrying a binary vector is infected with a plant, it is surrounded by a border sequence consisting of the LB and RB sequences on the vector. Partial DNA can be incorporated into plant nuclear DNA (EMBO Journal, 1 0 (3), 6 9 7-704 (1 99 1)). On the other hand, a pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Also, plant virus vectors such as force reflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV) and tobacco mosaic virus (TMV) can be used.

When using a binary vector-based plasmid, the target gene is inserted between the above-mentioned binary vector boundary sequences (LB, RB), and this recombinant vector is amplified in Escherichia coli. Next, the amplified recombinant vector is introduced into Agrobacterium tumefaciens C 58, LBA4404, EHA 1001, EHA 105, etc. using a method such as freeze-thaw method, electoral position method, etc. The agrobacterium is used for plant transformation. In addition to the above method, an agropacterium for plant infection containing the target gene is prepared by the three-part joining method (Nucleic Acids Research, 1 2: 8 7 1 1 (1 984)). be able to. That is, E. coli containing a plasmid containing the target gene, E. coli containing a helper plasmid (eg, pRK20 1 3 etc.), and agrobacterium are mixed and cultured on a medium containing rifampicillin and kanamycin. By culturing with, it is possible to obtain an agrobacterium for plant infection.

 In order to insert the target gene into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or multiple cloning site, and ligated to the vector. Adopted. ·

 In addition to the target gene, for example, a promoter, a terminator, a poly A addition signal, a selection marker gene, and the like can be placed in the vector. The promoter does not have to be derived from a plant as long as it is a DNA that functions in a plant or a plant cell and can induce expression in a specific tissue of a plant or a specific developmental stage. Specific examples include cauliflower mosaic virus (CaMV) 35 S promoter, promoter of nopaline synthase gene (P nos), corn-derived ubiquitin promoter, rice-derived actin promoter, tobacco-derived PR protein promoter, etc. .

 The terminator may be any sequence that functions in plants or plant cells and can terminate the transcription of the gene transcribed by the promoter. For example, the terminator of nopaline synthase (NOS) gene, octopine synthesis Examples include an enzyme (OC S) gene terminator and a CaMV 35 S terminator.

Examples of selectable marker genes include drug resistance genes (tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, spectinomycin resistance gene, chloramphenicol resistance gene, neomycin resistance gene, etc.) Fluorescent or luminescent reporter genes (such as luciferase, / 3-galactosidase, β_dulcronitase (GUS), green fluorescent protein (GFP)), neomycin phosphotransferase Enzyme genes such as ze II (NPT I 1) and dihydrofolate reductase.

 Alternatively, the selection marker gene may be ligated to the same plasmid together with the target gene as described above, or a recombinant vector may be prepared. Alternatively, a recombination obtained by ligating the selection marker gene to the plasmid. A vector and a recombinant vector obtained by ligating a target gene to a plasmid may be prepared separately. If prepared separately, co-transform each vector into the host.

An example of construction of a binary vector that can be used in the present invention is shown in Example 2 described later. That is, plasmid p SP 148 with 35 S promoter, spacer, NO S terminator inserted between 1 o XP of plasmid p DNR-1 (C LONTEC H) containing 2 1 o XP. (Figure 2) to produce, in the NOS terminator one and scan Bae between p o first and spacer first and _{3 5} s 2 or object DN a fragment opposite directions of the triggers of RNA i between the promoter Insert a trigger to place it (Figure 3, plasmid p SP 148_ # 1 049 1 FR), and insert this hairpin-type ds RNA expression cassette into Cre-1 ox recombination (US Pat. No. 6, 4 1 0, 3 1 7) can be recombined with the plasmid p SP 1 07 (Fig. 4) to produce the binary vector p SP 1 0 7_ # 1 049 1 (Fig. 5).

 The two triggers may be facing each other or facing each other back. The spacer sequence may be any sequence as long as it forms a hairpin loop. For example, a ras gene sequence or an intron sequence can be exemplified, and its size is, for example, about several to several hundred bases. It is.

3. Method for producing tobacco genus plant of the present invention

 The method for producing a tobacco genus plant of the present invention comprises controlling the ribonucleic acid method, antisense method, gene disruption method, artificial mutation method, ribozyme method, co-suppression method and transcription factor for tobacco cells or tissues. Expression of any one of the gene encoding the protein (a) to (c) and the gene containing the polynucleotide (d) to (g) using any one method selected from And regenerating plant bodies. -

Tobacco plants, for example, Nicotiana 'Tmentififolmis (N icoti anat ome ntosiformis), Nicotiana Ninocotiana 5 7.1 6 5 1: 15: Nicotiana * Noresti Force (N icotianarustica), Nicotiana 'Tanokaku (N icotianatabac um), Nicotiana Plumbagi Examples include Nifolia (N icotianap 1 u mb aginifolia, N icotianaxsanderae), but are not particularly limited, but Nicotiana Tabacam is preferred. Varieties of varieties used as cigarette products, cigars used as cigarette ingredients, oriental varieties grown in the Orient region, and tobacco seeds have been introduced to various parts of the world. Including native species adapted to the climate and differentiated.

 Plant materials for producing the tobacco genus plant of the present invention include plant bodies and plant tissues (eg, roots, stems, leaves, seeds, embryos, ovules, ovary, shoot tips, cocoons, pollen, etc.) and their Plant culture cells such as slices, cells, callus, proplasts that have been treated with enzymes to remove cell walls, and suspension culture cells.

 Various gene engineering techniques can be used to suppress the expression of the above genes, and are not particularly limited. For example, RNA interference method, antisense method, gene disruption method, artificial mutation method, ribozyme method , Co-suppression methods or methods of controlling transcription factors.

 RNA interference method is performed by the RNA interference induction vector, or double-stranded RNA homologous to, for example, the polynucleotide shown in SEQ ID NO: 5 or 7, or a polynucleotide having 90% or more identity to SEQ ID NO: 5 or 7. Among them, a continuous example, preferably a double-stranded RNA of about 15 to 35 bases) is introduced into a plant cell or tissue.

 In addition, the antisense method can be performed, for example, by introducing the antisense vector into a plant cell or tissue.

Examples of the introduction of these vectors include the agglomeration method, the electroporation method, the particle gun method, the PEG-calcium phosphate method, the re-posome method, and the microinjection method. Although not preferred, the agrobacterium method is preferred. When using the agro-battery method, In some cases, tissue pieces (leaf pieces, callus, etc.) may be used.

 When using protoplasts, co-cultivation with agrobacterium with T i plasmid, fusion with agroplast plasmosome

(Sufuguchi plast method), in the case of using cultured cells, the method of co-culturing with agro-pacterium having T i plasmid, the method of infecting the sterilized cultured leaf pieces (leaf disc) of the target plant in the case of using tissue pieces, This can be done by infecting callus.

 Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, DNA is prepared from transformed plants, DNA-specific primers are designed, and PCR is performed. After PCR, the amplified product is electrophoresed, stained with bromide zyme, SYBR Green solution, etc., and the amplified product is detected as a single band to confirm that it has been transformed. be able to. It is also possible to detect the amplification product by performing PCR using a primer previously labeled with a fluorescent dye or the like. In addition, the amplified product can be bound to a solid phase such as a microplate, and the amplified product can be confirmed by fluorescence or enzymatic reaction, or DNA can be quantified based on the amplification rate using the comparative Ct method in real-time PCR. Yo!

 Alternatively, a vector in which the above-mentioned various reporter genes are linked to the downstream region of the target gene is prepared, and a plant is transformed in the same manner as described above using agropacterime into which the vector is introduced, and the expression of the reporter gene You can confirm this by measuring.

 The gene disruption method can be performed using various transposons and T-DNA.

Gene disruption using a transposon is a method that uses the gene insertion mechanism of the transposon into the genome. In particular, the method using an endogenous transposon is superior in that a large number of gene disruption lines can be produced at a time as a non-recombinant. Such endogenous insertion factors include transpozo in tobacco plants. A retrotransposon T to 1 is known (WO 00/0 7 1 699).

 Gene disruption using T-DNA is performed by inserting a foreign gene such as a drug resistance gene into the target gene and inserting it between the right border (RB) and the left border (LB). This is a method for producing and transforming tobacco plants using the agrobacterium method.

 Artificial mutagenesis is, for example, by irradiating plants with various types of radiation (electromagnetic waves, ultraviolet rays, X rays, γ rays, particle rays, neutron rays, α rays, i3 rays, electrons, ion beams, etc.) This can be done by treating with various mutagenic chemicals (alkylating agents, nucleobase analogs, sodium azide, etc.). '

 Co-suppression is a phenomenon in which when a gene having the same or similar sequence as a target endogenous gene is introduced into a plant by transformation, the expression of the introduced foreign gene and target endogenous gene are both suppressed. Point to (Smy th DR: Cu rr B iol 7: R 793, 1 9 9 7, Ma rtienssen R: C urr B io 1 6: 8 1 0, 1 996). The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with that of RNAI. For example, in order to obtain plants in which the # 1 049 1 gene is co-suppressed, use the # 1 049 1 gene or a vector prepared so that it can express DNA having a similar sequence. Then, the target plant may be transformed. The gene used for co-suppression need not be exactly the same as the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the sequence identity Have sex. In addition, sequence identity can be determined by the method described above.

 The ribozyme used in the ribozyme method is a general term for RNA molecules having catalytic activity. In particular, in this specification, it refers to RNA molecules designed to cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and Ml RNA contained in RNase P. However, the hammerhead type has an active domain of about 40 nucleotides called hairpin type. Some things (Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzymes, 35: 2 1 9 1,

1 990). Ribozymes designed to cleave targets are translocated in plant cells. As shown, it is linked to a transcription motor and transcription termination sequence such as the 35 S promoter of cauliflower mosaic virus. At this time, if an extra sequence is added to the 5 'end or 3' end of the transcribed RNA, the ribozyme activity may be lost. In such cases, the RNA containing the transcribed ribozyme may be lost. It is also possible to place another trimming ribozyme that works in cis on the 5 'side or 3' side of the ribozyme part (T aira K, eta 1: Protein En g). 3: 733, 1 990, D zianott AM & B ujarski JJ: Proc Na tl Ac ad S ci US A 86: 4823, 1 989, Grossha ris CA & Cec hTR: Nucl Ac ids R es 1 9: 38 75, 1 99 1, T aira K, eta 1: Nuc 1 Ac ids Res 1 9: 5 1 25, 1 99 1). It is also possible to increase the effect by arranging such structural units in tandem so that multiple sites within the target gene can be cleaved (Yu ya ma N, eta 1: B ioch em B iophys R es C o mm un 1 86: 1 27 1, 1 9 92). Thus, by specifically cleaving the transcript of the target gene of the present invention using a ribozyme, the expression of the gene can be suppressed.

 The method of controlling a transcription factor is a method of indirectly increasing or decreasing the expression of a target gene by increasing or decreasing the expression of a transcription factor that regulates the expression of the target gene. A transcription factor controls the expression of a gene by binding to a specific sequence (cis element) in the promoter region of the target gene. For example, by expressing a transcription factor, it is possible to suppress the gene expression of a metabolic gene (P 1 ant Molec lar Al Biol og 2006 6 2: 809-82 3).

 When plant tissue or cells are used as a material in the above genetic engineering technique, an organ or an individual may be regenerated by using a known tissue culture method from the obtained plant tissue or cells. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. Regeneration from plant cells to plants can be performed, for example, as follows.

First, plant tissues or protoplasts are used as plant materials for transformation. When used, they are cultured in a sterilized callus-forming medium with the addition of inorganic elements, vitamins, carbon sources, sugars as energy sources, plant growth regulators (plant hormones such as auxin and cytokinin), etc. It forms dedifferentiated callus that grows in a regular shape (hereinafter referred to as “callus induction”). The callus formed in this way is transferred to a new medium containing a plant growth regulator such as oxine and further grown (subcultured).

 When callus induction is performed in a solid medium such as agar and subculture is performed in, for example, liquid culture, each culture can be performed efficiently and in large quantities. Next, callus grown by the above subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as “redifferentiation induction”), and finally regenerates a complete plant body. . Induction of regeneration can be performed by appropriately setting the kind and amount of various components such as plant growth regulators such as auxin and cytokinin, carbon sources, light, temperature, etc. in the medium. By such redifferentiation induction, somatic embryos, adventitious roots, adventitious buds, adventitious foliage, etc. are formed and further grown into complete plants.

 The tobacco genus plant produced by the method of the present invention is obtained from the seed of the plant using the 0th generation plant as a parent, in addition to the “TO generation” which is a regenerated generation that has undergone transformation treatment. The next generation (Τ1 generation), the next generation (Τ2 generation) that is obtained by self-pollination of plant flowers. ) And other progeny plants. Various tobacco products such as cigarettes, cigars and pipe tobacco can be produced using these tobacco plants or their leaves as a raw material for tobacco products.

The quality of dry leaf tobacco, the raw material for tobacco products, depends greatly on the maturity of the tobacco leaves being cultivated. It is believed that the fertilizer will wither in the tens of days from the centering to the harvest, and that tapaco leaves will gradually mature with increasing dry weight. During this period, the content of tobacco leaves changes greatly, including increased alkaloid content and chlorophyll degradation. In other words, components related to the taste of dried tobacco are generated and accumulated with dramatic metabolic changes during the mature period. Therefore, the quality of dry leaf tobacco can be controlled by genes expressed in relation to tobacco leaf maturity. High-quality dry tobacco is used to properly dry tobacco leaves that are reasonably mature. It is possible to produce only with. For example, the dry leaves of good quality yellow tapaco are finished in a bright yellow color, fragrant, and characterized by a mild and sweet taste. Color tone is an important factor in determining the quality of dried leaves. For yellow tobacco, vibrant colors such as lemon and orange have good quality, and those with mottled and green colors have poor quality. (O fficial Standard Gradesfortlue — Cu red 1 obacco US ypes 1 1, 1 2, 1 3, 14, & Foreign Ty pe 92, United States D epart me ntof Ag riculture). The quality of dried leaf tobacco is also greatly affected by the drying process. Leaf Tobacco This drying process is not just for the purpose of drying leaves, but is an extremely important step in leaf tobacco production by changing the components in the leaves and expressing the unique color and flavor of the tobacco. In addition, when there are problems with management in drying processing, such as temperature and humidity and leaf suspension density, dry tobacco leaves become abnormal dry leaves such as quick-drying leaves, mulled leaves, and belted green leaves. It will be greatly damaged. The appearance of abnormal leaves and the ease of drying in such a drying process depend not only on the maturity level but also on the variety and soil environment. For example, “easiness of drying” as a genetic trait is also used for breeding of varieties.

 The transgenic tobacco (# 1 049 1 tobacco) that suppressed the expression of the # 1 049 1 gene of the present invention had almost the browning observed in the control tobacco introduced with the vector control when the leaves were dried. It has a bright yellow color and can be a good quality raw material. In addition, the # 1 049 1 cigarette of the present invention was also recognized as having an early maturity compared to the control tapaco.

 It should be noted that the above-mentioned leaf tobacco production is well known to those skilled in the art, and D a v i s D L .e t a l., T o b a c c o: P r o d u c t i o n,

Ch em istryand T echno 1 ogy (1 999) (B lac kw ell P ublishing), Collins WK. Eta 1., P rinciplesof F lue— Cu red T obacco Pro roduction (1 st E dition, 1 9 93) ( No rth C arolina

State Un iversity), Kawada, leaf tobacco cultivation method (1 99 1) (day It is also written in books such as this tobacco industry.

 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples. However, these examples do not limit the present invention.

Example

Example 1

 [Identification of a gene whose expression level changes greatly during maturity (# 1 04 9 1 gene)] The present inventors have determined that the maturity level of tobacco leaves is the raw material for the purpose of identifying genes that affect the quality of the raw leaf tobacco. Focusing on the fact that the quality of leaf tobacco is greatly influenced, in the leaf of tobacco grown in soil cultivation, or in the leaf of hydroponically cultivated tobacco in which maturation was induced artificially by reducing the amount of nitrogen fertilization, We searched for genes whose expression level changes greatly. As a result, we identified the # 1 0 4 9 1 gene as one of the genes whose expression specifically increases during tobacco leaf maturation.

 (1) Cultivation of tobacco and extraction of RNA samples from leaves

 The soil-cultivated tobacco (Tsukuba No. 1) was transplanted to No. 4 clay pot (terracotta; diameter 1 24 mm, height 1 1 Omm) 6 weeks after sowing, and meteorological meteorological equipment (Cottron: Koito Industries) (Made by Co., Ltd.) Fertilizer for pots is soil: compost: red ball (large), red ball

(Small): Vermiculite = 3 ·: 4: 1: 1: 1 fertilizer with a composition of 1: 1 Barrel per 6 liters S 6 2 5 (fertilizer) 1 kg mixed was used. The meteorological device is in the light period: 6: 3 0—1 8:30, temperature 26 ° C, humidity 60% (R H), long period: 1 8: 3

0-6: 30, temperature 18 ° C, humidity 80% (RH). Port water was used once a day so that the soil would not dry, and the amount of water was adjusted appropriately according to the growth. All the above-ground parts of the seedlings were collected at the time of transplantation, and used as a sample on the 0th day of transplantation (N = 8). Furthermore, after transplantation 1,

On the 2nd, 4th, 5th and 5th day, at 9:00:00 am, the first leaf from the bottom was collected. (1 Day 4 N = 3 6, 2 8-5 Day 2 N = 6, 4 Day 5 N = 9).

Hydroponic tobacco (Tsukuba No. 1) was transplanted to a polypot (round, 1 2. O cm, 9.8 cm, 1 bottom hole) filled with Hydroball (small grain) on the 8th day of germination. and, water culture medium containing _{1 5 mM KN0 3 (2. 5} mM K pi: p H 5. 5, 2 m

M Mg SO ₄ , 2 mM CaCl ₂ , 50 μ 5 Fe-EDTA, 70 MH ₃ BO ₃ , 14 μΜ Mn C l ₂ , 0.5 μΜ Cu SO ₄ , 1 MZ n S 0 ₄ , 0.2 μΜ NaMo O ₄ , 1 0 / zM N a C l, 0.0 1 / MC o C 1 ₂ ) was irrigated from the bottom and grown in an artificial meteor like the soil-cultivated tobacco. The reduced hydroponics was replenished as appropriate to maintain the volume. On the 7th day after hydroponics (germination 35), some plants were transferred to a hydroponic solution containing 0.3 mM ₃ to induce maturation. Meanwhile, plants grown in hydroponic solution containing the or or 1 5mM KN0 ₃ were the control. From these plants, the seventh leaf from the bottom was collected at 9 am for several days after the seventh day of hydroponics.

 The collected leaves were frozen with liquid nitrogen and then ground to prepare samples for RNA extraction. The seedlings at the time of transplantation used the entire above-ground part as a sample. T o tal RN A was purified from about 4 g of sample using C o n c rt P l ant RNA r a ge nt (Inv ito r o ge ne). In addition, poly (A) RNA was purified using Total RNA 1, OOO ^ g force and oligo d T-cellulose affinity column method (Gen E 1 ute mRN A miniprepkit, Sigma). (The operation method described in the first place is carried out).

 (2) c DNA microarray analysis

 The po 1 y (A) RNA obtained as described above was analyzed using a BY-2 ES T microarray equipped with approximately 170,000 kinds of ES T derived from tobacco culture cells of RIKEN. RIKEN B Y_ 2-E For more information on S T microarray Ma t s u o k a et al (2 0 04, A c o m p r e h e n s i v e g e n e e x p r e s s i o n a n a 1 y s i s t o w a r d t h e u n d e r s t a n d i n g o f g r o w t h a n d d i f f e r e n t i a t i o n o f t o b a c c o BY- 2 e e l 1 s • P 1 a n t C e 1 1

Physio 1 4 5: 1 2 8 0-9) and G a 1 is et al. (2 0 0 6, A nove 1 R 2 R 3 MYB transcri Ption factor N t MYB JS 1 isamethy 1 jasmonate— dependentregu 1 atorofpheny 1 P ro P anoid— conjgatebiosynthesisi ntobacco. P 1 an t J., 46: 5 73-92.) and NCB I (National C enterfor Biotechno 1 ogy Information) GEO (Gene E xpression Omn ibus) homepage .

 (h t t p: www. n c b i. n 1 m • n i h. g o v / g e o Z q u e r y / a c c. c g i? a c c = G P 4 7 0 6)

 (h t t p: www. n c b i. n 1 m • n i h. g o v / g e o / q. u e r y / a c c. c g i? a c c = GP L 4 7 0 7)

 (h t t p: //www.n c b i .n 1 m-n i h. g o v / g e o / q.n e r y / a c c .c g i? a c c = G P L 4 7 0 8)

 (h t t p: //www.n c b i .n 1 m-n i h.g o v / g e o Z q u e r y / a c c .c g i? a c c = GP L 4 7 0 9)

 The po 1 y (A) RN A purified from each sample and the c RN A of the kanamycin resistance gene added as an internal control to confirm the processing procedure, linearity, sensitivity, and accuracy of the microarray analysis are made into a vertical type. An choredd T 2

Single-stranded cDNA probe using 5 primers (GE Healtheare), Random 9 mers primer (GE Healthcare) and Superscript III reverse transcriptase (Invitrogen) At the same time, a nucleic acid labeled with the fluorescent dye Cy5 (GE Healthcare) was incorporated into the cDNA probe. c DN A probe and oligo

The probe solution was prepared by mixing A80, Cy3 labeled vector and Express Hyb ^™ Hybridization Solution (Clontech). The probe high-pridization and scanning follow the method of Matsuoka et al. Analysis was performed using 7 f, .Gene Spring (Silicon Genetics). As a result of microarray analysis, an expression tag (EST) is one of the genes whose expression is specifically increased during tobacco leaf maturation.

A gene that is homologous to the 1 f 1 sequence (SEQ ID NO: 1) (G e n B a n k G I: 5282952 7) was found.

(3) Full length of # 1 049 1 gene Isolation of cDNA sequence The full-length cDNA corresponding to the 1049 1 f sequence (SEQ ID NO: 1) clarified as described above could be obtained from the cDNA library owned by the applicant by homology search. The applicant's cDNA library is a full-length cDNA clone of tobacco (Cultivar Tsukuba No. 1) isolated individually and arranged with terminal sequence information. Three clones (N o. 00 1 5_2_E 03, N o.

0052_2_F 08, No. 00 77— 1— F 0 7) is cultured overnight in LB (containing 50 mg ZL ampicillin) liquid medium, and the plasmid DNA contained in each clone is used as a template for sequence analysis. QIA prep S pin M

Purification was carried out using 1 n i p r e p K i t (Q I AG EN). # 1 049 1Sequence analysis of the undecoded region of the gene was performed based on the cDNA sequence information of the above three clones using Primer e xpress software (Applied Biosyst ems). It was performed using the designed SEQ ID NOs: 2 to 4, and primers such as Ml 3 Pri mer M4 (Takara Bio) and M 13 Pri mer RV (Takara Bio). Sequence analysis was performed using B iGDy e (registered trademark) Terminatorv 3.1 Cycle Sequencing K i "Ap plied B iosyst ems) and 3 700 DNA A na 1 yzer (Ap plied B iosyst em s) 1 049 1 f The sequence obtained as described above was obtained using a nucleotide sequence analysis software (ATGC Ver. 4, GENETYX; Genetics). The full-length cDNA sequence (SEQ ID NO: 5, # 1 049 1—c) of 1 720 bp by combining the sequence (SEQ ID NO: 1) and the c DN A terminal sequence of the above three clones. In addition, the amino acid sequence encoded by the # 1 049 1 gene (SEQ ID NO: 6, # 1 049 1—protein) was deduced. # 1 049 1 gene full-length cDNA sequence (SEQ ID NO: 5), a sequence comparison analysis by BLAST was performed. And it was estimated. Example 2

[# 1 049 hairpin ds RNA expression cassette to suppress the expression of 1 gene Construction of binary vector including

 The DNA sequence that triggers RNA i (SEQ ID NO: 7 # 1 049 1—triger) is the sequence of clone No. PCR was amplified from the same region shown in FIG. 1 using the 0 and 11 ply combinations. PCR amplification was performed using PfU Ultra ™ High-fidelity DNA Polymerase (Stratagene) and Gene Amp (registered trademark) PCR System 9 700 (Applied Biosyst ems). After 95 minutes at 95 ° C, the reaction was performed at 95 ° C for 30 seconds, 60 C for 30 seconds, 72 ° C for 60 seconds for 30 cycles, and 72 ° C for another 10 minutes. The two amplified DNA fragments are N 0 1 1 nose added by the primer at the 5 '/ 3' ends, respectively. It has 3 1 1 or 8 & mH I / S p I recognition sequences (Figure 1). The obtained amplified DNA was double-digested with the restriction enzyme added to the 5 ′ / 3 ′ end, respectively, and purified with MinE 1 utekit (Q I AG EN). .

 As a vector for constructing the inverted repeat sequence (that is, the repeat of the forward sequence and the reverse sequence), pSP1 48 (FIG. 2) modified from pDNR-1 (Clontech) was used. Between the two 1 o XP sites of p SP 148 (Figure 2), there are a 35 S promoter, a retic cloning site 1 (MC S 1), a spacer self-alignment, a reticulated cloning site 2 (MC It exists in the order of S 2) and NO S terminator. The amplified DNA fragment double-digested with the aforementioned restriction enzymes was introduced into the Pst I / Not I sequence introduction site of MC S-1 of this vector and the BamH I / Sph I sequence introduction site of MCS 2. By ligating each, p SP 1 48— # 1 049 1 FR containing a hairpin ds RNA expression cassette was prepared (Fig. 3).

Next, the hairpin ds RNA expression cassette constructed on p SP 148— # 1 049 1 FR uses Cre _ 1 ox recombination (US Pat. No. 6 4 1 0, 3 1 7). And replaced with the binary vector p SP 107 (Fig. 4). Binary vector PSP 1 07 (Fig. 4) is a modification of p BI 1 2 1 (Clontech) using Creator Acceptor Vector Configuration Kit (C 1 ontech). C re R ec Using omb inasekit (Clontech) and performing Cre-1ox P recombination according to the instructions, p SP 148— # 1 049 1 2 containing the hairpin ds RN A expression cassette on FR A binary vector, p SP 1 07— # 1 049 1, was prepared by replacing the 1 o XP fragment with the 1 o XP site on p SP 1 0 7 (FIG. 5). Example 3

 [Production of transgenic tobacco with suppressed expression of # 1 049 1 gene] Constructed binary vector p S P 1 07_ # 1 049 1 and p S P 1 0 7

(Control; vector control) and the bacterial strain L B A 4404 (Ho e kema e t a 1. (1 983)) of Ag ro b a c t e r i um t ume f a c i e n s

Na ture 303: 1 79— 1 80) by freezing and thawing and selecting for resistance to kanamycin, the target p SP 1 07— # 1 049 1 and p SP 1 07)) Ag robacteri um A colony introduced into the strain LB A4404 of t ume faciens was obtained.

 Tobacco leaf harvested from the most developed leaf of the tobacco variety Tsukuba No. 1 cultivated in a greenhouse for about 1.5 months, was converted to a sodium hypochlorite solution containing 2% effective chlorine (Twe e 2

The surface was sterilized for about 5 minutes with 3 drops of ZL), washed 3 times with sterilized water, and about 5 mm square leaf pieces were prepared using a mess. This leaf piece and p S P 1 07— # 1 049

1 or p SP 1 0 7 (control, agrobacteri um t ume faciens approximately 10 ⁸ cells transfected with vector J that expresses only GFP gene and npt II gene and L ins ma ierand S koog (1 965) 48 hours of co-cultivation in a liquid medium consisting of 30 g / L sucrose and 30 mg / L sucrose After washing the leaf pieces three times with sterile water containing cefotaxime 25 OmgZL and carpenicillin 250 mg / L After washing off the bacteria, Mu rashigeand S koog inorganic salts, sucrose 30 g / L, indoleacetic acid 0.

3mgz L, 6— (γ, γ—d i me t y l a l l y l— am i n o) p u r i n e 10 mg / L, kanamycin 100 mg L, cefotaxime 250 mg

/ L, Calpenicillin 250mgZL, and Gellan gum 0.3% primary selection It was placed on a medium (pH 5.8). After about 2 weeks of culture, callus-like cell mass showing kanamycin resistance was transformed into Mu rashigeand Skoog 1 2-inorganic salts, sucrose 15 gZL, kanamycin 10 Omg / L cefotaxime 250 mgZL and gellan gum 0.3% And placed on a secondary selection medium (pH 5.8).

 A callus-like cell cluster showing kanamycin resistance in a secondary selection medium was cultured for about 3 weeks, and then a 5 mm x 5 mm leaf fragment collected from a redifferentiated individual was used as a GU S assay (Hieieta 1., 1 994) I tried it. Individuals selected by G U sa s s a y were placed again in the secondary selection medium (plant box). Further, after culturing for about 3 weeks, the rooted individuals were transplanted into No. 4 clay pot (terracotta; diameter: 124 mm, height: 110 mm).

 In this way, finally, transgenic tobacco (hereinafter referred to as # 1 049 1 tobacco) transformed with the binary vector p S P 1 0 7— # 1 049 1

Twenty individuals, six transgenic tobacco (hereinafter referred to as control tobacco) transformed with pS P 107 were produced. Transgenic tobacco was cultivated in a closed greenhouse in which the temperature was adjusted to about 23 ° C day and night using a cooling device and a boiler device under natural light. Fertilizer for pots is soil: compost: red bean clay (dog), red bean clay (small): vermiculite = 3: 4: 1: 1: 1 fertilizer with composition of 1 barre per 100 liters S 625 (fertilizer) A mixture of 1 kg was used.

 In addition, # 1 049 1 Inhibition of # 1 049 1 gene expression in tobacco was reduced by real-time PCR (Wo nge t a l. (2005) B i o t e c h n i q u e s

39: 75-85) To confirm the above, punch out a leaf disk with a diameter of about 10 mm from the transgenic tobacco obtained as described above, about 2 months after potting, and RNA later (Amb ion The material stored in was used as the material for real-time PCR. RNA is extracted using RNe a s y P l a n t M i n i K i t (Q I AG EN), and Omn i s c r i p t RT

Reverse transcription reaction was performed using Kit (QI AGEN). A part of this reverse transcription reaction solution was subjected to real-time PCR. # 1049 1 and i3—actin TaqMan (registered trademark) (Roche Molecul for real-time PCR) ar System ems) Probe and primer were designed using PrimerExpress (Applied Biosyst ems) (SEQ ID NO: 1 2 — 1 7). For real-time PCR, use Qu anti Tect Multiplex PCR Kit (<31 0 £ 1 ^) and D up 1 e X real-time using the “Ding & 9] ^ & 11 probe attached to the kit. According to the “Quantitative PCR” protocol, the reaction was carried out on a 7500 real-time PCR system (Applied Biosyst ems) for 45 cycles at 95 ° C for 15 minutes, then 94 ° C for 1 minute and 60 ° C for 1 minute. went. Figure 6 shows the results of real-time PCR, analyzed by the comparative Ct method (U ser B'u lletin # 2, AB IPR I SM 7 700 Sequence Detection System A pp 1 ied B iosyst em s). . It was confirmed that the expression of the # 1 049 1 gene was suppressed to an average of 15% of the control in all 20 types of # 1 049 1 tobacco investigated. Example 4

 [Analysis of Transgenic Tobacco]

 (1) Transgenic cigarette form, leaf color

The Konica Minolta chlorophyll meter (S PAD) was used as an indicator of tobacco leaf maturity for the upper, middle, and lower leaves of Transgenic tobacco obtained as described above for about two months after planting. — 502) was used to measure the S PAD value (a numerical value indicating the amount of chlorophyll contained in the leaves of the plant). As shown in Table 1, the S PAD value of # 1 049 1 tobacco (n = 1 5) was the control. The value was significantly lower than that of tobacco (n = 6), suggesting that chlorophyll decreased and maturation progressed in # 1 049 1 tobacco. table 1

 Number of days after potting SPAD value

 # 10491 Tobacco Control tobacco

 Average value (n = 15) Standard deviation Average value (n = 6) Standard deviation Top 3 leaves Top 71 30.6 6.7 38.4 0.9

 Middle 71 29.4 6.5 36.5 2.3 Bottom 71 21.0 6.5 32.7 3.2 Middle 3 Leaf Top 63 25.4 5.0 38.4 1.7

 Medium 63 18.6 5.7 32.7 2.6 Lower 63 13.3 4.9 24.9 4.2 Lower 3 leaves Upper 55 21.7 4.8 32.8 1.9

 Medium 55 18.2 4.4 25.1 3.1 Bottom 55 10.2 4.5 18.4 4.9 Also, as shown in Figure 7, # 1 0 4 9 1 tobacco showed no significant difference in morphology compared to control tobacco.

 (2) Characteristics of dry tobacco tobacco from Transgenic tobacco

 Leaves were harvested from tobacco about 2 months after the above potting and dried in yellow to produce dry leaves. The yellow drying was performed using a thermo-hygrostat manufactured by ESPEC according to a program consisting of 13 steps. Each step is as follows.

Step 1: After the start of drying, heat up and humidify for 3 hours. After reaching a temperature of 38 ° C / relative humidity 1 • 0 0%, treat under these conditions for 8 hours. Step 2: Next, raise the temperature over a period of 1 hour, dehumidify, reach 40 ° C / 87% relative humidity, and then treat for 15 hours under these conditions. Step 3: Next, heat up and dehumidify over 1 hour. After reaching a temperature of 43% relative humidity of 63%, treat under this condition for 15 hours. Step 4: Next, the temperature is raised and dehumidified over 1 hour. After reaching a temperature of 45 ° CZ and a relative humidity of 52%, it is treated under this condition for 15 hours. Step 5: Next, heat up and dehumidify for 1 hour. After reaching a temperature of 48 ° C / relative humidity of 43%, treat under this condition for 15 hours. Step 6: Next, heat / humidify over 1 hour, temperature 50 ° C / relative humidity 37% After reaching, processing for 10 hours under this condition. Step 7: Next, heat it up over 1 hour, dehumidify, and reach a temperature of 53 ° CZ relative humidity 3 1%, then treat under this condition for 8 hours. Step 8: Next, heat up Z for 1 hour, dehumidify, reach 56 ° C at a relative humidity of 26%, and then process for 6 hours under this condition. Step 9: Next, heat up Z over 1 hour, dehumidify, and after reaching 60 ° C / 20% relative humidity, treat for 6 hours under these conditions. Step 10: Next, heat up and dehumidify over 1 hour, and after 6% of relative humidity reaches 16%, treat under this condition for 6 hours. Step 11: Next, the temperature is increased over 2 hours, and after reaching a temperature of 65 ° CZ relative humidity of 16%, it is treated for 2 hours under these conditions. Step 1 2: Next, the temperature is raised in 1 hour, and after reaching a temperature of 68 ° C and relative humidity of 16%, it is treated for 8 hours under this condition. Step 1 3: Over 5 hours, temperature is lowered to 30 ° C / relative humidity 60%.

 Comparing the dry leaf finish, which is an indicator of the quality of the raw tobacco, between the two types of transgenic tobacco, as shown in Figure 8, the dry leaf of the control tobacco is browned, but # 1 049 1 The dry leaf finish was very clean. This result was in good agreement with the theory of leaf tobacco production, where high-quality raw tobacco was produced from mature tobacco leaves. From these results, it can be concluded that the suppression of the # 1 049 1 gene expression promoted tobacco leaf maturation and improved the finish of dry leaf tobacco, giving dryness to tobacco leaves.

(3) Content of carbonyls in the combustion smoke of dry leaves of Transgenic tobacco

 The dried leaves of Transgenic tobacco produced as above are 40 ° C, 95%

Humidified at RH for 2 hours, chopped into tobacco, then conditioned at 25 ° C, 60% RH for over 1 week, and then an infrared image furnace with a quartz tube as the reactor (U 1 V a c _

The content of formaldehyde and acrolein in the smoke was measured in a pyrolysis test using R i k o I n c.). The carrier gas was 100% nitrogen gas, and the flow rate was adjusted to 1 000 m 1 Zm i n using SEC—B 40 ma s s f l o c o n t r o l l e r (Ho r i b a St e c I n c.). A sample of 150 mg of tobacco was used as a sample, and packed in a width of 12.5 mm in the center of the quartz tube.

After replacing the air in the quartz tube with carrier gas for 1 minute, the temperature was raised from room temperature to 800 ° C at a rate of 16.7 ° C / s, and the temperature was fixed at 800 ° C for 5 seconds. this For temperature control, TPC — 1 000 progr am emp erarurecontroller (U lvac -R iko I n c.) Was used. Smoke generated by thermal decomposition consists of an impinger containing 100 ml of 2,4-dinitrophenylhydrazine solution and a 44-nm filter (He inr.

B o r g w a 1 d t T e c h n i k). The above method followed the method of Tori k ai e ta 1. (2004). The 2, 4-d i n i t r o p h e n y l h y d r a z i n e solution was weighed 2, 4—d i n i t r o p h e n y l h y d r a z i ne (containing 50% water) 9.5 1 0 7 g

After dissolving in 50 OmL and adding 2.8 mL of 60% perchloric acid, ultrapure water was added to make a 1 L aqueous solution. 44 mL of smoke collected, 3 OmL

2, 4-d i n i t r o p h e n y l h y d r a z i n e solution was shaken for 30 minutes, smoke was collected and mixed with 2, 4-d i n t r o p h e n y l h y d r a z i ne e solution (impinger). 2,4-dinitrophenylhydrazine mixed solution and Trizma base solution were mixed at a ratio of 2: 3, filtered through 0.45_ / mpolytetrafluoroethylene membrane filter, and then analyzed by high performance liquid chromatography. . The above method is based on the above-mentioned T o r i k a i e t a 1.

(2004). The analysis method using high performance liquid chromatography is He a l t h C a n a d a (1 999) Me t h o d o f No. T-

Compliant with 1 04. The T r i ζ m a b a s e solution is 0.4 g L of T r i zma b a s e, dissolved in 4 OmL of ultrapure water, and added with acetonitrile.

A 0 mL aqueous solution was used.

 # 1 049 1 The results of infrared image furnace analysis of tobacco and control tobacco are shown in Figure 9. In the analysis, we measured # 1 049 1 cigarettes and 3 control cigarettes, and calculated relative values (%) when the amount of formaldehyde and acrolein produced in the control cigarettes was 100%. Calculated. As a result, # 1 049

In # 1 049 1 tobacco, which suppressed the expression of 1 gene, the formaldehyde and acrolein contents in the smoke were significantly reduced, averaging 45% and 68%, respectively, in the control. # 1 049 1 Content of carbonyls in combustion smoke in tobacco This reduction is thought to be due to the reduced production of some components in the leaves, which are precursors of carbonyls in the combustion smoke. Therefore, the control of the carbonyl group in the smoke of dry leaf tobacco can be realized by using tobacco leaves with controlled expression of the # 1 049 1 gene. Example 5

 [# 1 049 Study on sequences for producing transgenic tobacco with suppressed expression of 1 gene]

 A new binary vector p S P 204 (FIG. 10) was used to construct an inverted repeat. The binary vector p SP 204 is a modified version of PSP 20 1 (Fig. 11) derived from p BI 1 2 1 and is based on the use of Gate wa y (registered trademark) Technology (Invitrogen). Directly repeated sequences can be constructed directly on a binary vector.

 # 1 049 1 3 DNA sequence in the middle of the gene (SEQ ID NO: 1 8, called M_triger) and 3 'downstream DNA sequence (SEQ ID NO: 1) 9, 3 '_triger and R) were amplified by PCR (Fig. 12). PCR amplification is shown in Example 2 above using clones of clone No. 00 1 5_2—E 03 as a saddle and using the primer combinations of SEQ ID NOs: 20 and 21 or SEQ ID NOs: 22 and 23. Performed under conditions. The amplified DNA fragment was cloned using the p ENTR / D—TOPO cloning kit (Invitrogen) to produce a Gatway entry single vector. Next, the insert DNA on the entry vector was reversely transferred onto the binary vector p SP 204 by X att R recombination using Ga te wa y (registered trademark) LR clonase 11 1 A binary vector pSP204—M—triger and a binary vector pSP204—3'_triger were constructed by insertion in the form of repetitive sequences (Figure 13).

Transgenic tobacco was produced according to the method shown in Example 3 above, and finally transformed with the binary vector p SP 204_M_triger (hereinafter referred to as # 1 049 1—M and U) 1 2 individuals and a ba Inary vector p SP 204— 3 '— Transgenic tobacco transformed with triger (hereinafter referred to as # 1 049 1— 3') 1 1 individual, binary vector p SP 20 1 Two transgenic tobaccos (hereinafter referred to as Control 2) were prepared. In parallel with the production of the new transgenic tobacco, the T 2 generation of transgenic tobacco obtained from self-breeding of the # 1 049 1 tobacco produced in Example 3 above (hereinafter referred to as # 1 049 1-5) ), And the wild-type tobacco variety Tsukuba No. 1 was nurtured.

 The expression level of # 1 049 1 gene in transgenic tobacco was investigated using the method by real-time PCR described in Example 3 above. Figure 14 shows the results of real-time PCR analyzed by the comparative Ct method. In # 1 049 1_M and # 1 049 1— 3 ′, the expression level of # 1 049 1 gene was almost the same as # 1 049 1—5 ′, which was strongly suppressed compared to control 2. Industrial applicability

 ADVANTAGE OF THE INVENTION According to this invention, the tobacco plant and dry leaf tobacco characterized by the content of carbonyls in combustion smoke being reduced can be provided.

 In addition, transformed tobacco plants usually have morphological effects such as leaf dwarfing and fewer leaves, resulting in a decrease in yield. Surprisingly, tobacco plants that suppress the expression of the generated gene have no morphological effect, and mature more rapidly than non-transformed tobacco plants, and are easy to dry. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims.

1. A tobacco genus plant in which expression of a gene encoding any one of the following proteins (a) to (c) is suppressed.

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the content of carbonyls in plant combustion smoke

 (c) A protein comprising an amino acid sequence having an identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 6, and capable of controlling the carbonyl content in the combustion smoke of plants.

 2. The gene is

 (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 5,

 (e) a polynucleotide encoding a protein having 85% or more identity to the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke;

 (f) Coding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5 and capable of controlling the carbonyl content in plant combustion smoke Polynucleotide to

 (g) A protein that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 and that can control the carbonyl content in the combustion smoke of plants. Polynucleotide

The tobacco genus plant according to claim 1, comprising:

 3. The carbonyl compound is selected from the group consisting of formaldehyde, acetoaldehyde, acetone, acrolein, propion aldehyde, croton aldehyde, methyl ketylketone and butyl aldehyde. Tobacco genus plant.

4. The carbonyl compound is formaldehyde or acrolein. The tobacco genus plant according to claim 3.

 5. The tissue or cell of the tobacco plant according to any one of claims 1 to 4.

6. The tissue or cell according to claim 5, wherein the tissue is a leaf.

 7. A group consisting of the base sequence shown in SEQ ID NO: 5, the base sequence shown in SEQ ID NO: 7, a base sequence having 90% or more identity to these sequences, and a partial sequence of 20 bases or more of those base sequences A gene expression suppression vector comprising a base sequence selected from

 8. The vector according to claim 7, comprising a base sequence consisting of 20 or more consecutive base sequences in the antisense direction.

 9. The vector according to claim 7, comprising a sense strand of the base sequence and an antisense strand paired with the sense strand.

 10. The vector according to claim 9, wherein the sense strand and the antisense strand are composed of 20 bases or more in which the base sequence is continuous.

 1 1. The vector according to claim 9 or 10, which has a spacer between the sense strand and the antisense strand.

 1 2. The vector according to any one of claims 7 to 11, which has a promoter operable in plant cells.

 1 3. Any of the cells or tissues of the genus Tobacco selected from RNA interference method, antisense method, gene disruption method, artificial mutation method, ribozyme method, co-suppression method and method of controlling transcription factor The method according to any one of claims 1 to 4, comprising suppressing the expression of any of the genes (a) to (g) defined in claims 1 and 2 and regenerating a plant. A method for producing the tobacco genus plant according to claim 1.

 14. The method according to claim 13, wherein the vector according to any one of claims 7 to 12 is introduced into a plant cell or tissue to regenerate the plant body.

 1 5. The method according to claim 14, wherein a progeny is created using the plant body as a master.

1 6. A progeny of the tobacco plant according to any one of claims 1 to 4, wherein the expression of any of the genes (a) to (g) defined in claim 1 or 2 is suppressed. A progeny characterized by

1 7. Tobacco plant according to any one of claims 1 to 4 or claim 1 6 Tobacco products made from the progeny leaves described in 1.

 1 8. In a tobacco genus plant, including the suppression of the expression of any one of the genes (a) to (g) defined in claim 1 or 2, How to reduce.

 1 9. A method selected from the group consisting of an RNA interference method, an antisense method, a gene disruption method, an artificial mutation method, a ribozyme method, a co-suppression method, and a method of controlling a transcription factor for suppressing the expression of the gene. The method of claim 18, comprising:

 20. The method according to claim 18 or 19, wherein the vector according to any one of claims 7 to 12 is introduced into a plant cell or tissue.