JP3138452B2 - Genes involved in anther dehiscence - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to DAD1 (DEFECTIVE I
The present invention relates to a N ANTHER DEHISCENCE 1) gene and a plant in which the expression of the gene has been suppressed. The DAD1 gene is a gene that controls the cleavage of anthers and the maturation of pollen. By suppressing the expression of the DAD1 gene, it is possible to produce a male sterile plant having a flower organ structure as close as possible to the wild type. The male sterile plant thus produced can restore fertility by treatment with jasmonic acid and linolenic acid, and can obtain self-fertilized seeds. Therefore, it is possible to breed a first-generation hybrid variety without requiring a maintenance line.

[0002]

2. Description of the Related Art In recent years, breeding of hybrids of the first generation (F1) utilizing heterosis in many crops has been promoted. The advantage of this F1 cultivar is that the emergence of heterosis will lead to excellent agricultural traits such as yield, disease resistance,
It is easy to impart a single-factor dominant agricultural trait, has high uniformity, and protects the rights of breeders because the genetic traits are separated in the next generation.

[0003] In the F1 variety, it is necessary to secure a large amount of seeds of the first generation of hybrids, and as a technique therefor, it has been practiced to use those having genetic male sterility for mothers. In addition, there are a method of utilizing self-incompatibility, a method of artificially removing male flowers, a method of artificially crossing, and the like. However, these methods have limited available plant species. Methods utilizing genetic male sterility have been widely used to secure F1 seeds because they are the most versatile.

[0004] There are two types of genetic male sterility, male sterility (GMS) controlled by nuclear genes and cytoplasmic male sterility (CMS) involving both nuclear genes and cytoplasmic factors. . When breeding F1 varieties, for the following reasons
CMS has been used. In the case of GMS, only individuals homozygous for that gene show male sterility,
Heterozygous individuals usually have fertility. Efficient production of F1 seeds requires that all individuals used for mothers be male-sterile, which means that when GMS is used to produce F1 seeds, This means that all individuals used must be homozygous for the gene for male sterility. However, GMS cannot produce and maintain homozygous individuals by selfing because the pollen itself is not available.
Self-pollinating heterozygous individuals can produce homozygous individuals, but in this case, only 1/4 homozygous individuals are obtained and cannot be put to practical use.

[0005] However, in the case of GMS, if a technique for obtaining a population homozygous for the gene is developed,
Using this, the production of F1 seeds becomes possible. In fact, a method of selecting male-sterile homozygous individuals has been devised and used in some cases, but there has been no method for breeding a homozygous population.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a nuclear gene that controls anther dehiscence and pollen maturation, and that the gene expression is controlled to provide male sterility and male fertility. The purpose of the present invention is to provide a technique for controlling gender so that all individuals of a plant population used for mothers become male sterile.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the T-DNA tagging method does not cause anther dehiscence even after flowering, and Arabidopsis thaliana Mutant (dad1) was successfully created. The pollen of this mutant lost germination ability and was male sterile. When the buds of the dad1 mutant were treated with jasmonic acid or linolenic acid, the flowers that subsequently blossomed exhibited anther dehiscence and formed inbred seeds. this
Genomic and cDNA clones of the DAD1 gene were isolated from the dad1 mutant based on the T-DNA sequence.
When the isolated genomic clone was ligated to a plant transfer vector and transformed into a dad1 mutant, the regenerated plants showed anther dehiscence and self-seeded seed formation, and were restored to the wild-type phenotype. . This result indicates that the isolated gene is the DAD1 gene. Furthermore, it was found that by incorporating an antisense DAD1 gene reversely linked to the DAD1 promoter into plant genomic DNA, a plant body without anther dehiscence can be produced, similarly to the dad1 mutant. The present invention has been completed based on the above findings.

That is, the first aspect of the present invention is the following (a) or (b)
Having the promoter activity represented by the nucleotide sequence of
It is about A. (a) the base sequence of SEQ ID NO: 3 (b) the base sequence of SEQ ID NO: 3 in which one or more bases have been deleted, substituted or added

A second aspect of the present invention relates to a DNA encoding the following protein (a) or (b). (a) a protein represented by the amino acid sequence of SEQ ID NO: 2 (b) an amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are deleted, substituted or added, That have the function of controlling cleavage of DNA

A third aspect of the present invention relates to a DNA represented by a nucleotide sequence complementary to the DNA of the second aspect. A fourth aspect of the present invention relates to a male sterile plant, wherein the expression of the DNA of the second aspect is suppressed. A fifth aspect of the present invention relates to a method for producing a male sterile plant, which comprises suppressing the expression of the DNA of the second aspect. A sixth aspect of the present invention relates to a method for restoring fertility, comprising treating the male sterile plant of the fourth aspect with jasmonic acid, linolenic acid, or a derivative thereof.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) First invention The DNA of the first invention is a DNA having a promoter activity, wherein one or more bases are deleted or deleted in the base sequence of SEQ ID NO: 3 or the base sequence of SEQ ID NO: 3. It is represented by the added base sequence. Such a DNA represented by a nucleotide sequence different from SEQ ID NO: 3 can be obtained by a technique commonly used at the time of filing the present application, for example, a site-directed mutagenesis method (Zoller et al., Nucleic Aci.
d Res. 10: 6487-6500, 1982) and DNA represented by the nucleotide sequence of SEQ ID NO: 3.
And DNA isolated from a different gene source.

[0012] The DNA of the first invention can also be obtained by the method described in the Examples, but since its base sequence has already been determined, a primer is synthesized based on this sequence.
It can also be obtained by performing PCR using DNA containing this gene as a template. The primer used is not particularly limited. As the DNA used as a template, plasmid pGD
AD1-HS (a strain containing this plasmid has been deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-17299).

(2) Second invention The DNA of the second invention is a protein represented by the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or more amino acid sequences are deleted. Encodes a protein represented by a substituted or added amino acid sequence and having a function of controlling anther dehiscence. Such a DNA encoding an amino acid sequence different from SEQ ID NO: 2 can be obtained by a technique commonly used at the time of filing the present application, for example, site-directed mutagenesis (Zoll
er et al., Nucleic Acid Res. 10: 6487-6500, 1982)
And DNA isolated from a gene source different from the DNA encoding the amino acid sequence set forth in SEQ ID NO: 2, and the like. The DNA encoding the protein represented by the amino acid sequence set forth in SEQ ID NO: 2 was isolated from a genomic library of Arabidopsis thaliana of WS ecotype.
r DNA isolated from the ecotype Arabidopsis thaliana cDNA library is Val-Val-V at positions 13 to 15 of SEQ ID NO: 2.
It encoded a sequence in which another Val was added to al. Needless to say, such a DNA is also included in the DNA of the second invention from the above-mentioned purpose.

[0014] Whether a certain DNA has a function of controlling the cleavage of anthers is determined by introducing the DNA into a plant in which the expression of endogenous DAD1 is suppressed (for example, "dad1" described in Example 2). It can be determined by examining whether or not the anther dehiscence is restored. The DNA of the second invention can also be obtained by the method described in Examples, but since the base sequence has already been determined, a primer is synthesized based on this sequence, and PCR is performed using DNA containing this gene as a template. It can also be obtained by performing. The primer used is not particularly limited. Examples of the DNA used as a template include the above plasmid pGDAD1-HS.

(3) Third Invention The DNA of the third invention is represented by a nucleotide sequence complementary to the DNA of the second invention. Although the DNA of the third invention can be obtained by the method described in the Examples, since its base sequence can be easily obtained from the sequence described in the sequence listing,
It can also be obtained by synthesizing a primer based on this sequence and performing PCR using DNA containing this gene as a template. The primer used is not particularly limited. Examples of the DNA used as a template include the above plasmid pGDAD1-HS.

(4) Fourth Invention The male sterile plant of the fourth invention is characterized in that the expression of the DNA of the second invention is suppressed. The type of plant is not particularly limited as long as it has the DNA of the second invention, and includes, for example, plants of the Brassicaceae, Poaceae, Compositae, Acalyptaceae and the like.

The following two points can be cited as characteristics of the male-sterile plant. One is that the trait is almost identical to that of the wild type, except that the anthers do not cleave. F1 seed utilizing male sterility, for example,
There are only a few reports of cruciferous vegetables. This is because many of the conventional male sterile lines have abnormal flower organs, so that pollinating insects do not visit flowers and the seeding amount is small. The male sterile plant of the present invention has a flower organ as close as possible to the wild type, so that it is easy to visit flowers of pollinating insects such as bees and flies, and it is possible to increase the seeding amount. Another feature of this plant is that its fertility can be restored by treatment with linolenic acid or jasmonic acid.
The male sterile plant of the present invention can easily obtain self-fertile seeds from such properties, and can easily produce a population of male sterile gene homozygous. This male sterile plant can be produced, for example, according to the production method of the fifth invention described below.

(5) Fifth Invention The production method of the fifth invention is characterized in that the expression of the DNA of the second invention is suppressed. The method for suppressing the expression of the DNA of the second invention is not particularly limited, but preferred methods include the following antisense method, co-suppression method, gene disruption tagging method, gene targeting method, and mutagen source. Methods and the like can be exemplified.

I) Antisense method This method comprises the steps of constructing a vector containing an appropriate promoter and the DNA of the third invention (antisense DNA) arranged downstream thereof and introducing the vector into a plant. Two inventions
This is a method for suppressing the expression of DNA. As the promoter used here, the DNA (promoter) of the first invention is preferable, but another promoter may be used. As other promoters, known plant expression promoters such as the CaMV35S promoter can be used.

The introduction of the vector into the plant can be performed by a method commonly used when introducing a foreign gene into a plant cell, for example, a method using Agrobacterium. The Agrobacterium is not particularly limited as long as it is commonly used for introducing a foreign gene into a plant.
robacterium tumefacience strain EHA101 (Hood et al., J.
Bacteriol. 168: 1291-1301 1986) can be used.

The third invention introduced together with the promoter
The DNA acts as an antisense gene for the DNA of the second invention in the plant. That is, the transcript of the DNA of the third invention is paired with the transcript of the DNA of the second invention originally present in the plant, and inhibits the translation thereof.
DNA expression is suppressed. Whether or not the DNA of the third invention has been introduced into the plant can be confirmed by extracting genomic DNA from the plant and performing Southern hybridization.

Ii) Co-suppression method In this method, a vector having an arbitrary gene arranged downstream of the DNA (promoter) of the first invention is constructed and introduced into a plant to cause co-suppression. This is a method for suppressing the expression of the DNA of the second invention. The gene linked downstream of the DNA of the first invention is not particularly limited, and for example, a GUS reporter gene, the DNA of the second invention, and the like can be used. Vectors can be introduced into plants in the same manner as in the antisense method described above.

Iii) Gene disruption type tagging method This method uses the T-DNA or transposon to
This is a method for suppressing the expression of the DNA of the second invention by inserting it into NA. By introducing a vector containing a T-DNA or transposon into a plant, a mutant strain group in which the T-DNA or transposon is randomly inserted into the genome is created. Genomic DNA can be prepared from the plants of these lineage groups, and the DAD1 gene-disrupted strain can be screened using PCR. Alternatively, by observing the phenotypes of the plants of these lineages, selecting mutants without anther dehiscence, and treating buds with linolenic acid or jasmonic acid to select DAD1 gene-disrupted strains. it can.

The vector used for T-DNA tagging is not particularly limited, and for example, pBIH1-IG can be used. The transposon used for transposon tagging is not particularly limited, and for example, a DNA-type transposon such as an Ac-Ds system or a retrotransposon can be used.

Iv) Gene targeting method In this method, the DNA of the second invention is expressed by inserting another DNA into a part of the DNA of the second invention by homologous recombination and knocking out the DNA of the second invention. It is a method of suppressing.
As a method for knocking out the DNA of the second invention, for example, the method of Kempin et al. (Kempin et al. Nature
389: 802-803. 1997). That is, a targeting vector in which a kanamycin gene or the like is inserted into the DNA of the second invention is prepared, and this is introduced into a plant to cause homologous recombination to knock out the DNA of the second invention. Whether or not homologous recombination has occurred can be confirmed by PCR or Southern hybridization.

V) Method Using Mutagen Source This method comprises treating the mutagen and treating the DNA of the second invention.
This is a method for suppressing the expression of the DNA of the second invention by causing a mutation in the DNA. The generation (M1) in which the seed or plant body of the target plant is treated with the induction source and the current generation (M1) is selfed
A DAD1 gene-disrupted strain can be selected from the plant of 2). For example, after treating thousands of individuals with a mutagen, these inbred seeds are harvested. Several seeds per M1 generation are planted in a field and observed to select mutants without anther dehiscence. By treating the buds of the selected individual with linolenic acid or jasmonic acid, a DAD1 gene-disrupted strain can be selected.

The mutagen to be used is not particularly limited as long as it is commonly used as a mutagen to increase the mutation rate. For example, X-rays and gamma rays, ethyl methanesulfonate (EMS), N-nitroso- N-methylurea (NM
U) can be used. The plant that has been male-sterilized by the methods iii), iv) and v) restores fertility in the next generation (F1) by crossing with a plant that normally expresses the DNA of the second invention. It is effective for F1 seeding of plants that use plant seeds as agricultural products, for example, general crops such as rapeseed, rice, barley, and wheat.

(6) Sixth invention The method for restoring fertility according to the sixth invention is characterized in that the male sterile plant according to the fourth invention is treated with jasmonic acid, linolenic acid, or a derivative thereof. is there. Examples of the derivatives of jasmonic acid and linolenic acid include, but are not limited to, methyl jasmonate, sodium linolenate, 12-oxo-phytogenic acid, coronatine, and the like.

As a treatment method, a method of immersing the buds in a solution containing jasmonic acid or the like can be exemplified. Alternatively, the buds may be treated with a brush, spray, or the like. The concentration and the immersion time of the solution containing jasmonic acid and the like are not particularly limited, but in the case of methyl jasmonate, it is preferable to immerse in a 0.05 to 0.5 mM solution for several seconds.

[0030]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the examples do not limit the scope of the present invention. [Example 1] Production of dad1 mutant (1) Insertion of T-DNA into Arabidopsis genomic DNA To perform T-DNA tagging, a tagging vector pBIH1-IG
(Provided by Prof. Kenzo Nakamura, Nagoya University, Fig. 1) was introduced into Arabidopsis thaliana using Agrobacterium. Gene transfer into Arabidopsis was carried out as follows in accordance with the experimental protocol of a model plant (Shujunsha p99-104).
Cut off the hypocotyl from the seedlings of Arabidopsis thaliana (WS ecotype, Department of Botany, Kyoto University Graduate School of Science)
2,4-dichlorophenoxyacetic acid (0.5mg / L) and kinetin
(0.05 mg / L), and callus was induced at the cut end of the hypocotyl. This callus was infected with Agrobacterium EHA101 strain containing pBIH1-IG, and the T-DNA of pBIH1-IG was
Was introduced into the callus cell genome.

(2) Regeneration of Transformed Arabidopsis thaliana Callus into which T-DNA was introduced was transformed with kanamycin (50 mg / L),
Hygromycin B (20mg / L), vancomycin (500m
g / L) and claforan (200 mg / L)
Shoots were induced while selection on (mg / L 2-isopentenyl adenine and 0.15 mg / L indole acetic acid). Shoots were excised and transferred to a RIM medium containing 0.02 mg / L indolebutyric acid to induce rooting. The regenerated plant (T1 generation) thus produced was replanted in vermiculite and cultivated under fluorescent light. Seeds (T2 generation) were formed by self-pollination and collected.

Example 2 Identification and Analysis of dad1 Mutant (1) Identification of dad1 Mutant T2 generation seeds were sown on vermiculite and cultivated under fluorescent light. An individual whose anther does not cleave after flowering is found, and da
It was named d1 (Fig. 2). Pollen of the non-transformed Arabidopsis thaliana (WS ecotype) was first backcrossed to the pistil of the flower of dad1 individual to obtain F1 seed. The phenotype of the grown plants was wild type, and the dad1 mutant was found to be a recessive mutation. After collecting self-fertilized seeds (F2) from the F1 plants, the dad1 mutants that appeared in the F2 plant population were backcrossed a second time in the same manner to obtain F2 seeds. Individuals exhibiting the dad1 phenotype were selected from this F2 population and used for the following analyses.

(2) Recovery of Mutation by Linolenic Acid and Jasmonic Acid Among the inflorescences of the dad1 mutant, those portions with buds that have not yet bloomed are replaced with a 0.05% aqueous solution of sodium linolenate or a 0.5 mM aqueous solution of methyl jasmonate (0.05%). % Tween
20) (see Fig. 2), anther dehiscence was observed in about 10 flowers that subsequently blossomed (Fig. 2), and selfed seeds were formed. When these seeds were taken, sown on the soil and raised, all individuals exhibited the dad1 phenotype.

[Example 3] Cloning of DAD1 gene (1) Isolation of T-DNA flanking region by IPCR (inverse PCR) Genomic DNA prepared from dad1 mutant was cut with EcoRI and circularized with T4 ligase. . T-DNA inserted into the genome (TD derived from the binary vector pBIH1-IG
Two primers HP corresponding to the left border side of NA)
HR (5'-TTTGCCCTCGGACGAGTGCT-3 ') and L6 (5'-CAG
Perform PCR using CACATCCCCCTTTCGCCAG-3 ') and obtain T-DNA
Part of gene A (0.5 kb) adjacent to the left border of
DNA fragment) was isolated.

(2) Isolation of genomic clone The DNA fragment (part of gene A) isolated in (1) was labeled with ³² P and used as a probe, and used as a probe in lambda DASH II (Stratagene).
Genomic clone lambda 2-1a-1 containing genomic A
Was isolated (FIG. 3).

(3) Subcloning of genomic clone into plasmid and determination of nucleotide sequence (FIG. 3) 3.9 kb gene fragment obtained by cutting lambda 2-1a-1 with HindIII and 5.8 obtained by cutting with EcoRI. Each of the kb gene fragments was transferred to a plasmid vector pBluescript SK + (Stra
tagA) and a subclone pAKH3.9 at the 5 ′ side of gene A and a subclone pAKE5.8 at the 3 ′ side were prepared. Next, a fragment obtained by digesting pAKH3.9 with HindIII and EcoRI and a fragment obtained by digesting pAKE5.8 with EcoRI and SplI were ligated and the plasmid vector pBluescript SK + (Stra
tagene) and the region from HindIII to SplI of gene A was reconstructed on a plasmid. This plasmid was named pGDAD1-HS. Using this plasmid, the nucleotide sequence of the region from HindIII to SplI of gene A was determined. The plasmid pGDAD1-HS containing gene A is
Deposited in Escherichia coli and deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERN P-17299 (Deposit date: March 12, 1999).

(4) Isolation of cDNA Clone and Determination of Nucleotide Sequence A 3.6 kb D fragment obtained by digesting lambda 2-1a-1 with SpeI.
The NA fragment was labeled with ³² P and used as a probe.
A cDNA library of Arabidopsis thaliana (Ler ecotype) flower buds prepared using I (Stratagene) as a vector was screened.
da 5a3 was isolated. Analysis of its nucleotide sequence revealed that this clone lacked the 3 'side, and thus primer DAD1-FP3 (5'-CTCCTTGAGAAGCAAGGCACG
AAG-3 ') was used to perform the 3'-RACE method, and the missing portion was isolated. The resulting full-length cDNA clone contained an open reading frame encoding a protein consisting of 448 amino acid residues. From the comparison of the nucleotide sequence of the genomic clone and the cDNA clone, the gene A
Was found to contain no introns. Also,
The open reading frame of the cDNA clone is 3 bp from the open reading frame of the genomic clone.
It was found that one extra amino acid residue was inserted near the N-terminus of the encoded protein. It was assumed that the activity of the encoded protein was unrelated.

Example 4 Complementation Experiment (Gene A is D
Confirmation of AD1 gene) pGDAD1-HS was cut with SpeI, and the cut out 3651 bp D
The NA fragment (part of SEQ ID NO: 1) was ligated to the SmaI site of the pBI101-derived binary vector pARK5-MCS (partially modified from plasmid pARK5 provided by Meiji Seika Co., Ltd.), and the binary plasmid pTDAD1-SS ( FIG. 4) was produced. This plasmid is transferred to Agrobacterium tumefacience C5
8C1 (pGV2260) strain. Example 1- (1) from inbred seed of dad1 mutant obtained by linolenic acid treatment
Callus was prepared in the same manner as described above, and infected with the above Agrobacterium containing pTDAD1-SS, and Spe containing gene A was
The T-DNA into which the I fragment was inserted was introduced into the genome of the callus derived from the dad1 mutant. Use this callus for bialaphos (10 mg / L)
Example 1 while selecting by (provided by Meiji Seika Co., Ltd.)
The cells were cultured in the same manner as in (2) to form regenerated plants. When this regenerated plant was transferred to soil for cultivation and flowering, anther dehiscence and the formation of self-fertilized seeds were observed, confirming that the plant was restored to the wild-type phenotype. From this result,
Finally, gene A was identified as the DAD1 gene.

Example 5 Production of Male Sterile Plant (Part 1, Method Using Antisense DAD1 Gene) (1) Construction of Antisense DAD1 Gene Transfer Vector pGDAD1-HS was cut with SpeI and cut out 3651 bp D
Plasmid pGDAD1-SS in which the NA fragment was ligated to pBluescriptII SK + (Stratagene) was prepared. Using this plasmid as a template, primer DAD-p1 (5'-
GGATCGATAGTGTCCCACGTGGGTAAC-3 ') and primer DAD-p2 (5'-CGCGGATCCGTTGTATATAGAGAA with BamHI site inserted)
The promoter region was amplified by PCR using TGG-3 '). In addition, two primers with BamHI sites inserted
DAD-c1 (5'-CGCGGATCCACACACACTTTCTCAAC-3 ') and DAD-c2
(5'-CGCGGATCCACAAACCCGTCTACC-3 ') was used to amplify the DAD1 translation region by PCR. These amplified fragments were inserted into pBluescriptII SK- (Stratagene) to prepare an antisense DAD1 gene in which the DAD1 translation region was ligated in the reverse direction behind the promoter sequence.

Next, the antisense DAD1 gene was cut out from the plasmid vector, and the binary vector pGAH (Onou
chi et al. Nucleic Acids Res. 19: 6373-6378. 199
1.Cla I and Sac, provided by Prof. Yasunori Machida, Nagoya University)
The binary plasmid pADAD1 (FIG. 5) was prepared by insertion into the site of I.

(2) Transformation of Arabidopsis thaliana The above binary plasmid pADAD1 was replaced with Agrobacterium tu
Introduced into the mefacience EHA101 strain and infiltrated under reduced pressure (vacuum infi
Arabidopsis (Columbia ecotype, Plant Genetic Breeding Laboratory, Graduate School of Agriculture, Tohoku University) was transformed by the ltration method. Transformation into Arabidopsis thaliana by the vacuum infiltration method was performed according to an experimental protocol of a model plant (Shujunsha pp. 109-113.).

The transformed Arabidopsis thaliana did not undergo anther dehiscence even after flowering as in dad1 (FIG. 6, left), and did not form inbred seeds. Untransformed buds of the transformant
When immersed in a 0.5 mM aqueous methyl jasmonate solution, the flowers that had flowered thereafter showed anther dehiscence, and pollen was exposed on the surface of the anther (FIG. 6, right). In addition, as a result of pollinating this pollen with pistils, formation of self-fertilized seeds was observed. From this, it was clarified that by introducing the gene transfer vector described in Example 5- (1), a transformed plant showing male sterility similar to that of the dad1 mutant can be produced.

Example 6 Production of Male Sterile Plant (Part 2, Method Using Promoter-GUS Gene Transfer Vector) (1) Construction of Promoter-GUS Gene Transfer Vector pGDAD1-HS was cut with SplI, Was smoothed with a Klenow fragment. Next, it was digested with HindIII and a DNA fragment of 3512 bp was isolated. This DNA fragment was converted into a binary vector pBI101.2 (Clont
ec) between the HindIII site and the SmaI site, and a plasmid pBIDAD1-H (FIG. 7) in which the GUS reporter gene was linked to the DAD1 gene promoter.

(2) Transformation of Arabidopsis thaliana pBIDAD1-H was introduced into Agrobacterium tumefaciens strain GV3101, and the cells were infiltrated under reduced pressure (vacuum inf) as in Example 5- (2).
Arabidopsis (WS ecotype, Kyoto University Graduate School of Science, Department of Botany) was transformed by the iltration method.
In most of the transformants, Arabidopsis thaliana showed no phenotypic abnormality. Take inflorescences from these plants and X-
Gluc (5-bromo-4-chloro-3-indoril-beta-D-glucuronid
e, Wako Pure Chemical Industries) solution, strong staining was observed in the thread. On the other hand, in some of the transformants, a phenotype similar to that of the dad1 mutant was observed, in which the anther did not cleave even after flowering. Restoration of cleavage ability by jasmonic acid treatment was also observed. In these individuals, the staining with X-Gluc also tended to be weak.

Example 7 Southern Hybridization Using DAD1 Gene Probe (FIG. 8) In order to investigate whether a gene similar to the DAD1 gene is present in genomic DNA of plant species other than Arabidopsis thaliana,
Genome DN of Brassicaceae, Gramineae, Compositae, and Brassicaceae
Southern analysis was performed using A. Komatsuna varieties Osome (Takii Seed Co., Ltd.), broccoli varieties Green Comet (Takii Seed Co., Ltd.), rice (Yamahoushi, Sasanishiki), barley (Daikoku No. 1), corn (Sweetome 102, Fujii Nosan) , Lettuce varieties (V lettuce (Kaneko Co., Ltd. and Summer Red (Takii Seedling Co., Ltd.))) and sugar beet (provided by Hokkaido University Faculty of Agriculture) CTAB method (Murray and Thompson Nucl
eic. Acids Res. 8: 4321-4325,1980)
Was extracted. Genomic DNA is digested with restriction enzyme EcoRI, 0.7%
Electrophoresed on agarose gel. The DNA was transferred to a membrane, hybridized using the DAD1 gene as a probe, and then washed under conditions of 0.5 × SSC, 0.1% SDS and 65 ° C. As a result, it was confirmed that the present gene or a gene whose sequence is similar to the present gene was present in all the plants tested.

[0046]

Industrial Applicability The present invention provides a nuclear gene that controls the dehiscence of anthers of a plant and a male sterile plant in which expression of the gene is suppressed. This male sterile plant is extremely useful for F1 seed production because its flower organs are as close as possible to the wild type and its fertility can be restored with jasmonic acid or the like.

[0047]

[Sequence List] SEQUENCE LISTING <110> Research Institute of Seed Production Co., Ltd. <120> YAKU NO REKKAI NI KANYOSURU IDENSHI <130> P99-0082 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 < 211> 8133 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (3487) .. (4827) <400> agatattatc ttaaagatca acctttgtgt tgagtaattt 180 cgttgtatag gcttgtggaa tatatacttc aattcttatc agaccataga gtcagatcat 240 ttccacgaat ccttatagaa acatttagtt atgaaataaa acacatccga aataactagc 300 tattagatat aagaaaaaat ttaaaaatat tattgctcat cacagatttt ggaaaaatat 360 atattcatct aatcatgtta atagtcgcca tatatatacg cacattattg ttaaattgaa 420 caatctttgt aaaattatgt ttgttttgaa tattcttata aacatagaga aaattgcata 480 ttatctgttt gtaatgaaac gtatatttat tatatataga ccgatataca ataaagtagc 540 tagtaatagt attgaacaac aattgatcac caaaagaaag ttgttagctg gcttctgatg 600 gtttaacggc tcagttatga ct tattcgag ctgcaccaga tactgttagc ttgctttagt 660 cgactttttt ttttttttaa caaacaaatg aaaatagtcg ttaagaagga cttcattgtt 720 agctactaac atacattcat tcatttgggt tatattatgc atgtagaaaa ctcatatgat 780 aaaatgtgcc gaatcgagcc taacaaacag gggacatggc agtaaatata gttataaatt 840 ttcttacacc ctattacaga gaaaggtgaa ttttgttttt tttgtcacag tgaaatttgt 900 ttatatattg aaatatttat acatgcataa actataaact atagttataa gtgtaattgt 960 ctctctttgc cctttttctt tatttcaaga atggtagctt aagtatataa gttttacttg 1020 taaataagtt ttggtagtat atagaagaat ccgagtagaa aactcatact aagtactata 1080 gagcatgaga aataagaaat tcataatgta gtttatcgta tgagttgact ggatgataga 1140 taccataaat atattaagtt aaaagattct actaaacgaa gaccaactgc aaagctacgt 1200 atatatttat ccactgcgta aactaaactt tttctctaca tcgatctctc aatctttggt 1260 cggttcactt tacaaatcat aaatatataa gagtcggttt agttctttac aaaaaatcaa 1320 catcgatcaa agttgatgtc atggctatca ctccattctt ctctgttggc ctcctttaaa 1380 atttacctct taattttctt ttggtatgga aagaagaaat actctttaat gttttccgta 1440 acaacgtgga caaatggtcc cgaaagaaga aat ggccaat gtaaatgata ttgaaatagt 1500 cccatcggaa aaaagagtga agaaattaaa cattataaag aaccaattaa tggcttttgt 1560 ggtgtgtagt attgtgtcca caaatatatg aatggtcacg ttggtttgat atatccaaaa 1620 cgatgacatt taaatcgggg aatgtaagta aagtacgtgt cataaccaat agttctaaag 1680 tggtgcatgc aatttttgtt tattacaaaa caagaaattt gttcccaatc gattggaaca 1740 ccaaatcact ttaattgctt aagcactttg atcgtattcc taacgaagac attttccacg 1800 cgttccatgt gcatctcctc ctcagtatct atggtttatc gtgtaaatta aaactccttt 1860 cttcaagtta gttcatagtc tacataattc tttttactgc atataacata taatattctg 1920 taagatatca agaatctaaa agtgttttta ttactagtgt cccacgtggg taacatttga 1980 aatgcattca aacgatgtga taatgtgaaa tgcttaaaag tatatagagt taaaacagtc 2040 tttcagaaat cacgatggtt atcaatacaa tataaagaaa atattgcaaa acagctaaga 2100 ctctaatagg atttataaat gaaaagaggc tatatgttat taaaaagcac aagactaata 2160 tataaagagc tacgacatgt gatgtgaaat tcaaatagta agtaactcac gttgcacttc 2220 ttttattcgt cgttttgccc gaacaacaca tcactccaac ttatttaatt ggacttattc 2280 tgttacccgc gatataggct agcaagataa atttcatcg c gacatttcat atcttgatca 2340 ttctgcctta ttcaatggat tcattcaacc attctctgct catttggata tgatcattta 2400 cacttacatt ttatatttag attctaagtc gataactaat tttaagattg ttatatgttg 2460 cttttatacg tagacataaa acttgtcgtt aattgtataa aaacaatatg ctagaagcct 2520 agaagggcgt gggaatatag gaggtgtata cttcgtactt tccttgagca ttcaaattca 2580 gcaattggcg gcgaatggct aaagccgaaa gtaccaaatc cagccgatat gagccgagtc 2640 gagctggtga tagttcctca cgtgtcaaag cctttctgag ccgtattgtt tgagaactcc 2700 cacgtgtcca gtcccatcga aaacgacggt cagatattta tttcgataca cacttctttc 2760 tcttctcata tcatctcagc cgtccatttc tcttagtctc attatcatac atttacttct 2820 tctttttttt tttggtcacc aattcggtta aatccttttt tttttgtaaa tataagtcac 2880 attaaaaaag agagtaaaaa cgtgatgtca aaaacacaac taataacgta gaatccattc 2940 atgatcccaa atattgattt cacattgtct ttatggtcca tgttagttaa gtaaaagaaa 3000 gaaagagcaa acatagctta tgtgtaccac ataatcattt atatagtatt gaaactttgt 3060 caaagattgt actcattctt catagaataa aatatacaaa cccagctatt gttagctttc 3120 taatttggta catttattgc ttttacatac acgaaaacta cttg aaatcc tacatacaca 3180 aacgagtacc aatgtgtgta tttgggtatt agatataaat tcatatactt atataagcac 3240 tgtccacaat tttcagagtc gagtcctttc attttcaaca atctctttaa ctccactata 3300 gatcccaact tttctcacta taaaaggaac ccatttcctc tttccttcaa tcactttatc 3360 ttcttacacc aaatctatct tcaagacttc aacatactcc attctctata tacaaccttt 3420 ttaagaaaac tttgattaat cagatcaaat atttataaat taaaacacac acacactttc 3480 tcaaca atg aga ttc tct ctt tct ccc gta cgt ccg cat agt gta gta 3528 Met Arg Phe Ser Leu Ser Pro Val Arg Pro His Ser Val Val 1 5 10 gta cct tca cta cca aaa cag gac gtc gtt tct tat ata agt ggt acg 3576 Val Pro Ser Leu Pro Lys Gln Asp Val Val Ser Tyr Ile Ser Gly Thr 15 20 25 30 acg tcg aat cgt caa tgt cga tgt gta ctt aca ctt cct tct cct tca 3624 Thr Ser Asn Arg Gln Cys Arg Cys Val Leu Thr Leu Pro Ser Pro Ser 35 40 45 gtt tcc act tcc cga cca ccg gtt tta ccc aaa ccg gaa acc tgg gag 3672 Val Ser Thr Ser Arg Pro Pro Val Leu Pro Lys Pro Glu Thr Trp Glu 50 55 60 agt ttg ctg cta aac cat gat caa att cca ggc gaa ttc tca ccc ac t 3720 Ser Leu Leu Leu Asn His Asp Gln Ile Pro Gly Glu Phe Ser Pro Thr 65 70 75 ggt tcg agt atc ccg gtt aag ctt ggc cgg aga tgg atg gag tat cag 3768 Gly Ser Ser Ile Pro Val Lys Leu Gly Arg Arg Trp Met Glu Tyr Gln 80 85 90 ggg ctt caa aat tgg gac ggt ctt tta gac cca ttg gac gac aat ctc 3816 Gly Leu Gln Asn Trp Asp Gly Leu Leu Asp Pro Leu Asp Asp Asn Leu 95 100 105 110 cgg cga gag att ctc cgg tac ggt caa ttt gtc gaa tcg gct tat caa 3864 Arg Arg Glu Ile Leu Arg Tyr Gly Gln Phe Val Glu Ser Ala Tyr Gln 115 120 125 gca ttt gat ttc gat cct tcc tct cca acc tac ggg aca tgc cgg ttt 3912 Ala Phe Phe Asp Pro Ser Ser Pro Thr Tyr Gly Thr Cys Arg Phe 130 135 140 ccg agg agc acg ttg tta gag cga tcc ggt tta ccc aac tcc ggt tat 3960 Pro Arg Ser Thr Leu Leu Glu Arg Ser Gly Leu Pro Asn Ser Gly Tyr 145 150 155 cga cta acg aag aac ctt cgt gcc acg tca ggt att aac ttg cca cgt 4008 Arg Leu Thr Lys Asn Leu Arg Ala Thr Ser Gly Ile Asn Leu Pro Arg 160 165 170 tgg att gag aaa gcg cca agc tgg atg gct aca caa tc t agc tgg att 4056 Trp Ile Glu Lys Ala Pro Ser Trp Met Ala Thr Gln Ser Ser Trp Ile 175 180 185 190 ggt tac gtg gca gtt tgc cag gac aaa gaa gag atc tcg cgg ctt ggg 4104 Gly Tyr Val Ala Val Cys Gln Asp Lys Glu Glu Ile Ser Arg Leu Gly 195 200 205 cgt aga gac gtc gtc atc tcc ttc cgt gga acc gcc acg tgt ctc gag 4152 Arg Arg Asp Val Val Ile Ser Phe Arg Gly Thr Ala Thr Cys Leu Glu 210 215 220 tgg tta gag aac ctt cgc gcc acg ctg act cat ctc cct aat ggg cct 4200 Trp Leu Glu Asn Leu Arg Ala Thr Leu Thr His Leu Pro Asn Gly Pro 225 230 235 act gga gca aat cta aac ggg tct aac tct ggg ccc atg gtt gag agc 4248 Thr Gly Ala Asn Leu Asn Gly Ser Asn Ser Gly Pro Met Val Glu Ser 240 245 250 ggg ttt tta agc ttg tat act tca ggt gtt cac agt ttg aga gac atg 4296 Gly Phe Leu Ser Leu Tyr Thr Ser Gly Val His Ser Leu Arg Asp Met 255 260 265 270 gta aga gaa gag atc gca agg cta ctc caa tct tac ggc gac gag ccg 4344 Val Arg Glu Glu Ile Ala Arg Leu Leu Gln Ser Tyr Gly Asp Glu Pro 275 280 285 tta agt gta acg ata acc ggt cac agc ctc ggc gct gcg atc gcg aca 4392 Leu Ser Val Thr Ile Thr Gly His Ser Leu Gly Ala Ala Ile Ala Thr 290 295 300 cta gca gct tac gat atc aaa acg acg ttt aaa cgt gcg cct atg gtt 4440 Leu Ala Ayr Tyr Asp Ile Lys Thr Thr Phe Lys Arg Ala Pro Met Val 305 310 315 acc gta ata tct ttc gga ggt cca cgt gtc gga aac aga tgc ttt cgg 4488 Thr Val Ile Ser Phe Gly Gly Pro Arg Val Gly Asn Arg Cys Phe Arg 320 325 330 aaa ctc ctt gag aag caa ggc acg aag gtt cta aga atc gtg aac tcc 4536 Lys Leu Leu Glu Lys Gln Gly Thr Lys Val Leu Arg Ile Val Asn Ser 335 340 345 350 gac gac gtc atc acc aaa gtt cct gga gtt gtt tta gaa aac aga gag 4584 Asp Asp Val Ile Thr Lys Val Pro Gly Val Val Leu Glu Asn Arg Glu 355 360 365 caa gat aac gtt aag atg aca gcg tcg ata atg ccg agc tgg ata cag 4632 Gln Asp Asn Val Lys Met Thr Ala Ser Ile Met Pro Ser Trp Ile Gln 370 375 380 aga cgc gtg gag gag acg ccg tgg gtt tac gct gaa atc ggt aag gag 4680 Arg Arg Val Glu Glu Thr Pro Trp Val Tyr Ala Glu Ile Gly Lys Glu 385 390 395 395 ctt cgg ctg agt agc cgt gac tcg ccg cac ttg agc agc atc aat gtg 4728 Leu Arg Leu Ser Ser Arg Asp Ser Pro His Leu Ser Ser Ile Asn Val 400 405 410 gcc acg tgt cat gag ctg aaa acg tat tta cat ttg gta gac ggg ttt 4776 Ala Thr Cys His Glu Leu Lys Thr Tyr Leu His Leu Val Asp Gly Phe 415 420 425 430 gtg agc tcc acg tgt cca ttc aga gaa aca gct cgg aga gtt ctc cat 4824 Val Ser Ser Thr Cys Pro Phe Arg Glu Thr Ala Arg Arg Val Leu His 435 440 445 aga tgaagattca ttgatccgac attcgtaact aatttaattt tgtgcaaaga 4877 Arg aacactaaac cgggaagaaa atcaagaacc gcgtaacggt ttgattcggt taacaaatca 4937 atttcgtgac atatctctaa cttagcggta gaattagaaa agaaagtaaa gtagaagaca 4997 gctctaaagg gcgaaaaatg aaaaatgtat tgaagctcgt ataatctcaa attgtttata 5057 tactgtatag tgtttttaaa aaaaattgcc ggtaattatg aaaatgtggt cataaatcat 5117 aaaattttgt cgcacgagca tgtgctacta aaactcctaa aggcgtaaat tgtcttaact 5177 tggacagaaa aggaagtgaa aagacaaaaa tgtccctatc tccgtccaaa tatgagaaat 5237 gggaatgccc caatgatggt gccacgtaag caccgaccgt tcgaagaagt tgactggatc 5297 tctg tacttg gcaaaaaagc ttaaaagatt cagttgcttg caaaatctga atgcccaatg 5357 agaacacgca tcatgcgccg tccatttatt taccgtatag tatttacttt cctcgttacc 5417 aggaaaattc accatcaacg cgttattttg tgggtgtgtc atgtctgtgt tgttttaaaa 5477 ctctccataa aagttggttt tatgttaacg tactactaag aaagaccata aaataatggc 5537 caagaaatgg gatatatgat tctcgtgata atgcgttatt ctatgagtaa tataaagcaa 5597 actagtgttc agttccatgt gtattaatat aaaagtatca tgttgtataa gtaaatagaa 5657 tatttcaaaa taattataat atttctatat gtatgctaaa cataattaca tggttcattg 5717 gcgtagagaa aataacattt tatcatgttt tgtatatatc ttaatgcata acacattctc 5777 acttagaact atctattatc tatttatttt tattttataa tattgtgttt ttcatttaat 5837 ataaaaaatt cagacaatga tatatactca aaataatctt tcaaaaacac ataaaattat 5897 catcgaacag gaagggtaaa taatagaaga tcttttttgt ttgtgtgtgg gttaaatata 5957 tgacggactc taagcatatg attatgtagt cattgaaatt agatatagtt ttcaattttt 6017 catatagtcg ttccttcttt tctcaaaaag aaaccactta actgagaagt gcacgaaaac 6077 ggtcagcatc actgtttttg cggtgacttg aaagggagaa tatatcgcat gcaaagatcc 6137 aagccgtata cgtacatata caggcacgtc gtagtctcaa atatatatgc acaaaagtga 6197 atagatcgca taccgaccca tacctaaaat gctagctttg aagctataca tagctttaga 6257 aagtgaccat catcgtcatc gttacttgtt actttgttag acccacacca ttcctcgtcc 6317 aaaaatctat caaatatgga gaccgccaca tattttagat tattaagctt atattgaagg 6377 gttttatatt tctttcttgc gtacatttct ctttcgtttt ctctaatgat ttaagttaga 6437 ttagagaggt caataacgta tgcagttaac cttttccatt tcccgttatt gtggaaacct 6497 cagtttgact acatactccg accacttttt agtgatattt aacacaaaaa gaacttttga 6557 aaaactaatt tatcggtgtg actttttttg ttcgcttgtg catttttttc caacactggt 6617 ccataagttt accatgtaac atatattagt atttagagtt agtatatttc tacaaatatg 6677 tgcacagcta gctagcatta cttggaccct taaaggtgac atgtattgga tataggaaat 6737 caggacccct ttagcaaaaa aaaaaaaaag aaaaaaaaaa gaaaatcagg accatcttaa 6797 aactatttgt tcctctctat ataatggttt ataatcataa ttaagattta aagtacaaat 6857 tatagttatt aaaaaggtta gttctcacag ataataacag aaaatatttt aagttaccaa 6917 ttttagcaca taatatctaa agtttttaaa atactatgaa tttaatatca aaaatcttat 6977 ataagtattt gatgg acgct ttacgcgata ataatagaat catgatttca aattgtgatt 7037 aattttttac gtaattcggt gaagataagt aattcttgta taaatttcac taatgttaat 7097 tttaaaagtt tatggaagat atgatataat tgttcataaa acttgttata atgctattat 7157 aagatatttt tcttaaaaag acaagtaaat tactttgttc gtcgaacaca aactaactta 7217 cgcttgatca agctgtattc tacaaaagtt atacattatt aatttatcat tacaaaaatt 7277 ggaaaaaggt tactttaaca attcaaaaat atatcgaaat ccaaaaagat ttatcaactc 7337 cttttaaatt tcacgtttta cgtgaatcat gtgatataat catatgcata caaatgtcat 7397 ctgagagatt tcagccgact tttgaacaag gaccaaaaca gcctacgcca tgcggagacc 7457 acgaaaatag atattttatt ataaataaat ggattttttt ggacgtaatg aaacgagaag 7517 tggatcagaa gattaattac ataatctttt ctatttataa atttaaacta tttctttcta 7577 attcattaac aattttgata catcaggaca atggtaactt atgttactat aagacaatgg 7637 taatatctca atagaagttt ttttttgtaa aatcgcaata tatacatgaa ttaataatga 7697 catatgatgg tcatttggac ataatataaa aatataaata tttggcaaaa taaataagta 7757 aatgaagggg agaggaatgt gtgagtgtgt gtgggtgtgg atgcctttat gcctcctaca 7817 tctcaacatt gagcaaaatt gcggctgccc aacaaccacc caaaagagaa ttagtcaaaa 7877 ttattaaaat caatgcatgt cctaatctct ctcacccaac tctcaccttc atttcaactc 7937 ccatgcatct ttttggtcaa ttaatttatt aatattataa atatattttt tcaatgaatt 7997 ttgaaaagac tgtgttgcat attaaaataa tgcgttgtac tattaagagt caaattagaa 8057 aatgtacttt taactccaaa ttaatttaca acatcttaga ttatgttatt tttttttttt 8117 caagtcttca cgtacg 8133 <210> 2 <211> 447 <212> PRT <213 > Arabidopsis thaliana <400> 2 Met Arg Phe Ser Leu Ser Pro Val Arg Pro His Ser Val Val Val Pro 1 5 10 15 Ser Leu Pro Lys Gln Asp Val Val Ser Tyr Ile Ser Gly Thr Thr Ser 20 25 30 Asn Arg Gln Cys Arg Cys Val Leu Thr Leu Pro Ser Pro Ser Val Ser 35 40 45 Thr Ser Arg Pro Pro Val Leu Pro Lys Pro Glu Thr Trp Glu Ser Leu 50 55 60 Leu Leu Asn His Asp Gln Ile Pro Gly Glu Phe Ser Pro Thr Gly Ser 65 70 75 80 Ser Ile Pro Val Lys Leu Gly Arg Arg Trp Met Glu Tyr Gln Gly Leu 85 90 95 Gln Asn Trp Asp Gly Leu Leu Asp Pro Leu Asp Asp Asn Leu Arg Arg 100 105 110 Glu Ile Leu Arg Tyr Gly Gln Phe Val Glu Ser Ala Tyr Gln Ala Phe 115 120 125 Asp Phe Asp Pro Ser Ser Pro Thr Tyr Gly Thr Cys Arg Phe Pro Arg 130 135 140 Ser Thr Leu Leu Glu Arg Ser Gly Leu Pro Asn Ser Gly Tyr Arg Leu 145 150 155 160 Thr Lys Asn Leu Arg Ala Thr Ser Gly Ile Asn Leu Pro Arg Trp Ile 165 170 175 Glu Lys Ala Pro Ser Trp Met Ala Thr Gln Ser Ser Trp Ile Gly Tyr 180 185 190 Val Ala Val Cys Gln Asp Lys Glu Glu Ile Ser Arg Leu Gly Arg Arg 195 200 205 Asp Val Val Ile Ser Phe Arg Gly Thr Ala Thr Cys Leu Glu Trp Leu 210 215 220 Glu Asn Leu Arg Ala Thr Leu Thr His Leu Pro Asn Gly Pro Thr Gly 225 230 235 240 Ala Asn Leu Asn Gly Ser Asn Ser Gly Pro Met Val Glu Ser Gly Phe 245 250 255 Leu Ser Leu Tyr Thr Ser Gly Val His Ser Leu Arg Asp Met Val Arg 260 265 270 Glu Glu Ile Ala Arg Leu Leu Gln Ser Tyr Gly Asp Glu Pro Leu Ser 275 280 285 Val Thr Ile Thr Gly His Ser Leu Gly Ala Ala Ile Ala Thr Leu Ala 290 295 300 Ala Tyr Asp Ile Lys Thr Thr Phe Lys Arg Ala Pro Met Val Thr Val 305 310 315 320 Ile Ser Phe Gly Gly Pro Arg Val Gly Asn Arg Cys Phe Arg Lys Leu 325 330 335 Leu Glu Lys Gln Gly Thr Lys Val Leu Arg Ile Val Asn Ser Asp Asp 340 345 350 Val Ile Thr Lys Val Pro Gly Val Val Leu Glu Asn Arg Glu Glu Gln Asp 355 360 365 Asn Val Lys Met Thr Ala Ser Ile Met Pro Ser Trp Ile Gln Arg Arg 370 375 380 Val Glu Glu Thr Pro Trp Val Tyr Ala Glu Ile Gly Lys Glu Leu Arg 385 390 395 400 Leu Ser Ser Arg Asp Ser Pro His Leu Ser Ser Ile Asn Val Ala Thr 405 410 415 Cys His Glu Leu Lys Thr Tyr Leu His Leu Val Asp Gly Phe Val Ser 420 425 430 Ser Thr Cys Pro Phe Arg Glu Thr Ala Arg Arg Val Leu His Arg 435 440 445 <210> 3 <211> 3422 <212> DNA <213 > Arabidopsis thaliana <220> <221> promoter <222> (1) .. (3422) <400> 3 aagcttcttt attacccaat taagtaaata tcccaaacaa ttccattttg catgtaaact 60 taatgtcact attccctaaa ttaaattaca caaaattgca gtgtatgtta tgatgattgt 120 ttgatacttt attccctcct agatattatc ttaaagatca acctttgtgt tgagtaattt 180 cgttgtatag gcttgtggaa tatatacttc aattcttatc agaccataga gtcagatcat 240 ttccacgaat ccttatagaa acatttagtt atgaaataaa acacatccga aataactagc 300 tat tagatat aagaaaaaat ttaaaaatat tattgctcat cacagatttt ggaaaaatat 360 atattcatct aatcatgtta atagtcgcca tatatatacg cacattattg ttaaattgaa 420 caatctttgt aaaattatgt ttgttttgaa tattcttata aacatagaga aaattgcata 480 ttatctgttt gtaatgaaac gtatatttat tatatataga ccgatataca ataaagtagc 540 tagtaatagt attgaacaac aattgatcac caaaagaaag ttgttagctg gcttctgatg 600 gtttaacggc tcagttatga cttattcgag ctgcaccaga tactgttagc ttgctttagt 660 cgactttttt ttttttttaa caaacaaatg aaaatagtcg ttaagaagga cttcattgtt 720 agctactaac atacattcat tcatttgggt tatattatgc atgtagaaaa ctcatatgat 780 aaaatgtgcc gaatcgagcc taacaaacag gggacatggc agtaaatata gttataaatt 840 ttcttacacc ctattacaga gaaaggtgaa ttttgttttt tttgtcacag tgaaatttgt 900 ttatatattg aaatatttat acatgcataa actataaact atagttataa gtgtaattgt 960 ctctctttgc cctttttctt tatttcaaga atggtagctt aagtatataa gttttacttg 1020 taaataagtt ttggtagtat atagaagaat ccgagtagaa aactcatact aagtactata 1080 gagcatgaga aataagaaat tcataatgta gtttatcgta tgagttgact ggatgataga 1140 taccataaat atattaagt t aaaagattct actaaacgaa gaccaactgc aaagctacgt 1200 atatatttat ccactgcgta aactaaactt tttctctaca tcgatctctc aatctttggt 1260 cggttcactt tacaaatcat aaatatataa gagtcggttt agttctttac aaaaaatcaa 1320 catcgatcaa agttgatgtc atggctatca ctccattctt ctctgttggc ctcctttaaa 1380 atttacctct taattttctt ttggtatgga aagaagaaat actctttaat gttttccgta 1440 acaacgtgga caaatggtcc cgaaagaaga aatggccaat gtaaatgata ttgaaatagt 1500 cccatcggaa aaaagagtga agaaattaaa cattataaag aaccaattaa tggcttttgt 1560 ggtgtgtagt attgtgtcca caaatatatg aatggtcacg ttggtttgat atatccaaaa 1620 cgatgacatt taaatcgggg aatgtaagta aagtacgtgt cataaccaat agttctaaag 1680 tggtgcatgc aatttttgtt tattacaaaa caagaaattt gttcccaatc gattggaaca 1740 ccaaatcact ttaattgctt aagcactttg atcgtattcc taacgaagac attttccacg 1800 cgttccatgt gcatctcctc ctcagtatct atggtttatc gtgtaaatta aaactccttt 1860 cttcaagtta gttcatagtc tacataattc tttttactgc atataacata taatattctg 1920 taagatatca agaatctaaa agtgttttta ttactagtgt cccacgtggg taacatttga 1980 aatgcattca aacgatgtga taat gtgaaa tgcttaaaag tatatagagt taaaacagtc 2040 tttcagaaat cacgatggtt atcaatacaa tataaagaaa atattgcaaa acagctaaga 2100 ctctaatagg atttataaat gaaaagaggc tatatgttat taaaaagcac aagactaata 2160 tataaagagc tacgacatgt gatgtgaaat tcaaatagta agtaactcac gttgcacttc 2220 ttttattcgt cgttttgccc gaacaacaca tcactccaac ttatttaatt ggacttattc 2280 tgttacccgc gatataggct agcaagataa atttcatcgc gacatttcat atcttgatca 2340 ttctgcctta ttcaatggat tcattcaacc attctctgct catttggata tgatcattta 2400 cacttacatt ttatatttag attctaagtc gataactaat tttaagattg ttatatgttg 2460 cttttatacg tagacataaa acttgtcgtt aattgtataa aaacaatatg ctagaagcct 2520 agaagggcgt gggaatatag gaggtgtata cttcgtactt tccttgagca ttcaaattca 2580 gcaattggcg gcgaatggct aaagccgaaa gtaccaaatc cagccgatat gagccgagtc 2640 gagctggtga tagttcctca cgtgtcaaag cctttctgag ccgtattgtt tgagaactcc 2700 cacgtgtcca gtcccatcga aaacgacggt cagatattta tttcgataca cacttctttc 2760 tcttctcata tcatctcagc cgtccatttc tcttagtctc attatcatac atttacttct 2820 tctttttttt tttggtcacc aattcggtta aatccttttt tttttgtaaa tataagtcac 2880 attaaaaaag agagtaaaaa cgtgatgtca aaaacacaac taataacgta gaatccattc 2940 atgatcccaa atattgattt cacattgtct ttatggtcca tgttagttaa gtaaaagaaa 3000 gaaagagcaa acatagctta tgtgtaccac ataatcattt atatagtatt gaaactttgt 3060 caaagattgt actcattctt catagaataa aatatacaaa cccagctatt gttagctttc 3120 taatttggta catttattgc ttttacatac acgaaaacta cttgaaatcc tacatacaca 3180 aacgagtacc aatgtgtgta tttgggtatt agatataaat tcatatactt atataagcac 3240 tgtccacaat tttcagagtc gagtcctttc attttcaaca atctctttaa ctccactata 3300 gatcccaact tttctcacta taaaaggaac ccatttcctc tttccttcaa tcactttatc 3360 ttcttacacc aaatctatct tcaagacttc aacatactcc attctctata tacaaccttt 3420 tt 3422

[Brief description of the drawings]

FIG. 1 is a diagram of a gene transfer vector used for T-DNA tagging.

FIG. 2 is a photograph showing the morphology of wild type (WS) and dad1 mutant flowers and anthers.

FIG. 3 is a diagram showing an outline of DAD1 gene cloning.

FIG. 4 is a diagram showing the structure of a gene transfer vector used in a complementation experiment.

FIG. 5 is a diagram showing the structure of an antisense DAD1 gene plant introduction vector.

FIG. 6 is a photograph showing morphology of flowers and anthers of Arabidopsis thaliana transformed with antisense DAD1.

FIG. 7 shows the structure of a promoter-GUS gene transfer vector.

FIG. 8 is a diagram showing the results of Southern hybridization using the DAD1 gene as a probe.

Continuation of the front page (72) Inventor Akiko Oda 38, Saigo-naka, Meidaiji-cho, Okazaki-shi, Aichi Okazaki National Collaborative Research Organization (56) Reference JP-A-7-59573 (JP, A) Table 8-507691 (JP, A) Plant J. , 10 (4), 679-689 (1996) Sex. Plant Repro d. , 11 (6), 297-322 (1999) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] (1 ) a DNA represented by the following (a) or (b): (a) a DNA represented by the nucleotide sequence of SEQ ID NO: 3 (b) a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 3
And DNA having promoter activity,
Mutation is generated in the DNA of (a) by site-directed mutagenesis
DNA that can be obtained by 2. The DNA shown in (a) or (b) below. (a) a DNA encoding a protein represented by the amino acid sequence of SEQ ID NO: 2 (b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 A DNA encoding a protein having a function of controlling anther dehiscence, wherein the DNA of (a)
Should be mutated by site-directed mutagenesis.
DNA obtained by 3. A DNA represented by a nucleotide sequence complementary to the DNA according to claim 2. 4. A male sterile plant, wherein the expression of the DNA according to claim 2 is suppressed. 5. A method for producing a male sterile plant, which comprises suppressing the expression of the DNA according to claim 2. 6. A method for restoring fertility, comprising treating the male sterile plant according to claim 4 with jasmonic acid, linolenic acid, or a derivative thereof.