JP3532707B2 - Mitochondrial gene with a function to cause cytoplasmic male sterility - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mitochondrial gene present in cytoplasmic male sterile tomato plants.

[0002]

2. Description of the Related Art Male sterility refers to the sterility phenomenon of male organs found in many higher plants. Male sterility is known to be due to the cytoplasm, to the nuclear gene, and to which both are involved. Among these, cytoplasmic male sterility is
It is thought to be caused by incompatibility between the nuclear genome and the cytoplasm. A plant having a male sterility trait, particularly a cytoplasmic male sterility trait, is excellent in that it facilitates the production of hybrid seeds when heterosis breeding is performed using self-fertilizing plants. Widely used.

In order to produce a cytoplasmic male-sterile plant, generally, a crop into which the cytoplasmic male-sterile trait is to be introduced and a plant belonging to the same species (including subspecies) or a crossable genus as the crop are used. This is done by mating. For example, a cytoplasmic male sterile rice plant uses rice pollen as an Indian variety (Chin
surah Boro II) can be produced by repeated pollination and nuclear replacement by mating.Also, the Brassicaceae plants with cytoplasmic male sterility are, for example, radish-derived cytoplasmic male sterility called "Ogura cytoplasm". In the same way as in rice, by crossing a plant with a cytoplasm that causes cytoplasm with a plant to which the trait of cytoplasmic male sterility is added (nuclear replacement method), or cells with Ogura cytoplasm and cytoplasmic male sterility It can be produced by fusing with cells of a plant to be introduced (asymmetric cell method).

However, in tomato, the cytoplasm causing cytoplasmic male sterility has not been found so far in the species or in the genus capable of crossing. Further, the male sterility trait is clarified only by flowering, and it is difficult to identify the trait in a plant before the flowering period. Therefore, in order to solve the above problems and to easily identify the traits in the plant body before the flowering period, a molecular biological analysis on male sterility is required.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a DNA that is closely linked to a gene that causes cytoplasmic male sterility in tomato plants.

[0006]

Means for Solving the Problems As a result of intensive research based on the above problems, the present inventor has found that cytoplasmic male sterile tomato plants and normal tomato plants produce mitochondrial DN.
By isolating and comparing A, a DNA region closely linked to a gene causing cytoplasmic male sterility was found, and the present invention was completed. That is, the present invention is a mitochondrial gene substantially containing the nucleotide sequence represented by SEQ ID NO: 1. Here, "substantially" means that the mitochondrial gene of the present invention has a mutation such as deletion, substitution, or insertion in the nucleotide sequence of the gene of the present invention as long as it has a function of causing cytoplasmic male sterility in tomato plants. It means that it may occur.

Further, the present invention is a recombinant vector containing the above-mentioned mitochondrial gene. Further, the present invention provides
It is a mitochondria containing the mitochondrial gene. Furthermore, the present invention is a cytoplasm containing the mitochondria. Furthermore, the present invention is a cell containing the above cytoplasm. Examples of the cells include cells of tomato plants. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In a tomato plant, it is possible to impart male sterility by asymmetrically fusing cells of a tomato plant and cells of a wild species related to potato. The present inventor used this asymmetric cell fusion method to transduce cytoplasmic male sterility into maternal plants of tomato cultivars. In such maternal plants that have undergone sterility transduction, the redifferentiated plants are mixed with male fertile individuals and non-male fertile individuals. Therefore, the present inventor has investigated whether or not these male sterility is present and mitochondrial DNA.
Studied the relationship with. As a result, the DNA of the present invention could be obtained from the mitochondrial genome specific to male sterile plants.

(1) Production of Cytoplasmic Male Sterile Tomato Plant In accordance with the method of Melchers et al. (JP-A-2-138927), several types of tomato cultivars and wild type potato are asymmetrically cell-fused and re-produced. A cytoplasmic male sterile tomato plant is selected from among the differentiated plants. Examples of tomatoes include Lycopersicon e
sculentum Mill.) (for example, variety “world's best”), Lycopersicon esculentum var.cerasiforme, and the like, and as a wild species of potato, for example, Solanum acaule hawks (Solanum acaule). Hawkes)
Is mentioned.

Asymmetric cell fusion means inactivation of the nucleus of one cell and inactivation of the cytoplasm (eg, mitochondria) of the other cell in two types of plant cells to be subjected to cell fusion. It means the method of merging. One cell with the nucleus inactivated is referred to as a cytoplasm-providing cell, and the other cell with the cytoplasm inactivated is referred to as a nucleus-providing cell.

The cytoplasm-providing cell is a cell that imparts a male sterility trait to the nucleus-providing cell, and the true leaf of the plant used as a material is cut off and the nuclear gene is inactivated by γ-ray or X-ray irradiation. After that, a known method (Cell Culture and Somatic C
ell Genetics of Plants: vol1, 375, Acad.Press In
c., 1984) to obtain the cells by protoplasting and isolating the cells of the true leaf (Japanese Patent Laid-Open No. Hei 10 (1998)
2-138927 publication).

On the other hand, the nucleus-providing cell is a cell constituting a plant body, and germinated seeds of a plant as a material, cutting off the leaves of the seedling, and isolating protoplasts by the same method as above. Then, the cells can be obtained by inactivating the cytoplasm with iodoacetamide or the like (JP-A-2-138927). Cell fusion is carried out by the well-known polyethylene glycol method (Planta, 120,215-227,1
974), electrofusion method (Planta, 151, 26-32, 1981) and the like.

After cell fusion, the plant body is redifferentiated from protoplasts. That is, the callus obtained by the culture after the fusion treatment is transplanted to a redifferentiation medium, and the redifferentiated foliage is obtained by culturing for about 2 months under the condition of light irradiation. Among plants regenerated from the fused cells, those having male sterility and those not having male sterility coexist. Therefore,
It is necessary to select only plants having the male sterility trait.

Selection is performed by the following method. That is, in the regenerated seedlings, the chromosomes of the cells of the meristems of the root tip of the same morphologically normal tomato plants were measured, and the same diploid (2n = 24) of the normal tomato plants was measured. All plants are cultivated and flowered. Anthers were collected from the bloomed flowers, a part of the pollen in the anther was stained with carmine acetate, the morphology was observed under a microscope, and a part of the remaining pollen was artificial pollen germination medium (Ame.J.Botany, 50,859). -865,1963), about 1
After a period of time, the germination elongation of the pollen tube is observed under a microscope. Further, the remaining pollen in the collected anthers is pollinated by a normal tomato plant grown by selfing and seed germination. From these, spherical pollen whose cells were dyed in bright red by carmine acetate was observed, germination elongation of pollen tubes on artificial pollen germination medium was not seen, and seeds by self-crossing could not be obtained. Tomato plants are selected as male sterile plants (R0 generation). Further, this male-sterile tomato plant is pollinated with pollen of a normal tomato plant, seeds are obtained, and its progeny (R1 generation) are cultivated. Generation) is confirmed to be cytoplasmic male sterile.

(2) Construction of Mitochondrial DNA Library of Cytoplasmic Male Sterile Tomato Plant Mitochondrial DNA is prepared from the green leaves of the cytoplasmic male sterile tomato plant selected as in the above (1). Mitochondrial DNA can be prepared by a conventional method (Proc. Natl. Acad. Sci. USA, 89, 6832-6836, 1992).
First, fully developed green leaves are homogenized in a buffer solution for mitochondrial isolation, the homogenate is filtered through three layers of miracloth, and then centrifuged at 1400 × g. 2200 more supernatant
Centrifuge at xg for 10 minutes and the supernatant at 15000 xg for 10 minutes. Add a buffer containing DNase I to the precipitate and suspend it at 0 ° C.
Let stand for 30 minutes. Add DNase I and add 30 at 0 ℃
Let stand for a minute. Add EDTA to stop the activity of DNase I, add a buffer for washing and centrifuge, suspend the precipitate again in the buffer and centrifuge. By repeating this several times, mitochondrial granules are obtained. Gently degrade mitochondrial granules and convert mitochondrial DNA using standard methods such as phenol /
Extract with chloroform / isoamyl alcohol extraction method, precipitate with ethanol and purify with cesium chloride ultracentrifugation.

The DNA obtained by the above method is partially digested with an appropriate restriction enzyme (eg, Sau3AI) and then treated with alkaline phosphatase to dephosphorylate the DNA fragment. This is ligated with a vector cleaved with a restriction enzyme (for example, BamHI) to prepare a library. As the vector, a phage or a plasmid that can autonomously grow in a host microorganism is used. Examples of the phage vector include EMBL3 and M13, and examples of the plasmid vector include pBR322 and pUC18. Furthermore, in addition to a vector capable of autonomous growth in two or more host microorganisms such as Escherichia coli and Bacillus brevis, various shuttle vectors can be used.
Such a vector can also be cleaved with the above restriction enzyme to obtain a fragment thereof.

To ligate the DNA fragment and the vector fragment, a known DNA ligase is used. And DNA
The fragment and the vector fragment are annealed and then ligated to produce a recombinant vector. The recombinant vector can be introduced into the host microorganism by a known method. For example, when the host microorganism is E. coli, the calcium method (Lederberg, EM et al., J. Bacteriol. 119, 1072 (197
4)) etc. can be adopted and the host microorganism is phage D.
In the case of NA, the in vitro packaging method (Horn, B. M
ethods in Enzymology, 68,299 (1979), etc. can be adopted. In the present invention, an in vitro packaging kit (GIGAPACK GOLD; manufactured by STRATAGENE) is used.

(3) Identification of mitochondrial genomic region strongly linked to cytoplasmic male sterility: The cytoplasmic male sterile tomato plant selected in the above (1) and the raw diet Otama-type tomato variety "World No. 1"")) Green leaves. All of the DNA extractions are performed by the CTAB method (Lichtenstein a
nd Draper (1985) DNA cloning vol.1 67-119).

The total DNA obtained is treated with a restriction enzyme (eg BamH
Cleave at I) and fractionate the fragments by agarose gel electrophoresis. Then, each fraction is transferred to a nylon membrane (pearl). Then, using the clone containing the mitochondrial DNA fragment obtained in (2) as a probe, Southern hybridization (Southern (197
5) J. Mol. Bio. 98: 503-517). For the probe, [α- ³² P] dCTP was added to each fragment in the library.
Is used and radiolabeled (Feinberg, et al.
al. (1983) Anal. Biochem. 132: 6-13), autoradiography is performed to detect bands.

(4) Screening When the desired fragment is obtained by the Southern hybridization, the desired fragment is treated with an appropriate restriction enzyme (for example, Ba
A plasmid clone can be obtained by cutting with mHI) and introducing the resulting fragment into a plasmid.
The plasmid used here is pBlueskript SK
+ Etc. are mentioned.

(5) Production of pollen non-germinating male sterile tomato plants Non-germinating cytoplasmic male sterile tomato plants are used as cytoplasm-providing parents to perform asymmetric cell fusion with any tomato variety or line, A pollen non-germinating cytoplasmic male sterile tomato plant having the characteristics of tomato varieties or strains is produced. The above
The pollen non-germinating male-sterile tomato plant obtained in (1) is pollinated with fertile tomato pollen to obtain seeds. In order to introduce a part of the mitochondrial genome of non-germinated cytoplasmic male sterile tomato plants into tomato cells with male fertility, the cotyledonary cytoplasm and male fertility of cotyledon cells obtained by germination of this seed Tomato cotyledon cells are fused (asymmetric cell fusion) to obtain a plant. The asymmetric cell fusion method is the same as above.

The plant body after cell fusion is re-differentiated to select an individual having a non-germination type male sterility trait. The CTAB method (Lichtenstein and Draper (198
5) Total DNA is extracted by DNA cloning vol.1 67-119), cleaved with restriction enzyme BamHI, fractionated by agarose gel electrophoresis, and transferred to nylon membrane (pearl). Southern hybridization is performed using the phage clone as a probe.

(6) Analysis of nucleotide sequence of DNA fragment specifically present in pollen non-germinating cytoplasmic male sterile tomato plant The determination of the nucleotide sequence of BamHI fragment obtained by the method of (4) above is known. This can be done by either method. Examples of the method include the dideoxy method. In the present invention, the Sequenase kit (UBC) is used. Once the base sequence is determined, thereafter, by chemical synthesis, or by PCR using a primer synthesized from the determined base sequence, or by hybridizing a DNA fragment having the base sequence as a probe, The desired gene can be obtained.

The gene of the present invention thus obtained is a gene closely linked to a gene directly involved in male sterility. Therefore, the gene of the present invention stably exists even after generations, and the gene of the present invention is introduced from one mitochondria into the other mitochondria even if cell fusion is performed multiple times.

(7) Mitochondrial Granules, Cytoplasm Containing the Mitochondria and Cells Containing the Cytoplasm The mitochondria of the present invention contain a DNA sequence resulting from the recombination of mitochondrial DNA arbitrarily generated by cell fusion. The cytoplasm of the present invention has X
It is obtained by irradiating with gamma rays or γ rays to disrupt the nuclear genetic material so that it does not function normally, and then isolating protoplasts. It is also possible to separate the cytoplasm by centrifugation. Further, since the cell of the present invention is characterized by containing mitochondrial granules containing the mitochondrial DNA fragment of the present invention, the cell containing the mitochondria of the present invention can be identified by a method such as in situ hybridization. it can.

[0026]

[Example 1]

(1) Production of Cytoplasmic Male Sterile Tomato Plant Aseptically germinated the seeds of the best tomato in the world, cut cotyledons from the seedlings, and modified Shahin's modified TSE-2 enzyme solution (Cell Cultu
re and Somatic Cell Genetics of Plants: Vol.1, (Vasi
ed.) pp375, Acad. Pre. Inc., 1984) was used to isolate protoplasts. For this isolation, cellulase Onozuka R-11 was added to 2.0% and macerozyme R-10 in TSE-2 enzyme solution.
Was modified to 0.2% (modified TSE-2 enzyme solution) and used.

The protoplasts were purified, suspended in a 10 mM iodoacetamide solution and left at 4 ° C. for 15 minutes to inactivate the protoplast cytoplasmic factors. On the other hand, Soranum acule hawks was aseptically propagated by cutting. Cut off the true leaves and give 10 gamma rays or X rays.
After inactivating the nuclear gene by irradiation with 0krad, the modified TS
Protoplasts were isolated using E-2 enzyme solution. Equal amounts of iodoacetamide-treated tomato protoplasts and gamma- or X-ray-irradiated solanum Akaure Hawks protoplasts were mixed, and the method of Kao et al. (Planta, 12
0,215-227,1974).

That is, 5 drops of the protoplast suspension was dropped on a plastic petri dish, and 12 minutes later, polyethylene containing 25% (W / W) polyethylene glycol 1540, calcium chloride 10.5 mM and potassium dihydrogen phosphate 0.7 mM was added. Twelve drops of glycol solution were dropped and left for 20 minutes. Next, 20 drops of high-pH high-calcium solution containing 50.0 mM of glycine-sodium hydroxide buffer (pH 10.5), 50.0 mM of calcium chloride and 0.2 M of mannitol was added twice at intervals of 20 minutes, while removing an appropriate amount of the mixed solution. A washing solution containing 0.75 mM calcium chloride and 0.4 M mannitol was added twice at 15-minute intervals. Finally TM-2 (Theor.
Appl. Genet., 69, 235-240, 1985) was added twice at 15-minute intervals.

Cultivation and regeneration of protoplasts is performed by Shahin
(Theor. Appl. Genet., 69, 235-240, 1985). That is, 2 weeks after the fusion treatment, small callus obtained from the liquid medium was transplanted to a solid medium, and the temperature was set to 500 Lx at 24 ° C.
Cultured for 6 days at
The cells were further cultured at 24 ° C. and 4500 to 5000 Lx for 2 months. Regenerated stems and leaves are TM-5 (Theor.Appl.Genet., 69,235-240,1985)
Then, the cells were cultured for 1 month and a half. The rooted stems and leaves were aseptically propagated by cutting, and about 4 months after the fusion treatment, they were put out in pots and cultivated in a greenhouse.

World's first tomato as starting material and number of chromosomes (2n =
24) Regenerated plants with the same morphology of stems and leaves were made to flower, and pollen collected from these flowers was stained with carmine acetate.
Then, almost 100% red is stained, and the pollen of the regenerated plant similar to the tomato in the world is also sown in an artificial pollen germination medium (Ame.J.Botany: 50,859-865,1963), and germination of pollen is performed. I observed. In the world's number one tomato, 50 ~
80% germinated and extended the pollen tube, whereas self-fertilization of regenerated plants with pollen that did not germinate at all resulted in no seeds. Further, this pollen was pollinated by the best tomato plant in the world, but no seed was obtained, and only when the pollen was pollinated by the best tomato in the world, seeds were obtained.

When seeds obtained by crossing this world's best tomato were sown, a tomato plant having no difference in morphology and growth including pollen from the world's best tomato was obtained. However, like the mother plants of these plants, i.e., the regenerated tomato plants of the present generation, pollen germination was not seen on the artificial pollen germination medium and on the stigmas of tomatoes, and therefore seeds could not be obtained. . As described above, a pollen non-germinating type cytoplasmic male sterile tomato plant was produced by asymmetric cell fusion between the world's best tomato and Solanum acaurea hawks.

(2) Construction of mitochondrial DNA library of pollen non-germinating cytoplasmic male sterile tomato plant MSA1 A buffer solution for mitochondrial isolation of green leaves of pollen non-germinating cytoplasmic male sterile tomato plant MSA1 (0.44M Sucrose / 50mM T
ris-HCl, pH7.59 / 3mM EDTA / 0.1% polyvinylpyrrolidone /
The mixture was homogenized in 0.2% bovine serum albumin / 1 mM 2-mercaptoethanol), the homogenate was filtered through three layers of Miracloth, and then centrifuged at 1400 × g. Supernatant at 2200 xg
After centrifugation for 10 minutes, the supernatant was centrifuged at 15,000 × g for 10 minutes. Buffer containing 200 μg / ml DNase I in the precipitate (0.44M Sucrose / 5
0mM Tris-HCl, pH7.5 / 25mM MgCl) was added and suspended,
And let stand for 30 minutes. Stop the activity of DNase I by adding EDTA to a final concentration of 50 mM and wash buffer (50 mM Tris
-HCl, pH 7.5 / 50 mM EDTA / 0.1% polyvinylpyrrolidone) was added and the mixture was centrifuged. The precipitate was suspended again in a buffer solution and centrifuged.
By repeating this several times, mitochondrial granules were obtained.

Next, the resulting mitochondrial granules were placed in a buffer solution (1% sarkosyl / 20 mM EDTA / proteinase K (200 μg / m 2
l) / 50mM Tris-HCl, pH8.0), gently dissolve at 37 ℃ for 1 hour, and then mix mitochondrial DNA with phenol and phenol / chloroform / isoamyl alcohol (24: 24: 1).
(vol / vol) mixture and extracted with ethanol and purified by cesium chloride density gradient ultracentrifugation (140,000 × g). The DNA obtained by the above method was digested with the restriction enzyme Sa
After partial digestion with u3AI, alkaline phosphatase treatment is performed to dephosphorylate the DNA fragment, which is then digested with the restriction enzyme Ba
Ligation was performed with the vector EMBL3 cut with mHI, and a library was prepared by in vitro packaging.

(3) Identification of mitochondrial genomic region strongly linked to cytoplasmic male sterility From the cytoplasmic male sterile tomato plant selected in (1) above and the green leaf of the tomato in the world, the CATB method (Lichtenstein and Drape)
r, (1985) DNA cloning vol.1,67-119)
Was extracted, cleaved with the restriction enzyme BamHI, fractionated by agarose gel electrophoresis, and transferred to a nylon membrane (pearl).

Southern hybridization (Southern (1975) J. Mol. Bio. 98) was carried out using the phage clone containing the mitochondrial DNA fragment of pollen non-germinating cytoplasmic male sterile tomato plant MSA1 obtained in (2) above as a probe. :Five
03-517). Random primer method for probe
(Feinberg, et al. (1983) Anal. Biochem. 132: 6-13) [α- ³² P] dCTP was incorporated and labeled, and the band was detected by autoradiography.

As a result, a 6.6 kbp BamHI fragment showing homology to the phage clone A641 was used as a nucleus donor cell in all pollen non-germinating cytoplasmic male sterile tomato plants produced by the asymmetric cell fusion examined. It was found regardless of the tomato cultivar or line tested (Fig. 1). In FIG. 1, lane 1 is DNA from the world's tomato, lanes 2 to 9 are DNA from regenerated tomato plants, and lane 10 is solanum.
Acaure Hawks-derived DNA, M is a molecular weight marker. A band of 6.6 kbp appeared in the plants in lanes 7, 8 and 9, and these plants were non-germinated pollen cytoplasmic male sterile. The regenerated plants in lanes 2 to 6 had male fertility.

(4) Screening and determination of nucleotide sequence Clone A641 was digested with BamHI and the 6.6 kbp fragment was pBlueskr.
Plasmid clone A641B6.6 was obtained by introducing into the BamHI site of ipt SK +. The nucleotide sequence of A641B6.6 was determined using the Sequenase kit (UBC). As a result, the base sequence represented by SEQ ID NO: 1 was obtained.

(5) Introduction of mitochondrial genome of non-germinated cytoplasmic male sterile tomato plant into tomato plant cells with male fertility Part of mitochondrial genome of non-germinated cytoplasmic male sterile tomato plant Was introduced into plants with male fertility and transformed into pollen non-germinating cytoplasmic male sterility, in order to examine whether the cytoplasm of pollen non-germinating cytoplasmic male sterile tomato plants was used as cytoplasmic donor cells, Asymmetric cell fusion with fertile tomato cells was performed. CTAB method from leaves of regenerated plants (Lichtenstein and Draper, (1985) DNA clonin
gvol.1,67-119) to extract total DNA, and use the restriction enzyme Bam
It was cut with HI, fractionated by agarose gel electrophoresis, and transferred to a nylon membrane (pearl). Southern hybridization was performed using the phage clone A641 as a probe.

As a result, the 6.6 kbp BamHI fragment was found only in all pollen non-germinating cytoplasmic male sterile tomato plants, and not in male fertile tomato plants (FIG. 2). ). In FIG. 2, lanes 1 to 12 are DNA derived from regenerated tomato plants, lane 13 is DNA derived from the world's most tomato, and lane 14 is DN derived from MSA1 tomato.
A and M represent molecular weight markers. Lanes 1, 5, 7, 8
A band of 6.6 kbp appeared in plants of 11 and 11. The plants in these lanes 1, 5, 7, 8 and 11 were non-germinated pollen cytoplasmic male sterility.

From the above, it was shown that the DNA of the present invention causes pollen non-germinating cytoplasmic male sterility of tomato plants. INDUSTRIAL APPLICABILITY The present invention provides a method for identifying a trait of pollen non-germinating cytoplasmic male sterility in a tomato plant before flowering. Tomato plant and Solanum ac
aule or non-pollen non-germinated cytoplasmic male sterile tomato plants by asymmetric cell fusion with tomato plants, as a probe for Southern hybridization, as a probe of Southern hybridization, non-pollen non-germinated cytoplasmic male It is possible to select fertile plants.

[0041]

INDUSTRIAL APPLICABILITY The present invention provides a DNA that is closely linked to a gene that causes cytoplasmic male sterility in tomato plants. The DNA of the present invention is used for producing non-pollen non-germinating cytoplasmic male sterile tomato plants and for improving tomato varieties.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 6623 Sequence shape: Nucleic acid chain number: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Lycopersicon esculentum Mill. Cv. Sekai-
ichi MSA1 Cell type: true leaf organelle name: mitochondrial direct origin library name: MSA1 mitochondrial DNA library clone name: A641B6.6 sequence: GATCCGAATC GGACCTAAAT GGGAATGACA TGAAAAGTCT TTTTCAAAAC CTAGCATTAA 60 GGGCGTTACG ACGATATACG AGAGGATAG AGAAGACAATG. TCGTCTCATT TCCTTACTTC TTTATAATTC 180 ATTCACTTCA AGACTGGTTC TGAACGCCGC GTAAGATCAT CTTCAACCAA CCTCCACCGT 240 ACTTTCGGTG CGACCACTAA AGAATTGCTT ATACACTAAA GAGCTGCTTA TATACGTTTA 300 CTAAAAGACT GACTTTCATT CATTATCATT AGTCCAGCGT GAAACTAAGA AAGAGAGATG 360 TGCCTAAGGC GGCATGTAAG CAAACTGTGC ACGCTAAGAG AAGAGGAGAG ATGTTCGCAA 420 TTACTACCAC AAGAAAGCCG CCCCCTATCT TCTAAAGGGG CCCGTCACTT CGTTCGTACC 480 TTCTTGGCTT AGGCTTCCAA CTGAACAACA TGGCCTTGCC GCCCCGCCAC TAGCAGAAGC 540 TAAGCGCTTG AAAACGCTAA GAAAGGTTGC TTCTGTCGCC CTTCTGTGCA ACAGTCCACC 600 AGTAGCGGAA GAGAGGGTTA CCGTACATAC AAGAGTCTTC CAGTTCTTAC GGAAGCTCTT 660 TCTTACCAGT CAATCAAGTA GTACA AGAAC TGTATCTTAT CTTATAGCCC GGCGCGGCGG 720 GTCGCTTTTA AATTAATTAA GAAGATAGAA GAAAGTATTA AATAGATAAG GAGTAGATGA 780 GTGAGTGAGT CCTCACTGAC CTGAGCGATT TCGATCACCT AGCAGGCCTT TTATTTGGTA 840 GGTAACATCG CCAAAGCGTA TCATCAGATT TGACTACGAA GGAAGGAACT CGAAGTGAAA 900 GGGCACCGTC TTGTGCAAGG CAACGATAGT TGGCCCTCTA TATCTTCTAT CTGGTAGACT 960 TGGTAAAAGG GCCCCGACTT ATTTCAGAAT TTGAGTTCGA CCGCGGCTTC CCCCTTTTTT 1020 TGGGGGGGAT CAAGTTCCTT GCCAACTGCC ACCTATCGAC CCCGATCTCT TTTCATTTGT 1080 TTTGGATATT ACCAAACCTA GCCTAGCCTT CGTAAATCAC ACTAAAGCGG ACACCCAACT 1140 ATGGAAAGCC TCCTTAAGGG TGGGTTAGGT TAGTCAATCC GGCCCGAGAC GGTTCGTTGA 1200 CAAAACCGCC GTAGAATTCC TACTTTTCAA TAGAGCGGAG CAGGACTGAT CAACGAGAAA 1260 GAGGCCCTAC TCACTACAAA CGAATACACC ACAAGATTGA ACTGCACTCA TGCCTCTCCC 1320 GCATCAAGTT GACCGCCCCC CCCTACGTAG TGATTCTATC AATCATGTAC GTAGGTAGGA 1380 CCCTCCATTC TGATTGGTAT ATCATGAAAA AAGAAAGCCA TTTGCACCAG GCGCACTGCG 1440 TTTCCCTTGC CCAGATGTTC GTCTTTCTAA GTCAAGTAAT GGTGACCCAC CGAAGACTGG 1500 AGCTTCGTAA CATGTTGACG AAGGCCCCCG AACTG AGGGT TCGGTCAGTA GAAGGGACGA 1560 CTTTGTCTCC CCGGAAGAAG AATAGCCCCG CTCTCTAGCC GACTGATCCC GTTGACCCTA 1620 TAACTGGGAG GGAACGTGTC ACATTAGAGG TGAACGATCA GAGGTGTCTC TAATCACCAC 1680 GACGAGGCGG GTCCCTCTTT TAGTAGACTC AGCTATGAAT ATTCATTCAG AAATGAATGC 1740 CGGGCTCTTT CTTTCACTGC TAACCCCATT TTATTTTCAA CATGCTGAAA CTTTCTGTTT 1800 CGTCGAGCCC CGAGCTTGTG GTCCTCCTTT GAGTGTGGGT TCAATTGAAT GAATCTGTCA 1860 AGTCAAGGAA ACCGTACGTA GCAGCAAGGA TGGTGCATCG CCTATTTATC GTTCTCGCTC 1920 GGGAGCCAAA ACGAACACGC CTGCTACCTA CTGTACTGGA CCTTCGACCA GGCATTCTAC 1980 CTTTGTCGAA TCGGAACCAA CACCACTGCA TTGCGCTCGA ACAGTTGAGC GGCCGCCGGC 2040 AGCAACTTCC GTGCACAGAT ATAGATTCAT TCCTCGTACC TGGTCCAGCC TCACAACCCT 2100 TCGCGGGTTG GTAAGAAGTC AGTCTTGTTT GGTAAGACGG AATTGGACAA AGAAAAGAGA 2160 GTGCTCGACC GGAACAACGG CCAACAAAGA CCGTAATTAC ATAGATAACT GCTCCTCCCC 2220 TTCTCCTTAA GGCTTTAAGG CGAACCGTAT GGCGGCTGGA AAGAAAGACG CCCTCACCTT 2280 CTCTGGTGTC AGTGAGAGCA ATTTGAGTAT CCCCCGTTGA CCTTCTTTTG TAGATAGAAT 2340 CTTCAATGAG TGGAAACAAG CAACCTTACT AAGAAAGGTG CTTGTTGAGT AAGGCCGGCT 2400 TTCGAGCCCA AGCCCCTTGT ATAGGTTGGG CGCTTGAGCG ATCTTTCACA TATCGATCAA 2460 GGCTTTCCTT GAGTTTGAAA TAAGGGGCAA GAAGCAAGGG CGTCAAGGCT GCGCGAAACG 2520 GCTTGCGCCT TAGCGCATCC CTTTTCTTGC AGGAGTGCTA ACGCGCGACC TTACGTTTTC 2580 TTCCGCTTGC TCTGCAGTAC GAGCCTCATA CAGAGCCGCG CTCCTTTAAT ATGATGAGAA 2640 GAAGGGGGGC CGGCCTCTTT TCCGTACCAA TCCTTATTTA CTACTACATC TTTTTTTGGG 2700 TGTATAGCTC AGTTGGTAGA GCATTGGGCT TTTAACCTAA TGGTCGCAGG TTCAAGTCCT 2760 GCTATACCCA AACCTACCTT ACACTCTACT ATGAATAAGG ATGCATAGTA GCCTTCAAAG 2820 ATGACTCTTT GGCCTTTCGA CTCGCTTCGG CGACTTCGCC TTTAAGTGAC AAGGGGGTGA 2880 GCCGCTCCAA GCAAAGCATA GTGAATCCCG AACTTGCCCA CGCTCATCCA AACCGGCCTC 2940 AAAAAGCGAT CGGAAAGCGA AGCCCTTCCT TCCAATCCAA GCCGGCGAAG CCGCCCCTAT 3000 ATCTAACACC GCTCACCCCG ATCACATATT GGCACGTTCA TGATGCATCA TAATCAAAAG 3060 GCCGTAAAGT AAAGAAGACC TATGCATCAG TGTACTGTCA TGATCACATA TAGCTCGATT 3120 TTATTGACGG CTTGAAGGCG AACCGCTATT TCTTAGGGCA AAGGGCGCTC CATCCTCCTA 3180 ACTCCAACCA CTTCTCCCAG GGACAGGAAC TACGGCTTGA CCTTCG GGGA AGAAGTCCGT 3240 GGGACCCCTC CTTCGAGGCG CCTAGATAGT GAAAGGTTAT CCCTTTGCAA GGCTGGAAGC 3300 TAAGCAAAGT CACGAAGCTG GCTGACTACC CTCGAGCAGA GCCCAGACCC GGAGTGGTGG 3360 CTGAACTCGC CGCAGAGTTC AGGAGCGGAA AAGCCACTTG ACTGTAAGGA AAGGAGCGAG 3420 CAAGACTATC TTGGTGAATG CAATAAGAAC CGGCTGCTCG CCGCAGCAGA GTGCTTTGCC 3480 CATGCTGCTA TCCGCCGCCG AAGCGCTCAA GGGATTCGTG CTTGAATGTC GAGATCAGGG 3540 GACTCTATCC AATCCATGCG GCAAATCTGT TTGTGGTGGA AAAAAGTCAA CAATAAAGAA 3600 AAAAGAAACA AACAGAATAT AGAAAATCAT TGAATTTGTT TGCTCCGATA CAAGAGATCG 3660 GCCCCGAAGC AAGATATGGT GGGTGCCAGC AACTTTCACT TGTTAGGAGT AGGGGCTGAA 3720 AAGAGCTCTT TGTATAGGTT GGGTTTTCTG GGCTTTGATG CTTACCTTCT TATTATTAAA 3780 AGGGGAGGGT TTAATAAAGG AGCTGAAAGC CACTTGACTG TAAGGAGAGG AGGTCAATAA 3840 AAAAAAAAGA GATGTTAATT AATCAAAGGT CCAACTGATC ACCACGCCTG TATTGTAAAT 3900 ATGCAGTTAG GAATAATCCA GCCAAAGTAA TAGGAATTAG GCCTAACACG ATTCCAAATA 3960 GAAAAACTTC AACCATTTTT CTTTGTTTGA AAGGAGAAAA AAAAAGAGGT AATATCTATA 4020 CCTAAATATT ATCCGAATTG ACATGAATCT CAATGACAAT GACCAAGAAT T GACAATTGG 4080 TAACGTAAGT AAATATAGAA TACACGTAAT CCCACAGAAA GATTTCTTTT TTAGAATTGT 4140 TCATTTTGAA TATCAGTTTA AATAAGTCGT ATCTTGCTCA GACCAATAAA TAGAGCTGAG 4200 GTTATAGTTA AAGCCGCTAA TAGAAAACCG AAAGAACTAG TTATAGTAAG CATGAAAGGG 4260 CTAAATTAAA TGAAATATGT TCTTTTTATA CATATGTTTA GAAAGCACTT ACCTAAGTTT 4320 CCATTTTTGC AAAATAGGAG GATAGGCAAA AATCTCAATT TCAATTGAAA GAAGAAGTTG 4380 AATTAAATTT TATTCATCTA TCATTATAGG CAGACAGCAC TAACAAAGAT AAGTGACAGA 4440 CATATAAAAG AAATTGATTC ATCATCTGTA ACATCTATCC ATCCCGCAAT TCCAATCAAC 4500 CGATTCATTG AGTAACTCTT GGGTAAACTA TTAAACTAAT AAGAGCTGTA TCGTCTAACT 4560 TCATTCAAAA ATGAAACTGG AATTTTTATA GAATAAAATT CGAAAATCAT TTCTATTTTG 4620 ATCATTTCTT AGATTCTGTT GAATTGTATC GAATCCATTT TCTATTTTAA AGCGAACACT 4680 TATAAAACTA GAAAACTTTT TCTTTTTACT TTAATTTAAG TTTAACTCAT TAGATTCAGT 4740 AATATATTAA TTCACATAAT ATCTTGAATG ATTGAGAAGA AGAAAAAGTT ATCACACAAT 4800 CGCTTGAAAA AATCTTTTTT TTGGGATCAA TATCAGCAGT TCATCCAACG ATAAACAACA 4860 CCCGAATTAT AGAGCTACGA CACAATCAAA CCCGAACGAA CAAAATGTTG AATTTCA TCG 4920 TACCAGTCTC TACTGGGGGT TATTACTCAT TTTTGTACTT GCTTTTTTAT TTTCCAATTA 4980 TTTCTTCAAT TAACAAAACG AAAGAGAATA AATAAGATTC TCTTAGCCTC GTTCGGAAGG 5040 ATCTCATAAT TCAATTATCC AAGGCTGTTT ATGTCTCTAG CATGACCACT TGATGAAATC 5100 TGGAGGGAAG TGGGGTAAAT GGCCGATACT ACTGGAAGGA TTCCTCTTTG GATAATAGGT 5160 ACTGTAACTG GTATTCTTGT AATCGGTTTA ATAGGTATTT TCTTTTATGG TTCATATTCC 5220 GGATTGGGTT CATCCCTGTA GTAATCGAAT GAATTGAGTT GTCAACATGA AAGCGTAAGA 5280 ACTCAACGGG ATTCCCAGCA TCTCGTAGAA AAAGAGTATG TAATGTAGAT TTTTCAGAAG 5340 GGTCTAAATA AGACAAAGGA GTTTTCATAA TTTCATTTTT TTTATCATAA ATAATCGCTA 5400 ACAAGAATTT GGTTCTTTTT ACGAACTTGA TGTCGATAAA TCTGCGAATC TATAAATTCA 5460 TTTCGGCCAA TTGAACCTTC TCGAACTCTT TCTTTTTAAG AACCAAAAAG ATTTGTGCCA 5520 AAATAACAGA TGCCAAGAAG AACAAAAGTC CTTGGACACG TAATGGATCT TGAAGTACTA 5580 TTTCTGCATC TCCTGACCAA ATCCGCCTAC ATTAGGATTA CTCGTTAATC CTTGATCAAA 5640 TTTGATAGAT TCGCCCCCGG AAACAAGAAG TTCTGGTCCG GGAGGGATAA TATCAACCAC 5700 TTGACGCCCA TCCGACGCAT CCGTTATGGT TATCTCATAT CCCCCTTTGT CTTTTCGTAT 57 60 GATTTTGCTT ACTATACCTG CTGCTGTACG ATTATAAACT GCATTCTTAC TCTTGCTGCC 5820 GTCGGGATAA ATCTGAACCC TTCCCCTGTT CCCGCCTACG TATATAGGAT ATTTTTCGAA 5880 GTGAACATCC TTCTTAGTAG CAGGGTCCGG GGAAAGAATA GGGAAGGTTA ATTTCACTAT 5940 ATTTTTTACC AGGGACAGGG CCTACCACAA GAATCTTTTT TTTATTGGGG CGATAGCTCT 6000 TAAAAGAAAA ATTGCCAATC TTTTCTTTCA TCTCGGGAGG GATACGATCG GGAGGAGCTA 6060 ATTCAACCCC CTCCGGTAAA ATAAGAACAA GCCCCCACGT TCAACCCCCC TTTTTTACCC 6120 ATTAGCAAGA ACTTGTTTCA GTTGCATATC ATAAGGAATT CGAACAACTG CCTCAAATAC 6180 AGTATCCGGA AGTACCGCTT GTGGAACCTC AATCTCCACG GGCTTATTAG CTAAATGGCA 6240 ATTGGCACAT ACAATACGCC CAGTCGCTTC TCGTGGATTT TCATAACCCT GCTGTGCAAA 6300 AGTGGGATAT GCACTTGCAA TAGATGTCCG AGTGTTGATA TATATCATGA GCGATACGGA 6360 AATAGATCGA GTAACTCTTT CTTTAGCCAA GAAAAAGCAT TTCTAGTTTG CATGGTCCAA 6420 TCATTGATCG CGAAAATTTG ACAATAAATT GGGTAGGCTC ACTAGTATAG TTCCTAGCCA 6480 CGATTCTGCT ATTTGCTGGA ATAATCTAGG ATGAATCTCC CCGATGGTTA GAAATAGAAA 6540 GAGTAAATAC TATTTTCAAT ACTTTGCTTA TGTACAACTA CAAAGTACCA AGCATTCTAA 6600 TTG GATTAGA TTAATATCAA TGG 6623

[Brief description of drawings]

FIG. 1 is an electrophoretic photograph showing the results of Southern hybridization.

FIG. 2 is an electrophoretic photograph showing the result of Southern hybridization.

─────────────────────────────────────────────────── ─── Continuation of the front page (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 5/00-5/14 A01H 1 / 00,5 / 00 JSTPlus (JOIS ) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG) PubMed

Claims (4)

(57) [Claims] 1. A mitochondrial gene comprising the following nucleotide sequence (a) or (b): (A) the nucleotide sequence represented by SEQ ID NO: 1 (b) one or several nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 have been deleted, substituted or inserted, and cytoplasmic male sterile in tomato plants Sequence having the function of causing 2. A male-sterile tomato plant characterized in that the cells containing the mitochondrial gene according to claim 1 are fused with any tomato plant-derived cells to re-differentiate the plant from the resulting protoplasts. How to make. 3. From the protoplasts obtained, claim 1
The method of claim 2, further comprising selecting protoplasts containing the described mitochondrial gene. 4. A male-sterile tomato plant obtained by the method according to claim 2 or 3.