Cytoplasmic male sterility gene, male sterility restored plant, method for restoring fertility of cytoplasmic male sterility in Solanaceae plants, method for producing male sterility restored plant, male sterile plant, and method for producing male sterility plant

Abstract

To provide cytoplasmic male sterility genes of solanaceous plants that can be used to induce and restore male sterility and can greatly improve the efficiency of plant mating and seed collection, and to provide male sterility restorer plants, methods of restoring fertility of the cytoplasmic male sterility of solanaceous plants, methods of producing a male sterility-restoring plant, male sterile plants, and methods of producing male sterile plants.SOLUTION: There is provided a cytoplasmic male sterility gene of a solanaceous plant encoding a protein selected from the group comprising of: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a), and exerting the cytoplasmic male sterility in plants; and (c) a protein having an amino acid sequence having 80% or more identity with the amino acid sequence of (a) and exerting the cytoplasmic male sterility in plants.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a gene for cytoplasmic male sterility in plants of the Solanaceae family, a male sterility-restored plant obtained by manipulating the expression of the gene, and a male sterile plant. [Background technology]

[0002] In the breeding of vegetables and other crops, F1 seed production involves crossing two different parent lines, a seed parent and a pollen parent. To prevent self-pollination from the seed parent line, a process called emasculation is performed, in which the anthers are removed from the seed parent. However, this emasculation process is currently primarily performed by hand, which requires labor and is costly, resulting in a rise in the price of seeds obtained through seed production. Furthermore, the need for a large number of workers poses a risk of the leakage of valuable intellectual property, such as parent lines. Manual emasculation also has a certain risk of failure, which can lead to contamination with pollen from the seed parent, resulting in seeds that do not represent the desired cross-breeding.

[0003] As a countermeasure to these problems, the seed parent can be made male sterile, i.e., to stop producing pollen or to produce sterile, i.e., inactive, non-cross-fertilizing pollen, thereby eliminating the possibility of pollen from the seed parent being pollinated without the need for manual male sterilization.

[0004] For example, in rice, a breeding technique that utilizes cytoplasmic male sterility (CMS) is known. One example of cytoplasmic male sterility is a phenomenon in which the action of mitochondrial genes in the mitochondria within the cytoplasm causes incompatibility between the cell nucleus and mitochondrial gene products, resulting in the inactivation of pollen, failure to mate, and the failure to produce seeds or fruit. In many plant species, cytoplasm is transmitted only from the female side, so CMS lines are produced using cytoplasm replacement lines in which heterologous cytoplasm has been replaced.

[0005] Patent Document 1 discloses a simple method for producing male-sterile tomato plants, which involves fusing protoplasts isolated from tomato plants and treated to be incurable with cytoplasmic factors with protoplasts isolated from Solanum plants and treated to be incurable with nuclear genetic material, and then regenerating male-sterile tomatoes from the fusion product. This technique aims to create a line with male sterility by protoplast fusion, and to efficiently render the target tomato plants male-sterile in a short period of time without affecting any traits of the target tomato plants other than male sterility.

[0006] Patent Document 2 describes a gene containing a specific DNA that causes RT-type cytoplasmic male sterility in rice, and a method for identifying sterility using the gene. This technology identifies a mitochondrial gene that causes RT-type cytoplasmic male sterility in rice, and by using this gene as a DNA marker, it can also be used to identify rice lines that are cytoplasmic male sterile.

[0007] Patent Document 3 discloses a method for producing plants with modified traits, which utilizes a plant expression cassette containing a promoter containing either DNA with a specific sequence number or a partial sequence thereof, and DNA that exhibits specific promoter activity in microspores and, optionally, anther dehiscence tissue, and a heterologous gene operably linked to the promoter. This technology aims to provide a genetic engineering technique that utilizes pollen-specific genes, which is useful for modifying plant traits, such as male sterility, in horticultural plants of the Solanaceae family, such as petunia. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 2824841 [Patent Document 2] International Publication No. 2014 / 027502 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-92937 Summary of the Invention [Problem to be solved by the invention]

[0009] Although the technology of Patent Document 1 can obtain a line with male sterility in tomato, a member of the Solanaceae family, the gene responsible for this cytoplasmic male sterility has not been identified. Therefore, although it is possible to produce a plant that does not bear fruit in the Solanaceae family using the aforementioned sterile line, it is unable to produce seeds and therefore cannot be propagated. In order to apply male sterility lines, it is desirable to identify the gene responsible for cytoplasmic male sterility and be able to control its on / off state.

[0010] The technology of Patent Document 2 clarifies the gene responsible for cytoplasmic male sterility in grasses, but the gene responsible for cytoplasmic male sterility in plants of the Solanaceae family has not been clarified.

[0011] The technology in Patent Document 3 attempts to genetically manipulate sterility in a type of plant in the Solanaceae family, but the gene manipulated is a gene contained in the nucleus, changing the plant's traits. This technology cannot manipulate sterility on / off by manipulating mitochondrial cytoplasmic genes without changing nuclear genes, as is the case with cytoplasmic male sterility. The technology in Patent Document 3 manipulates plants in the Solanaceae family, such as petunias, in the horticultural field, and is not intended for use in the food field.

[0012] The present invention has been made in consideration of the above-mentioned circumstances, and its object is to provide a cytoplasmic male sterility gene in a Solanaceae plant, which can be used to induce and restore male sterility and can greatly improve the efficiency of plant breeding and seed production, a male sterility-restored plant, a method for restoring cytoplasmic maleness in a Solanaceae plant, a method for producing a male sterility-restored plant, a male sterile plant, and a method for producing a male sterility plant. [Means for solving the problem]

[0013] In order to solve the above problems, the present invention has the following aspects. [1] (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a protein that has the amino acid sequence of (a) in which one or more amino acids are deleted, substituted, or added, and that exerts cytoplasmic male sterility in a plant; (c) a protein that has an amino acid sequence that is 80% or more identical to the amino acid sequence of (a) and that exerts cytoplasmic male sterility in plants; A cytoplasmic male sterility gene encoding a protein selected from the group comprising: [2] A male sterility restored plant, which is a Solanaceae plant in which the cytoplasmic male sterility gene has been deleted or the function or expression of the cytoplasmic male sterility gene has been inhibited. [3] The male sterility restorer plant, wherein the Solanaceae plant is a plant of the genus Solanum or Capsicum. [4] The male sterility restorer plant as described above, wherein the Solanum plant is eggplant, potato, or tomato. [5] The male sterility restorer plant, wherein the Capsicum plant is a chili pepper or a bell pepper. [6] The male sterility restored plant, in which the mitochondrial genome DNA has been edited to delete the cytoplasmic male sterility gene, or to inhibit the function or expression of the cytoplasmic male sterility gene. [7] A male sterility restored plant in which the mitochondrial genomic DNA has been edited by the mitoTALEN method, The male sterility restored plant described above, which uses a mitoTALEN expression vector whose target genes are the base sequences shown in SEQ ID NOs: 9 and 12, respectively. [8] A method for restoring cytoplasmic male sterility to a Solanaceae plant, comprising deleting the cytoplasmic male sterility gene in the cytoplasmic male sterility plant or inhibiting the function or expression of the cytoplasmic male sterility gene. [9] A method for restoring cytoplasmic male sterility to the Solanaceae plant, comprising editing the mitochondrial genome DNA of the cytoplasmic male sterile Solanaceae plant, thereby deleting the cytoplasmic male sterility gene, or inhibiting the function or expression of the cytoplasmic male sterility gene.

[10] A method for restoring fertility to a cytoplasmic male-sterile plant of the Solanaceae family, comprising editing the mitochondrial genomic DNA by the mitoTALEN method, A method for restoring fertility to cytoplasmic male sterility in the Solanaceae plant, using a mitoTALEN expression vector with the base sequences shown in SEQ ID NOs: 9 and 12 as target genes, respectively.

[11] A method for producing a male sterility-restored plant, which restores cytoplasmic maleness to a Solanaceae plant by the method for restoring fertility of cytoplasmic male sterility in a Solanaceae plant.

[12] A male sterile plant, which is a Solanaceae plant in which the cytoplasmic male sterility gene is expressed.

[13] The male sterile plant, wherein the Solanaceae plant is a plant of the genus Solanum or Capsicum.

[14] The male sterile plant, wherein the Solanum plant is a tomato.

[15] A method for producing a male sterile plant, which comprises expressing the cytoplasmic male sterility gene in a Solanaceae plant. [Effects of the Invention]

[0014] According to the present invention, the cytoplasmic male sterility gene of Solanaceae plants has been identified, and it can be used to induce and restore male sterility, thereby greatly improving the efficiency of plant breeding and seed production. There are thus obtained a cytoplasmic male sterility gene of Solanaceae plants, a male sterility restored plant, a method for restoring cytoplasmic maleness of Solanaceae plants, a method for producing a male sterility restored plant, a male sterile plant, and a method for producing a male sterility plant. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing the steps of identifying a cytoplasmic male sterility gene in a Solanaceae plant according to this embodiment. [Figure 2] Figure 1 shows the results of RNA-seq for candidate genes for cytoplasmic male sterility: (a) orf137, (b) orf265, and (c) orf265. [Figure 3] FIG. 1 shows RT-PCR results for candidate genes for cytoplasmic male sterility genes. [Figure 4] Schematic diagram showing (a) the structure of the vector used to inhibit expression, and (b) and (c) the sequence of the target gene for candidate genes for cytoplasmic male sterility. [Figure 5] This figure shows the results of examining mitochondrial DNA expression in tomato CMS lines in which the target gene was disrupted. [Figure 6] FIG. 1 is a photograph showing the restoration of male sterility in a line in which the orf137 gene has been disrupted. DETAILED DESCRIPTION OF THE INVENTION

[0016] The cytoplasmic male sterility gene for solanaceous plants, the male sterility restorer plant, the method for restoring cytoplasmic maleness in solanaceous plants, the male sterility restorer plant, the male sterile plant, and the method for producing the male sterility plant according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0017] (Cytoplasmic male sterility gene in Solanaceae plants) The cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a protein that exerts the cytoplasmic male sterility in a plant, which has an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a); (c) a protein that has an amino acid sequence that is 80% or more identical to the amino acid sequence of (a), and that exerts the cytoplasmic male sterility in a plant; The gene sequence encoding a protein selected from the group comprising:

[0018] Solanaceae plants broadly include plants of the Solanaceae family, such as plants of the Solanaceae genus, including plants of the Solanum genus and Capsicum genus. Solanaceae plants also include plants of the Solanum genus, including eggplant, potato, and tomato. Capsicum plants include peppers and bell peppers.

[0019] It is particularly preferred that the Solanaceae plant is a crop, that is, a plant used in the agricultural field, particularly as a food raw material. In this embodiment, the presence or absence of male sterility is manipulated by suppressing the expression of mitochondrial genome DNA.

[0020] The cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is typically (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 It is a gene that encodes In particular, the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 was discovered by the inventors and is designated orf137. SEQ ID NO: 2 shows the gene sequence of orf137. As will be explained in the Examples below, this gene was discovered in the mitochondrial genes of a CMS (cytoplasmic male sterility) line of tomato and confers cytoplasmic male sterility. The protein that confers cytoplasmic male sterility is called an S factor, and the cytoplasmic male sterility gene is considered to be a gene encoding the S factor, and will hereinafter also be referred to as the S factor gene. Furthermore, as described below, by deleting this gene in a Solanaceae plant or inhibiting the function or expression of the cytoplasmic male sterility gene, it is possible to restore a CMS line from a state in which it has male sterility to a state in which it does not, i.e., to restore male sterility. Furthermore, inducing the expression of this gene can confer cytoplasmic male sterility, particularly in Solanaceae plants, and by transferring this gene orf137 to other Solanaceae plants, male sterility can be induced. Until now, no gene that confers cytoplasmic male sterility has been found in plants of the Solanaceae family, but the present inventors have now discovered the gene and a method for its application.

[0021] In addition, the cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is (b) It is also preferable that the gene has an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a), and encodes a protein selected from the group including proteins that exert the cytoplasmic male sterility in plants. Also, (c) It is also preferable that the protein has an amino acid sequence with 80% or more identity to the amino acid sequence of (a) and exerts the cytoplasmic male sterility in plants.

[0022] Specifically, the gene may encode an amino acid sequence in which amino acids have been deleted, substituted, or added to the amino acid sequence of (a) or (b). These deletions, substitutions, or additions may be introduced by known methods for creating mutant proteins, such as site-directed mutagenesis. The mutations may be artificially introduced or may be similar mutations that occur naturally. Genes into which such modifications or mutations have been introduced can also be used depending on the plant species and the purpose. Furthermore, it is also preferable that the gene encodes a protein consisting of an amino acid sequence having 90% or more homology to the amino acid sequence of (a) or (b) above. The means for searching and determining the sequence identity can be a search using FASTA or BLAST, for example.

[0023] It is also preferable that the cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is a gene whose base sequence has 80% or more, preferably 90% or more, homology to the base sequence of a gene encoding the amino acid sequence of (a) or (b). The cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is also preferably a nucleic acid complementary to the gene sequence of sequence (a) or (b). This nucleic acid may be one in which a deletion, substitution, or addition has been introduced into the base sequence of the nucleic acid complementary to the gene sequence of sequence (a) or (b).

[0024] (Male sterility recovery plant) The male sterility-restored plant of this embodiment is a Solanaceae plant in which the cytoplasmic male sterility gene has been deleted or the function or expression of the cytoplasmic male sterility gene has been inhibited. This plant is a male sterile plant, specifically, a CMS lineage plant in which the cytoplasmic male sterility gene is expressed, which has been restored from male sterility and has activated pollen.

[0025] "Making the cytoplasmic male sterility gene defective" refers to a state in which the cytoplasmic male sterility gene is deleted from within the plant by disruption, loss, or other means. "Inhibiting the function or expression of the cytoplasmic male sterility gene" includes suppressing the function or expression of the DNA, RNA, or protein of the gene. The deletion, function, or expression of these genes can be suppressed by external application of chemicals or the like that can suppress the function or expression. These methods broadly include methods for eliminating or reducing the function of the protein encoded by the gene. For example, methods can be used to prevent the protein from localizing to mitochondria, or, even if it can localize to mitochondria, to prevent it from functioning. These suppression methods can be appropriately selected from known or future similar means.

[0026] In this embodiment, the deletion of the cytoplasmic male sterility gene includes editing the mitochondrial genomic DNA to delete the gene, since the gene is present in the mitochondrial genomic DNA. In this embodiment, mitochondrial genomic DNA refers to genes located in mitochondria, whereas genes present in the nucleus of a cell are sometimes called nuclear genes.

[0027] The male sterility restorer plant of this embodiment is a male sterility restorer plant in which mitochondrial genomic DNA has been edited using the mitoTALEN method, and it is also preferable to use a mitoTALEN expression vector in which the base sequences shown in SEQ ID NOs: 9 and 12 have been introduced into the vector as target genes.

[0028] The mitoTALEN method is a means for editing mitochondrial genome DNA structure and is disclosed, for example, in Japanese Patent Application Laid-Open No. 2018-130043. Specifically, a double-strand break is introduced into a target sequence region present in each molecular species of mitochondrial genome DNA in a plant cell, thereby inducing a structural change in the mitochondrial genome. Here, the target sequence region refers to the region in each molecular species of mitochondrial genome DNA where deletion / loss is desired to be induced or a region adjacent thereto. The structural change in the mitochondrial genome is induced by DNA recombination that occurs between a sequence present near the target sequence region and a homologous sequence present in another region. In particular, the double-strand break is introduced by TALEN (transcription activator-like effector nucleases). In particular, according to the above publication, a double-strand break is introduced into a gene or a region adjacent to the gene present in each molecular species of mitochondrial genomic DNA in a plant cell, thereby causing the gene to be deleted, and the deletion of the gene is induced by DNA recombination occurring between a sequence present in the gene or the region adjacent to the gene and a homologous sequence present in another region. This method is used for the purpose of deleting a gene responsible for male sterility in a plant cell.

[0029] Specifically, for example, a tandem expression plasmid with an added mitochondrial localization signal is used to simultaneously express two TALENs, such as a TALE left and a TALE right, in one plasmid and localize them to mitochondria. As the target sequence region, for example, the base sequences shown in SEQ ID NOs: 9 and 12 are preferably introduced into the vector as target genes. SEQ ID NOs: 9 and 12 are part of the orf137 gene, and are used to delete or disrupt the orf137 gene in the mitochondrial genome DNA, thereby deleting the orf137 gene. LEFT and TALE that binds to the latter part of the sequence RIGHTThese sequences are paired and introduced into a vector, and the vector is then introduced into mitochondria. The TALEN sequence is preferably, for example, 12 to 20 bases. The TALEN sequence can be, for example, the TALE shown in Figure 4(b) described below. LEFT-1 , TALE RIGHT-1 or the TALE shown in Figure 4(c) LEFT-2 , TALE RIGHT-2 You can select areas such as:

[0030] (Method for restoring cytoplasmic masculinity in Solanaceae plants) The method for restoring cytoplasmic male sterility in a Solanaceae plant according to this embodiment involves deleting the cytoplasmic male sterility gene in the cytoplasmic male sterile Solanaceae plant or inhibiting the function or expression of the cytoplasmic male sterility gene. This method can be carried out, for example, by editing the mitochondrial genomic DNA of the cytoplasmic male sterile Solanaceae plant. Mitochondrial genomic DNA can be edited, for example, by the mitoTALEN method, which can delete the cytoplasmic male sterility gene.

[0031] (Method for producing male sterility-restored plants) The method for producing a male sterility-restored plant of this embodiment involves restoring the cytoplasmic male sterility of the Solanaceae plant by the method for restoring cytoplasmic male sterility of the Solanaceae plant described above. In this production method, the male sterility-restored plant and the method for restoring the male sterility can be selected from those described above.

[0032] (male sterile plants) The male sterile plant of this embodiment is a Solanaceae plant in which the cytoplasmic male sterility gene is expressed. The Solanaceae plant of this embodiment can be selected from the Solanaceae plants described above, but can also be selected from plants or lines that do not naturally possess a cytoplasmic male sterility gene. Specifically, the Solanaceae plant can be selected from plants of the genus Solanum or Capsicum, and the Solanaceae plant can be selected from, for example, tomato. CMS lines have been found in tomatoes created by cell fusion with other Solanaceae plants, such as potato, and male sterile plants can be similarly produced.

[0033] Methods for expressing a cytoplasmic male sterility gene in a Solanaceae plant can be achieved by known methods for introducing genes into plants, particularly into mitochondrial DNA. For example, a vector for expressing ORF137 in mitochondria can be created and introduced into other Solanaceae plants to induce male sterility. Alternatively, genes can be introduced by modifying and reconstructing mitochondrial DNA using, for example, the mitoTALEN method. In this case, it is preferable to modify and reconstruct mitochondrial DNA at sites other than essential genes.

[0034] (Method for producing male sterile plants) The method for producing a male sterile plant of this embodiment involves expressing the cytoplasmic male sterility gene in a Solanaceae plant. The Solanaceae plant and the method for expressing the cytoplasmic male sterility gene can be selected from those described above, such as a method of creating a vector for expression in mitochondria and introducing it into another Solanaceae plant to induce male sterility.

[0035] (Effects of this embodiment) When the cytoplasmic male sterility gene of this embodiment is induced and expressed in a Solanaceae plant, pollen is produced, but the pollen is inactive and does not undergo cross-pollination, thereby controlling so-called male sterility. In a plant line that has cytoplasmic male sterility, i.e., expresses a cytoplasmic male sterility gene, maleness can be restored to the plant by deleting the cytoplasmic male sterility gene or inhibiting the function or expression of the cytoplasmic male sterility gene, thereby allowing the plant to produce viable pollen, fruit, and seeds. On the other hand, by making a plant strain that does not have cytoplasmic male sterility express the cytoplasmic male sterility gene of this embodiment, the possibility of pollen from the seed parent pollinating can be eliminated without cross-pollination and without performing the process of slow maleing. In other words, by turning the expression of this gene on and off, the work involved in crossbreeding can be made significantly more efficient and less costly.

[0036] This embodiment identifies a cytoplasmic male sterility gene for Solanaceae and provides a male sterility restorer plant, a method for restoring cytoplasmic maleness to a Solanaceae plant, a method for producing a male sterility restorer plant, a male sterile plant, and a method for producing a male sterile plant. Solanaceae plants include species whose fruits are edible, such as eggplant, tomato, chili pepper, and bell pepper, as well as species whose tubers are edible, such as potato. They also cover a wide range of species, including those cultivated as luxury crops, such as tobacco, and ornamental plants used in horticulture, such as physalis or petunia. The technology of this embodiment can be applied to phylogenetic manipulation, such as crossbreeding, of these Solanaceae plants, and therefore has a wide range of applications. [Example]

[0037] Examples are shown below, but the present invention is not limited to these examples.

[0038] (Identification of the S-factor gene for cytoplasmic male sterility) In order to search for the cytoplasmic male sterility gene in Solanaceae plants, we searched for mitochondrial genes and extracted genes that are only present in CMS (cytoplasmic male sterility) lines. In summary, the genome sequences of normal cultivated tomatoes and CMS line tomatoes were constructed and the gene structure was predicted. Next, expression analysis was performed to select expressed genes. Next, the expressed genes were compared between cultivated tomatoes and CMS tomatoes using RNA derived from anthers, and genes specifically expressed only in CMS tomatoes were selected.

[0039] Specifically, as shown in Figure 1, (1) First, we predicted open reading frames (ORFs) encoding more than 70 amino acids from the genes of three tomato CMS lines known as CMS[PF], CMS[MSA1], and CMS[OF209], and extracted 831, 1025, and 969 ORFs, respectively. (2) Next, we compared the orfs of the mitochondrial genes (mitochondrial donors) of the CMS strains (PF, LA1673, Sekai-ichi, LA1670, OF209, S. pennellii, and N. tabacum) with those of the original strains, and eliminated orfs that were not specific to the CMS strains. The remaining orfs were 183, 272, and 140 genes for CMS[PF], CMS[MSA1], and CMS[OF209], respectively. (3) Next, we identified common ORFs among the CMS strains, which consisted of 36, 41, and 33 genes in CMS[PF], CMS[MSA1], and CMS[OF209], respectively. (4) From these, we performed RNA-Seq on the anthers to detect expressed orfs. As a result, three orfs, orf137, orf193, and orf265, were extracted. Two copies of orf137 were obtained from two CMS lines, CMS-PMt002g07240 and CMS-PMt005g13392, one copy of orf193 was obtained from the CMS line, CMS-PMt002g06465, and one copy of orf265 was obtained from the CMS line, CMS-PMt010g15739.

[0040] The results of the RNA-seq analysis described in (4) above are shown in Figure 2. The sequence of orf137 in Figure 2(a) is 55% identical to the CMS protein of the pepper CMS line, the sequence of orf193 in Figure 2(b) is 93% identical to cytochrome f, and the sequence of orf265 in Figure 2(c) is 92% identical to ATP synthase subunit 8. These results suggest that orf137, which shows partial homology to genes in the pepper CMS line, is particularly likely to be a gene related to CMS.

[0041] RT-PCR was performed to examine RNA expression in each tissue of the CMS[P] lineage for the three orfs described in (4) above. For orf137, the forward primer (5'-3') was SEQ ID NO: 3 and the reverse primer (5'-3') was SEQ ID NO: 4. For orf193, the forward primer (5'-3') was SEQ ID NO: 5 and the reverse primer (5'-3') was SEQ ID NO: 6. For orf265, the forward primer (5'-3') was SEQ ID NO: 7 and the reverse primer (5'-3') was SEQ ID NO: 8. SEQ ID NO: 3: cgattgagaa agcggcaggc SEQ ID NO: 4: gttattttcg ctgcaacggc g SEQ ID NO: 5: ggggaatcgg ccttctttag tc SEQ ID NO: 6: ggggagggtt taataaagga gctg SEQ ID NO: 7: cggagtgaag ctgtattgag gg SEQ ID NO: 8: gaggagagga acgaagaacg aaac The results are shown in Figure 3. Orf137 was particularly highly expressed in pollen.

[0042] (Male sterility restoration by gene inhibition) Next, we tested the restoration of male sexuality by actually attempting to inhibit the S element of these three mitochondrial genes. We edited the mitochondrial genome DNA to delete the DNA. We used the mitoTALEN method to edit the plant mitochondrial genome. Specifically, we designed the Ti plasmid expression vector as shown in Figure 4(a). In the figure, MLS represents the mitochondrial localization signal, and TALE left and TALE right indicates the corresponding sequence that binds to the target gene. This target sequence created two targets in different regions for three genes: orf137, orf193, and orf265. left and TALE right Regarding the target genes to which the first target binds, the first target is shown in Figure 4(b) (SEQ ID NOs: 9 to 11) (Equivalent to Sequence #8, #9, #10 in the diagram) The second target is shown in Figure 4(c) (SEQ ID NOs: 12 to 14). (Equivalent to Sequence #11, #12, and #13 in the diagram) From each of these target sequences, two sites were further selected before and after the sequence shown in the figure, and each was used as a TALE. left and TALE right The vector was incorporated into an expression vector as the sequence shown below. Gene introduction into the vector was performed using the Acrobacterium method, in accordance with the method described in "A Highly Efficient Transformation Protocol for Micro-Tom, a Model Cultivar for Tomato Functional Genomics." Plant and Cell Physiology, Volume 47, Issue 3, March 2006, Pages 426-431. The vector was created at the request of the Arimura Shinichi Laboratory at the University of Tokyo.

[0043] Figure 5 shows the mitochondrial DNA expression in tomato CMS lines in which this vector was introduced and the target gene was disrupted. Lanes #127-1, #127-2, #140-1, #140-2, and #140-3 represent lines in which the orf137 gene was disrupted using the mitoTALEN method. The "Positive" lanes represent the original CMS line. The upper band representing the conserved genome represents the original mitochondrial DNA, while the lower band represents orf137. In the CMS lines, both the conserved genome and orf137 bands are visible. However, in lines #127-1, #127-2, #140-1, #140-2, and #140-3, only the conserved genome was visible, with no orf137 band, indicating that the orf137 mitochondrial DNA was disrupted.

[0044] As shown in the photograph in Figure 6, when the orf137 gene in the mitochondria of the #127-1 strain was disrupted, tomato fruit was produced regardless of whether the strain was a CMS strain. These results demonstrate that inhibiting the orf137 gene completely restores pollen fertility to CMS strains, demonstrating that orf137 is the gene responsible for male sterility and that inhibiting orf137 can restore male fertility to male-sterile strains. [Industrial Applicability]

[0045] According to the present invention, the cytoplasmic male sterility gene of Solanaceae plants has been identified, and it can be used to induce and restore male sterility, thereby greatly improving the efficiency of plant breeding and seed production. There are thus obtained a cytoplasmic male sterility gene of Solanaceae plants, a male sterility restored plant, a method for restoring cytoplasmic maleness of Solanaceae plants, a method for producing a male sterility restored plant, a male sterile plant, and a method for producing a male sterility plant.

Claims

1. (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a protein that exhibits cytoplasmic male sterility in a plant, having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a); (c) a protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence of (a) and that exerts cytoplasmic male sterility in plants; A male sterility restored plant, which is a Solanaceae plant in which a cytoplasmic male sterility gene encoding a protein selected from the group comprising:

2. The male sterility restorer plant according to claim 1 , wherein the Solanaceae plant is a plant of the genus Solanum or Capsicum.

3. 3. The male sterility restorer plant according to claim 2, wherein the Solanum plant is eggplant, potato, or tomato.

4. The male sterility restorer plant according to claim 2 , wherein the Capsicum plant is a chili pepper or a bell pepper.

5. The male sterility restored plant according to any one of claims 1 to 4, wherein the mitochondrial genomic DNA is edited to delete the cytoplasmic male sterility gene.

6. A male sterility restored plant in which the mitochondrial genomic DNA has been edited by the mitoTALEN method, A male sterility-restoring plant as described in claim 5, which uses a mitoTALEN expression vector with the base sequences shown in SEQ ID NOs: 9 and 12 as target genes, respectively.

7. A method for restoring cytoplasmic male sterility to a Solanaceae plant, comprising deleting a cytoplasmic male sterility gene in the Solanaceae plant, the method comprising: The cytoplasmic male sterility gene is (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a protein that exhibits cytoplasmic male sterility in a plant, having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a); (c) a protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence of (a) and that exerts cytoplasmic male sterility in plants; A method for restoring fertility to a cytoplasmic male sterility plant of the Solanaceae family, wherein the cytoplasmic male sterility gene encodes a protein selected from the group consisting of:

8. 8. The method for restoring fertility to a cytoplasmic male sterile Solanaceae plant according to claim 7, comprising editing the mitochondrial genome DNA of the cytoplasmic male sterile Solanaceae plant to delete the cytoplasmic male sterility gene.

9. A method for restoring fertility to a cytoplasmic male-sterile plant of the Solanaceae family, in which the mitochondrial genomic DNA is edited by the mitoTALEN method, comprising: A method for restoring fertility to cytoplasmic male sterility in a Solanaceae plant described in claim 8, using a mitoTALEN expression vector with the base sequences shown in SEQ ID NOs: 9 and 12 as target genes, respectively.

10. A method for producing a male-sterility-restored plant, which comprises restoring cytoplasmic male sterility of a Solanaceae plant by the method for restoring fertility of cytoplasmic male sterility according to any one of claims 7 to 9.

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