JP7760118B2 - 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 - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act On March 5, 2021, the research paper "Organelle genome assembly uncovers the dynamic genome reorganization and cytoplasmic male sterility associated genes in tomato" was posted to the preprint server bioRxiv operated by Cold Spring Harbor Laboratory in the United States, with the following public URL: <https://www.biorxiv.org/content/10.1101/2021.03.03.433741v1> <https://www.biorxiv.org/content/10.1101/2021.03.03.433741v1> The results were published at: org/content/10.1101/2021.03.03.433741v1.full.pdf+html>.

The present invention relates to a gene for cytoplasmic male sterility in Solanaceae plants, and male sterility-restored plants and male sterile plants produced by manipulating the expression of that gene.

When crossbreeding crops such as vegetables, F1 seed production involves crossbreeding 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 carried out, in which the anthers are removed from the seed parent. However, this emasculation process is currently mainly done by hand, which requires a lot of labor and is costly, causing the price of seeds obtained through seed production to rise. Furthermore, the need for a large number of workers poses a risk that valuable intellectual property, the parent line, may be leaked. Manual emasculation also has a certain degree of risk of failure, which could result in contamination with pollen from the seed parent, resulting in seeds that do not result in the desired crossbreeding.

To address these issues, the seed parent can be rendered male sterile, i.e., so that it does not produce pollen, or so that it produces sterile, inactive pollen that does not cross-pollinate, thereby eliminating the possibility of pollen from the seed parent being fertilized without the need for manual sterilization.

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 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.

Patent Document 1 discloses a simple method for producing male-sterile tomato plants, which involves fusing protoplasts isolated from tomato plants and treated to render them inoperable for cytoplasmic factors with protoplasts isolated from Solanum plants and treated to render them inoperable for nuclear genetic material, and then regenerating male-sterile tomatoes from the fusion product. This technology creates a line with male sterility through protoplast fusion, and aims 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.

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

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, useful for modifying plant traits, such as male sterility, in horticultural plants such as petunia of the Solanaceae family.

Patent No. 2824841 International Publication No. 2014/027502 Japanese Patent Application Laid-Open No. 2003-92937

The technology described in Patent Document 1 makes it possible to obtain male-sterile lines in tomatoes, which belong to the Solanaceae family, but the gene responsible for this cytoplasmic male sterility has not been identified. Therefore, while it is possible to produce fruitless Solanaceae plants using the aforementioned sterile lines, they are unable to produce seeds and therefore cannot be propagated. In order to apply male-sterile lines, it is desirable to identify the gene responsible for cytoplasmic male sterility and be able to control its on/off state.

The technology in 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 yet been identified.

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 characteristics. 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 horticultural plants such as petunias in the Solanaceae family, and is not intended for use in the food industry.

The present invention was made in light of the above-mentioned circumstances, and its purpose is to provide a cytoplasmic male sterility gene for Solanaceae plants that can be used to induce and restore male sterility, greatly improving the efficiency of plant breeding and seed production, a male sterility-restored plant, a method for restoring cytoplasmic maleness in Solanaceae plants, a method for producing a male sterility-restored plant, a male sterile plant, and a method for producing a male sterility plant.

In order to solve the above problems, the present invention has the following aspects.
[1] (a) a protein consisting of the amino acid sequence shown 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 a 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 as described above, 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 as described above, wherein the Capsicum plant is a chili pepper or a bell pepper.
[6] The male sterility restored plant, wherein the mitochondrial genome DNA has been edited to delete the cytoplasmic male sterility gene, or the function or expression of the cytoplasmic male sterility gene has been inhibited.
[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 with the base sequences shown in SEQ ID NOs: 9 and 12 as target genes, respectively.
[8] A method for restoring fertility to a cytoplasmic male sterile Solanaceae plant, comprising 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.
[9] A method for restoring fertility of a cytoplasmic male sterile Solanaceae plant, comprising editing the mitochondrial genomic DNA of the cytoplasmic male sterile Solanaceae plant to delete 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 as described above, wherein the Solanaceae plant is a plant of the genus Solanum or Capsicum.
[14] The male sterile plant as described above, wherein the plant of the genus Solanum is a tomato.
[15] A method for producing a male sterile plant, which comprises expressing the cytoplasmic male sterility gene in a Solanaceae plant.

The present invention identifies the cytoplasmic male sterility gene in Solanaceae plants, which can be used to induce and restore male sterility, greatly improving the efficiency of plant breeding and seed production. It also provides a cytoplasmic male sterility gene in Solanaceae plants, a male sterility restored plant, a method for restoring cytoplasmic maleness in Solanaceae plants, a method for producing a male sterility restored plant, a male sterile plant, and a method for producing a male sterile plant.

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 1 shows the results of RNA-seq for candidate genes for cytoplasmic male sterility, (a) orf137, (b) orf265, and (c) orf265. FIG. 1 shows RT-PCR results for candidate genes for cytoplasmic male sterility genes. 1A and 1B are schematic diagrams showing the structure of a vector used to inhibit expression of candidate genes for cytoplasmic male sterility, and (b) and (c) the sequence of the target gene. This is a diagram showing the results of examining mitochondrial DNA expression in a tomato CMS line in which a target gene was disrupted. FIG. 1 is a photograph showing the restoration of male sterility in a line in which the orf137 gene has been disrupted.

The following describes, with reference to embodiments, the cytoplasmic male sterility gene for solanaceous plants, male sterility restored plants, methods for restoring cytoplasmic maleness in solanaceous plants, methods for producing male sterility restored plants, male sterile plants, and methods for producing male sterile plants according to the present invention. However, the present invention is not limited to the following embodiments.

(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 shown 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 exhibits the cytoplasmic male sterility in a plant, the protein having an amino acid sequence that has 80% or more identity with the amino acid sequence of (a);
The gene sequence encoding a protein selected from the group comprising:

Solanaceae plants broadly include plants of the Solanaceae family, such as plants of the Solanaceae genus or Capsicum. Solanaceae plants also include, for example, eggplant, potato, or tomato. Capsicum plants include chili peppers or bell peppers.

Solanaceae plants are particularly preferably crops, i.e., plants used in the agricultural field, particularly as food raw materials. In this embodiment, the presence or absence of male sterility is manipulated by suppressing the expression of mitochondrial genome DNA.

The cytoplasmic male sterility gene of the Solanaceae plant of this embodiment is typically
(a) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
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. The gene sequence for orf137 is shown in SEQ ID NO: 2. As will be explained in the Examples below, this gene was discovered in the mitochondrial genes of the CMS (Cytoplasmic Male Sterility) strain 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.

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 that is 80% or more identical to the amino acid sequence of (a) and that exerts the cytoplasmic male sterility in plants.

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.
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). The sequence identity can be searched and determined by searching using FASTA or BLAST, for example.

In addition, it is also preferable that the cytoplasmic male sterility gene of a 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).

(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.

"Deleting the cytoplasmic male sterility gene" refers to a state in which the cytoplasmic male sterility gene is deleted from within the plant by destruction or loss. "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.

In this embodiment, the deletion of the cytoplasmic male sterility gene includes a procedure of deleting the gene by editing the mitochondrial genomic DNA, 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.

The male sterility restorer plant of this embodiment is a male sterility restorer plant whose 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 as target genes into the vector.

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. The target sequence region here 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 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 using 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.

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 portions of the orf137 gene and are used to delete or disrupt the orf137 gene in mitochondrial genomic DNA, thereby deleting the orf137 gene. From the base sequences of the above SEQ ID NOs: TALE _LEFT, which binds to a portion of the first half of the sequence, and TALE _RIGHT, which binds to a portion of the second half of the sequence, are selected, and these sequences are paired and introduced into a vector, which is then introduced into mitochondria. The TALEN sequence preferably has, for example, 12 to 20 bases. The TALEN sequence can be located, for example, in the TALE _LEFT-1 and TALE _RIGHT-1 regions shown in Figure 4(b) or the TALE _LEFT-2 and TALE _RIGHT-2 regions shown in Figure 4(c).

(Method for restoring cytoplasmic masculinity in Solanaceae plants)
The method for restoring cytoplasmic male sterility in a Solanaceae plant of 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 performed, for example, by editing the mitochondrial genomic DNA of the cytoplasmic male sterile Solanaceae plant. The mitochondrial genomic DNA can be edited, for example, by the mitoTALEN method, which can delete the cytoplasmic male sterility gene.

(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.

(male sterile plants)
The male sterile plant of this embodiment is a Solanaceae plant in which the cytoplasmic male sterility gene described above 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 tomato produced by protoplast fusion with other Solanaceae plants, such as potato, and male sterile plants can be produced in a similar manner.

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 then introduced into other Solanaceae plants to induce male sterility. Alternatively, genes can be introduced by modifying and reconstructing mitochondrial DNA, for example, using the mitoTALEN method.
In this case, it is preferable to modify and reconstruct mitochondrial DNA at a site other than the essential gene.

(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.

(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 the cytoplasmic male sterility gene of this embodiment expressible in a plant strain that does not have cytoplasmic male sterility, 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 on/off the expression of this gene, the efficiency and cost of crossbreeding operations can be greatly improved.

This embodiment identifies a cytoplasmic male sterility gene for the Solanaceae family 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 potatoes. 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 genetic manipulation, such as crossbreeding, of these Solanaceae plants, and has a wide range of applications.

The following are examples. Note that the present invention is not limited to these examples.

(Identification of the S-factor gene for cytoplasmic male sterility)
In order to search for cytoplasmic male sterility genes in Solanaceae plants, we searched for mitochondrial genes and extracted genes that are only found in CMS (cytoplasmic male sterile) lines.
In summary, the genome sequences of normal cultivated tomatoes and CMS line tomatoes were constructed and the gene structures were predicted. Expression analysis was then 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.

Specifically, as shown in FIG.
(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.

The results of the RNA-seq 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.

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.

(Male sterility restoration by gene inhibition)
Next, we tested the restoration of male sexuality by actually attempting to inhibit the S elements of these three mitochondrial genes. We then edited the mitochondrial genome DNA to delete the DNA. We used the mitoTALEN method to edit plant mitochondrial genomes.
Specifically, we designed the Ti plasmid expression vector shown in Figure 4(a). In the figure, MLS indicates the mitochondrial localization signal, and TALE _left and TALE _right indicate the corresponding sequences that bind to the target gene. Two distinct target sequences were created for each of the three genes, orf137, orf193, and orf265. Regarding the target genes to which the TALE _left and TALE _right bind, the first target is shown in Figure 4(b) (SEQ ID NOS: 9-11) (corresponding to Sequences #8, #9, and #10 in the figure) , and the second target is shown in Figure 4(c) (SEQ ID NOS: 12-14) (corresponding to Sequences #11, #12, and #13 in the figure) . From each of these target sequences, two sites were selected before and after the sequences shown in the figure, and these were incorporated into the expression vector as the TALE _left and TALE _right sequences, respectively. The vector was incorporated by gene transfer using the Acrobacterium method, and the gene transfer method was performed in accordance with "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.

Figure 5 shows the results of examining mitochondrial DNA expression in tomato CMS lines in which this vector was introduced to disrupt the target gene. 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, while "Positive" represents the original CMS line. The upper conserved genome band in the figure represents the original genome of the mitochondrial DNA, and the lower band represents orf137. In the CMS lines, both the conserved genome and orf137 bands are visible, but in lines #127-1, #127-2, #140-1, #140-2, and #140-3, only the conserved genome is visible, with no orf137 band, indicating that the mitochondrial DNA at orf137 has been disrupted.

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 it 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.

The present invention identifies the cytoplasmic male sterility gene in Solanaceae plants, which can be used to induce and restore male sterility, greatly improving the efficiency of plant breeding and seed production. It also provides a cytoplasmic male sterility gene in Solanaceae plants, a male sterility restored plant, a method for restoring cytoplasmic maleness in Solanaceae plants, a method for producing a male sterility restored plant, a male sterile plant, and a method for producing a male sterile plant.

Claims (10)

(a) a protein consisting of the amino acid sequence shown 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 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: The male sterility restorer plant according to claim 1, wherein the Solanaceae plant is a plant of the genus Solanum or Capsicum. The male sterility restorer plant according to claim 2, wherein the Solanum plant is eggplant, potato, or tomato. The male sterility restorer plant according to claim 2, wherein the Capsicum plant is a chili pepper or a bell pepper. The male sterility restored plant according to any one of claims 1 to 4 , wherein the mitochondrial genomic DNA has been edited to delete the cytoplasmic male sterility gene. 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. A method for restoring fertility to a cytoplasmic male sterile Solanaceae plant , comprising deleting a cytoplasmic male sterility gene in the cytoplasmic male sterile Solanaceae plant,
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 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 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. 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 . 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. A method for producing a male-sterility-restored plant, in which cytoplasmic male sterility of a Solanaceae plant is restored using the method for restoring cytoplasmic male sterility of any one of claims 7 to 9.