WO2021244007A1 - 水稻雄性育性调控基因、水稻雄性育性调控基因突变体、其应用以及调控水稻育性的方法 - Google Patents

水稻雄性育性调控基因、水稻雄性育性调控基因突变体、其应用以及调控水稻育性的方法 Download PDF

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WO2021244007A1
WO2021244007A1 PCT/CN2020/137145 CN2020137145W WO2021244007A1 WO 2021244007 A1 WO2021244007 A1 WO 2021244007A1 CN 2020137145 W CN2020137145 W CN 2020137145W WO 2021244007 A1 WO2021244007 A1 WO 2021244007A1
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plant
rice
gene
sequence
seq
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PCT/CN2020/137145
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English (en)
French (fr)
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龙湍
唐杰
李佳林
吴春瑜
刘昊
李燕群
韩晓斌
曾翔
李新鹏
安保光
吴永忠
黄培劲
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海南波莲水稻基因科技有限公司
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Priority claimed from CN202010491115.0A external-priority patent/CN113754747B/zh
Priority claimed from CN202010491100.4A external-priority patent/CN113754746B/zh
Application filed by 海南波莲水稻基因科技有限公司 filed Critical 海南波莲水稻基因科技有限公司
Priority to US17/927,343 priority Critical patent/US20230220413A1/en
Publication of WO2021244007A1 publication Critical patent/WO2021244007A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to the field of biotechnology, in particular to a plant fertility regulating gene GMS2, GMS2 encoding protein, gene knockout mutants of GMS2, the application of GMS2 genes, proteins and mutants in cross breeding; and plant fertility related proteins GMS2 mutant, its coding gene, its molecular markers and its application in cross breeding.
  • Hybrid rice is the first generation of offspring obtained after crossing the parental parent. Its yield is often more than 15% higher than that of the conventional rice parent, and its resistance and adaptability are far better than that of the parental parent. Therefore, the application and promotion of hybrid rice is an important way to increase rice yield.
  • Male sterile line is the key node of hybrid rice breeding technology.
  • Male sterile line refers to a plant line with abnormal development of male gametes and loss of fertility, and normal development of female gametes. It can only accept pollen from its male parent as its mother, and cannot bear fruit by itself.
  • nucleocytoplasmic interaction type and light-temperature sensitive type.
  • the sterility gene of the nucleocytoplasmic male sterile line is in the cytoplasm, but there is no fertility restorer gene in the nucleus. When a restorer line with a fertility restorer gene in the nucleus is crossed with its mating group, a fertile hybrid can be produced.
  • nucleocytoplasmic sterile line is the first sterile line to be used on a large scale in hybrid rice breeding and production, laying a material foundation for the establishment and development of the hybrid rice industry.
  • nucleoplasmic sterile lines are restricted by the genotype of the restorer lines, only about 5% of the germplasm resources can be used.
  • the cytoplasmic sterility gene has the potential risk of poor rice quality and the epidemic of specific diseases and insect pests.
  • Photothermosensitive male sterile line is a sterile line whose fertility is regulated by light and temperature environment. Under certain light and temperature conditions, this sterile line remains sterile and can be used for hybridization. When the conditions change, the sterile line restores fertility and can be used for the reproduction of the sterile line. Since the photo-temperature-sensitive male sterile line realizes the combination of sterile line and maintainer line, only the male parent and its matching group are required to produce the first hybrid of offspring. Therefore, the corresponding breeding technology is often referred to as the "two-line method". ". The genes that regulate light-temperature-sensitive male sterility are in the nucleus.
  • the currently cloned genes include PMS3, TMS5, CSA, and TMS10 (Chen and Liu, 2014, Male sterility and fertility restoration in crops, Annu Rev Plant Biol, 65:579- 606; Zhou H, et al, 2014, RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice, Nature Communications, 5:4884-4892).
  • the photo-temperature-sensitive sterile line has a simple reproduction procedure, and the matching group is more free due to the widespread existence of restorer genes.
  • the large-scale application of photo-thermo-sensitive sterile lines has greatly consolidated and promoted the development of the hybrid rice industry. However, because the fertility of this type of sterile line is affected by the light and temperature environment, the risk of seed production is high, and the seed production area is restricted.
  • Nuclear male sterility is caused by nuclear gene mutations, including dominant mutations and recessive mutations, sporophyte gene mutations and gametophytic gene mutations. Dominant mutations and gametophytic gene mutations can only be inherited through female gametes, while recessive mutations can be inherited through both female gametes and male gametes, and follow Mendel's law.
  • the present invention provides a plant fertility-related regulatory gene and a male sterile line of recessive nuclear sterility type based on mutation of the gene.
  • the sterile line has stable fertility and is only regulated by a single gene encoded by the nucleus, and is not affected by the light and temperature environment.
  • the fertility restoring genes of the sterile line are widely present in rice germplasm resources, and fertility can also be restored by transferring wild-type genes.
  • the gene and the sterile line generated by the mutation of the gene provide elements for the development of a new type of hybrid breeding technology for rice, and lay a foundation for solving the problems in the existing technology.
  • An object of the present invention is to provide a plant fertility-related protein, its coding gene, and its application in regulating plant male fertility by manipulating the gene.
  • any of the methods described below can be used together with the corresponding nucleotide sequence of the plant fertility-related protein provided by the present invention, for example, to make the endogenous plant fertility-related protein in the plant
  • Mutations in the coding sequence, introduction of the antisense sequence of the sequence into the plant, use of hairpin format, or linking it with other nucleotide sequences to regulate the phenotype of the plant, or are known to those skilled in the art and can be used to affect the plant Any of many methods of male fertility.
  • the present invention finds a pollen development regulating gene GMS2 with male fertility regulating function in rice.
  • GMS2 is located on rice chromosome 4, and its genomic nucleotide sequence in the japonica rice variety Nipponbare is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 3.
  • indica rice variety 9311 its genome nucleotide sequence is shown in SEQ ID NO: 4
  • its CDS sequence is shown in SEQ ID NO: 69
  • its amino acid sequence is the same as that of the japonica rice variety Nipponbare.
  • the amino acid sequence of the fertility gene in Arabidopsis lyrata is shown in SEQ ID NO: 9; in the banana (Musa acuminata) the amino acid sequence of the fertility gene is shown in SEQ ID NO: 10;
  • the amino acid sequence of the fertility gene in Oryza glaberrima is shown in SEQ ID NO: 11; the amino acid sequence of the fertility gene in Oryza brachyantha is shown in SEQ ID NO: 12;
  • the amino acid sequence of the fertility gene in barley (Hordeum vulgare) is shown in SEQ ID NO: 13; the amino acid sequence of the fertility gene in sorghum (Sorghum bicolor) is shown in SEQ ID NO: 14; in maize (Zea mays)
  • the amino acid sequence of the fertility gene is shown in SEQ ID NO: 15; the amino acid sequence of the fertility gene in Setaria italica is shown in SEQ ID NO: 16.
  • the above-mentioned fertility genes can be isolated and obtained from various plants.
  • the highly homologous functionally equivalent sequence includes a DNA sequence that can hybridize with the nucleotide sequence of the GMS2 gene disclosed in the present invention under stringent conditions.
  • the "stringent conditions" used in the present invention are well known, including hybridization solutions containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA at 60°C for 12-16 hours, and then at 65°C. Wash with a washing solution containing 0.1% SDS and 0.1 ⁇ SSC for 15-60 minutes.
  • the functionally equivalent sequence also includes at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence of the GMS2 gene disclosed in the present invention, and has fertility
  • the DNA sequence regulating function can be isolated from any plant.
  • the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithms (Bioinformatics, 4(1): 1117, 1988), Needleman-Wunsch global alignment method (J Mol Biol, 48( 3): 443-453, 1970), Smith-Waterman local comparison method (J Mol Biol, 147: 195-197, 1981), Pearson and Lipman similarity search method (PNAS, 85(8): 2444-2448, 1988), Karlin and Altschul's algorithm (Altschul et al., J Mol Biol, 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.
  • the first aspect of the present invention is to provide a plant male fertility related protein
  • the plant male fertility related protein is the following protein (1) or (2):
  • the present invention provides a nucleic acid encoding the plant male fertility-related protein.
  • the nucleic acid of the present invention can be isolated and obtained from any plant, including but not limited to Brassica, maize, wheat, sorghum, Brachypodium aureum, African cultivated rice, Brachypodium, Capsicum, White mustard, Strawberry, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus vulgaris, peanut, Yinsu, oats, rapeseed, barley, oats, rye (Rye), millet, milo, triticale, single grain Wheat, Spelt, Diecorn, Flax, Grammagrass, Frictiongrass, Fake milo, Fescue, Perennial Wheatgrass, Ganlian, Cranberry Moss, Papaya, Banana , Safflower, oil palm, cantaloupe, apple, cucumber, stone gladiator, gladiolus, chrysanthemum, lily family, cotton, school, sunflower, brassica, sugar beet, coffee, ornamental plants and pine etc
  • the plants include corn, millet, Arabidopsis thaliana, Brachypodium dilatum, soybean, safflower, mustard, wheat, barley, rye, wild rice, African cultivated rice, cotton and sorghum.
  • sequence of the nucleic acid is any one of the following:
  • a DNA fragment with the sequence described in any one of (1) and (2) has 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity and encodes a rice male DNA fragments of fertility-related proteins.
  • the present invention provides an inhibitor of nucleic acid encoding the plant male fertility-related protein, and the introduction of the inhibitor into a plant can reduce the expression level of the nucleic acid encoding the plant male fertility-related protein, not express or mutate and lose it live.
  • the inhibitor can be a protein or nucleic acid (including but not limited to antisense gene, siRNA and its DNA, dsRNA and its DNA, sgRNA and its DNA, etc.).
  • the present invention provides a biological material containing a nucleic acid encoding the plant male fertility-related protein, or an inhibitor containing a nucleic acid encoding the plant male fertility-related protein, and the biological material is an expression cassette, a vector, and a host Cells, transgenic cell lines or transgenic plants.
  • the present invention provides a plant, plant tissue or plant cell, which exhibits male sterility and is caused by a mutation in a nucleic acid encoding the plant male fertility-related protein, and the mutation is one or more nucleotides Deletion, insertion or substitution mutations, or mutations produced by the transfer of antisense genes, co-suppression, or introduction of hairpin structures; the mutations cause the expression of the plant male fertility-related protein to be reduced or not expressed Or inactivation.
  • the plant, plant tissue or plant cell can be obtained by natural mutation or artificial mutagenesis, and can be a transgenic plant, plant tissue or plant cell or a non-transgenic plant, plant tissue or plant cell.
  • the artificial mutagenesis includes physical and chemical mutagenesis, insertion mutation, gene targeting knockout, antisense gene transfer, co-suppression or introduction of hairpin structure, etc.
  • the plants include, but are not limited to, Brassica, corn, wheat, sorghum, wild rice, African cultivated rice, Brachypodium, Capsicum, white mustard, sesame, soybean, Arabidopsis, Phaseolus vulgaris , Peanuts, yinsu, oats, rapeseed, barley, oats, rye (Rye), millet, milo, triticale, einkorn wheat, spelt wheat (Spelt), dimer wheat, flax, gram Horsegrass (Grammagrass), friction grass, false milo, fescue, perennial wheatgrass, Ganlian, cranberry moss, papaya, banana, safflower, oil palm, cantaloupe, apple, cucumber, stone gladiator, gladiolus, Chrysanthemum, Liliaceae, cotton, sunflower, brassica, sugar beet, coffee, ornamental plants and pines, etc.
  • it includes corn, millet, Arabidopsis
  • the plant, plant tissue or plant cell is obtained by using the CRISPR-Cas9 method, and the target sequence used in the CRISPR-Cas9 method is located in the sequence of the nucleic acid encoding the plant male fertility-related protein, and the target sequence is The reverse complementary sequence has a 5'-(N)X-NGG-3' structure, where N represents any one of A, T, C, and G, and X is any nucleotide sequence of 19 or 20 nt.
  • the plant, plant tissue, or plant cell-derived line is a target site or a target site obtained by using the CRISPR-Cas9 method with GCGGTCGGTGGCGGCCATGG (SEQ ID NO: 17) and CGCCTCCCTCGCCGTCGCG (SEQ ID NO: 18) as target sites. Plants with mutations in the area adjacent to the target site.
  • the second aspect of the present invention provides the plant male fertility related protein or the nucleic acid encoding the plant male fertility related protein or the inhibitor of the nucleic acid or the biological material or the plant, plant tissue or plant cell Any of the following applications:
  • regulating the male fertility of the plant may be reducing or losing the male fertility of the plant. Specifically, it can be achieved by regulating the development of plant male reproductive cells and pollen. Wherein, the male fertility of the plant can be reduced or lost by mutating the gene encoding the plant male fertility-related protein to reduce its expression or not expressing it, or by encoding the plant male fertility-related protein The introduction of inhibitors of nucleic acids into plants is achieved.
  • the male sterile plant is a recessive nuclear sterile line with a homozygous mutation of the nucleic acid encoding the plant male fertility-related protein.
  • a recessive genic sterile line carrying a homozygous mutation of the nucleic acid encoding the plant male fertility-related protein is used for cross breeding and seed production.
  • the improvement includes yield improvement, quality improvement, resistance to diseases and insect pests, stress resistance, lodging resistance and the like.
  • the above-mentioned plants are self-pollinated or cross-pollinated crops, including but not limited to rice, corn, wheat, and sorghum.
  • the third aspect of the present invention is to provide a method for affecting plant fertility by affecting the plant male fertility-related protein or the sequence of the nucleic acid encoding the protein, or by affecting the transcription and translation of the nucleic acid.
  • influencing plant fertility refers to changing the fertility of the plant, such as causing male sterility of the plant.
  • a variety of methods can be used to influence the sequence of the plant male fertility-related protein or the nucleic acid encoding the protein or its expression and translation in the plant, so as to achieve regulation of plant male fertility. Effect.
  • sequence affecting the plant male fertility-related protein or the nucleic acid encoding the protein or its expression and translation in plants can be performed using many tools available to those of ordinary skill in the art, for example, by physical and chemical mutagenesis , Insertion mutation, gene targeting knockout, antisense gene transfer, co-suppression or introduction of hairpin structure, etc., can all be used to disrupt the normal expression of the plant male fertility-related proteins, thereby obtaining male sterile plants .
  • the fourth aspect of the present invention is to provide a mutant of the plant male fertility-related protein, which is the insertion, and/or deletion, and/or substitution of several nuclei in the gene encoding the plant male fertility-related protein. Utilized acid is obtained, and the mutant can cause male sterility in rice.
  • the present invention provides a target site suitable for targeted knockout of nucleic acid encoding the plant fertility-related protein by the CRISPR-Cas9 method, which is target site 1: GCGGTCGGTGGCGGCCATGG (SEQ ID NO: 17) and/or target Site 2: CGCCTCCCTCGCCGTCGCGG (SEQ ID NO: 18).
  • the present invention also provides sgRNA that specifically targets the aforementioned target site 1 and target site 2.
  • the CRISPR-Cas9 targeting vector containing the above-mentioned sgRNA DNA sequence also belongs to the protection scope of the present invention.
  • the fifth aspect of the present invention is to provide any of the following applications of the target site or the CRISPR-Cas9 targeting vector targeting the target site or the CRISPR-Cas9 DNA containing the sgRNA:
  • regulating the male fertility of the plant may be reducing or losing the male fertility of the plant. Specifically, it can be achieved by regulating the development of plant male reproductive cells and pollen. Wherein, the male fertility of the plant can be reduced or lost by mutating the gene encoding the plant male fertility-related protein to reduce its expression or not expressing it, or by encoding the plant male fertility-related protein The introduction of inhibitors of nucleic acids into plants is achieved.
  • the male sterile plant is a recessive nuclear sterile line with a homozygous mutation of the nucleic acid encoding the plant male fertility-related protein.
  • the inhibitor using the nucleotide is the inactivation of the fertility regulatory protein, thereby creating a recessive male sterile plant for cross breeding and seed production.
  • the improvement includes yield improvement, quality improvement, resistance to diseases and insect pests, stress resistance, lodging resistance and the like.
  • the present invention also provides a method for preparing a male sterile plant, which is to reduce, not express or inactivate the expression of the plant male fertility-related protein in the plant.
  • the present invention provides a method for preparing male sterile rice using CRISPR-Cas9 technology, which is to use CRISPR-Cas9 technology to knock out or mutate the nucleic acid encoding the plant fertility-related protein in rice.
  • the CRISPR-Cas9 technology uses the target site GCGGTCGGTGGCGGCCATGG (SEQ ID NO: 17) and/or the target site CGCCTCCCTCGCCGTCGCGG (SEQ ID NO: 18) as the target site, so that the target site or the target site and its neighbors Nucleotide sequence mutation.
  • the present invention also provides a method for obtaining orthologous gene fragments of GMS2 gene in plants, and using the method to obtain Arabidopsis thaliana, banana, African cultivated rice, short-medicine wild rice, barley, sorghum, corn and millet.
  • the method for obtaining orthologous gene fragments of GMS2 gene in plants provided by the present invention includes: using the aforementioned DNA fragments of GMS2 gene to perform a blastx search in a nucleotide database; More than or equal to 50% is an orthologous gene fragment of the GMS2 gene.
  • Another object of the present invention is to provide a mutant of plant fertility-related protein GMS2, the coding gene of the mutant, its molecular markers and the application in cross breeding.
  • the present invention has discovered a mutant of rice fertility-related protein GMS2, which lacks three amino acids of asparagine, serine and tyrosine at positions 40 to 42 relative to the wild-type GMS2 protein.
  • GMS2 is located on rice chromosome 4, and its genomic nucleotide sequence in the japonica rice variety Nipponbare is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 3. Show. In the indica variety 9311, its genome nucleotide sequence is shown in SEQ ID NO: 4, and its CDS sequence is shown in SEQ ID NO: 69, and its amino acid sequence is the same as that of the japonica rice variety Nipponbare.
  • the amino acid sequence of the fertility gene in Arabidopsis lyrata is shown in SEQ ID NO: 9; in the banana (Musa acuminata) the amino acid sequence of the fertility gene is shown in SEQ ID NO: 10;
  • the amino acid sequence of the fertility gene in Oryza glaberrima is shown in SEQ ID NO: 11; the amino acid sequence of the fertility gene in Oryza brachyantha is shown in SEQ ID NO: 12;
  • the amino acid sequence of the fertility gene in barley (Hordeum vulgare) is shown in SEQ ID NO: 13; the amino acid sequence of the fertility gene in sorghum (Sorghum bicolor) is shown in SEQ ID NO: 14; in maize (Zea mays)
  • the amino acid sequence of the fertility gene is shown in SEQ ID NO: 15; the amino acid sequence of the fertility gene in Setaria italica is shown in SEQ ID NO: 16.
  • the above-mentioned fertility genes can be isolated and obtained from various plants.
  • the highly homologous functionally equivalent sequence includes a DNA sequence that can hybridize with the nucleotide sequence of the GMS2 gene disclosed in the present invention under stringent conditions.
  • the "stringent conditions" used in the present invention are well known, including hybridization solutions containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA at 60°C for 12-16 hours, and then at 65°C. Wash with a washing solution containing 0.1% SDS and 0.1 ⁇ SSC for 15-60 minutes.
  • the functionally equivalent sequence also includes at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence of the GMS2 gene disclosed in the present invention, and has fertility
  • the DNA sequence regulating function can be isolated from any plant.
  • the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithms (Bioinformatics, 4(1): 1117, 1988), Needleman-Wunsch global alignment method (J Mol Biol, 48( 3): 443-453, 1970), Smith-Waterman local comparison method (J Mol Biol, 147: 195-197, 1981), Pearson and Lipman similarity search method (PNAS, 85(8): 2444-2448, 1988), Karlin and Altschul's algorithm (Altschul et al., J Mol Biol, 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.
  • another aspect of the present invention is to provide a plant male fertility-related protein mutant, which contains the following amino acid mutations relative to the wild-type plant male fertility-related protein: NxYL occurs in the NxYL conservative sequence. The deletion of at least one of the three amino acids of, x and Y;
  • the wild-type plant male fertility-related protein is the protein described in (1) or (2) below:
  • the mutant contains the following amino acid mutations relative to the wild-type plant male fertility-related protein: three amino acid deletions of N, x, and Y occur in the NxYL conservative sequence; where x is S or N.
  • the mutant contains the following amino acid mutations relative to its wild-type male fertility-related protein: amino acids at positions 40, 41, and 42 are deleted, and its wild-type male fertility-related protein has SEQ ID NO: 3
  • the sequence or SEQ ID NO: 3 has been substituted and/or deleted and/or added with one or several amino acid residues to obtain a protein with the activity of regulating plant male fertility.
  • the present invention provides a mutant of rice GMS2 protein, which has an amino acid sequence as shown in SEQ ID NO.8.
  • the present invention also provides a nucleic acid encoding the mutant of the plant male fertility-related protein.
  • the nucleic acid of the plant male fertility-related protein mutant of the present invention can be isolated and obtained from any plant, including but not limited to Brassica, maize, wheat, sorghum, Brachypodium, African cultivated rice, Brachypodium, Capsicum, white mustard, sesame, soybean, Arabidopsis, Phaseolus vulgaris, peanut, yinsu, oats, barley, oats, rye (Rye), millet, milo, triticale, einkorn wheat, Sri Lanka Spelt, Dimer wheat, Flax, Grammagrass, Frictiongrass, Fake milo, fescue, perennial wheatgrass, Ganlian, cranberry moss, papaya, banana, safflower , Oil palm, cantaloupe, apple, cucumber, stone gladiator, gladiolus, chrysanthemum, lily family, cotton, sunflower, beet, coffee, ornamental plants and pines, etc.
  • any plant including but not limited to
  • the plants include corn, millet, Arabidopsis thaliana, Brachypodium dilatum, soybean, safflower, mustard, wheat, barley, rye, wild rice, African cultivated rice, cotton and sorghum.
  • the nucleic acid encoding the mutant relative to the nucleic acid encoding the wild-type plant male fertility-related protein contains the following nucleotide mutations: corresponding to positions 118 to 126 in the coding region of the LOC_Os04g48490 gene
  • the AACAGCTAC base is missing.
  • the genomic nucleotide sequence of the mutant GMS2 is shown in SEQ ID NO: 6, and the CDS sequence is shown in SEQ ID NO: 7.
  • the present invention also provides a biological material, which is an expression cassette, a vector, a host cell, a transgenic cell line or a transgenic plant containing a nucleic acid encoding the plant male fertility-related protein mutant.
  • Another aspect of the present invention is to provide any one of the following applications of the mutant or the nucleic acid or the biological material:
  • regulating the male fertility of the plant may be reducing or losing the male fertility of the plant. Specifically, it can be achieved by regulating the development of plant male reproductive cells and pollen. Wherein, the male fertility of the plant can be reduced or lost by mutating the gene encoding the plant male fertility-related protein to reduce its expression or not expressing it, or by encoding the plant male fertility-related protein The introduction of inhibitors of nucleic acids into plants is achieved.
  • the male sterile plant is a recessive nuclear sterile line with a homozygous mutation of the nucleic acid encoding the plant male fertility-related protein.
  • a recessive genic sterile line carrying a homozygous mutation of the nucleic acid encoding the plant male fertility-related protein is used for cross breeding and seed production.
  • the improvement includes yield improvement, quality improvement, resistance to diseases and insect pests, stress resistance, lodging resistance and the like.
  • the above-mentioned plants are self-pollinated or cross-pollinated crops, including but not limited to corn, wheat, sorghum, and rice.
  • Another aspect of the present invention is to provide a plant, plant tissue or plant cell, which has male sterility traits, and the protein encoded by the gene of the wild-type plant male fertility-related protein in its genome sequence contains the following amino acid mutations: At least one of the three amino acids N, x, and Y is missing in the NxYL conservative sequence, and the wild-type plant male fertility-related protein is as described above.
  • the protein encoded by the gene of the wild-type plant male fertility-related protein in the genome sequence of the plant, plant tissue or plant cell contains the following amino acid mutations: N, x, and Y three amino acids occur in the NxYL conservative sequence Missing.
  • the plant is rice
  • the plant tissue is rice tissue
  • the plant cell is a rice cell.
  • the present invention provides a rice whose genome or transcriptome contains the following mutations: AACAGCTAC bases at positions 118 to 126 in the coding region of the LOC_Os04g48490 gene are deleted, resulting in positions 40, 41 and 42 of the protein encoded by the LOC_Os04g48490 gene The asparagine, serine, and tyrosine are deleted; the sequence of the coding region of the LOC_Os04g48490 gene is shown in SEQ ID NO.69, and the sequence of the protein encoded by the LOC_Os04g48490 gene is shown in SEQ ID NO.3.
  • the present invention also provides a rice tissue whose genome or transcriptome contains the following mutations: AACAGCTAC bases at positions 118 to 126 in the coding region of the LOC_Os04g48490 gene are deleted, resulting in the 40th and 41st of the LOC_Os04g48490 gene encoding protein. And the asparagine, serine and tyrosine at position 42 are deleted.
  • the present invention also provides a rice cell whose genome or transcriptome contains the following mutations: the AACAGCTAC bases at positions 118 to 126 in the coding region of the LOC_Os04g48490 gene are deleted, resulting in the 40th and 41st positions of the LOC_Os04g48490 gene encoding protein. And the asparagine, serine and tyrosine at position 42 are deleted.
  • the present invention further provides that the sequence mutation of the LOC_Os04g48490 gene in the genome sequence is as shown in SEQ ID NO: 6, resulting in the CDS mutation of the LOC_Os04g48490 gene as the sequence shown in SEQ ID NO: 7, and the encoding protein mutation is as shown in SEQ ID NO: Rice, rice tissue or rice cell with the sequence shown in 8.
  • the present invention provides rice mutant material gms2, gms2 exhibits male sterility, the sequence of the LOC_Os04g48490 (GMS2) gene in its genome sequence is mutated as shown in SEQ ID NO: 6, and the CDS mutation of LOC_Os04g48490 gene is as shown in SEQ ID NO The sequence shown in :7, the amino acid sequence of the encoded protein is mutated to the sequence shown in SEQ ID NO: 8; the genomic sequence of the LOC_Os04g48490 gene is shown in SEQ ID NO: 4.
  • GMS2 LOC_Os04g48490
  • the genomic sequence of the rice mutant material gms2 is deleted in the AACAGCTAC bases at positions 118 to 126 in the LOC_Os04g48490 gene (GMS2) coding region (sequence is shown in SEQ ID NO: 69), resulting in the LOC_Os04g48490 gene encoding protein (sequence shown in SEQ ID NO: 3)
  • GMS2 LOC_Os04g48490 gene
  • nucleotide sequence shown in SEQ ID NO: 6 can be introduced into the recipient plant by hybridization, backcrossing or transgenic methods to obtain new male sterile mutant materials.
  • Another aspect of the present invention is to provide a molecular marker for detecting the mutant or the mutant material, and the molecular marker is amplified by primers whose nucleotide sequence is shown in SEQ ID NO. 19-20.
  • the present invention provides specific primers for amplifying the molecular markers, the nucleotide sequence of which is shown in SEQ ID NO: 19-20.
  • the present invention provides detection reagents or kits containing primers with nucleotide sequences as shown in SEQ ID NO: 19-20.
  • the present invention also provides any one of the following applications of the molecular marker or the detection reagent or kit:
  • the rice when using the primers shown in SEQ ID NO: 19-20 to amplify rice genomic DNA, if only a 140 bp band can be amplified, the rice expresses the plant male fertility-related protein mutant and has LOC_Os04g48490 gene encoding
  • the homozygous genotype of the AACAGCTAC bases at positions 118 to 126 in the region is deleted and exhibits male sterility traits; if only one band of 149bp can be amplified, rice does not express plant male fertility-related protein mutants, The genotype without AACAGCTAC at positions 118 to 126 of the coding region of LOC_Os04g48490 gene is deleted and shows male fertility; if two bands of 140bp and 149bp can be amplified at the same time, then rice expresses plant male fertility Related protein mutants, a heterozygous genotype in which the AACAGCTAC bases at positions 118 to 126 in the coding region of LOC_Os04g48490 gene
  • Another aspect of the present invention is to provide a method for preparing male sterile plants, which comprises: mutating wild-type plant male fertility-related proteins, and the mutations include the following amino acid mutations: N, x occur in the NxYL conservative sequence The deletion of at least one of the three amino acids of Y; the wild-type plant male fertility-related protein is the same as described above.
  • the plant is rice
  • the method comprises: mutating the genome or transcriptome of rice, the mutation comprising the following mutation: AACAGCTAC bases at positions 118 to 126 in the coding region of the LOC_Os04g48490 gene are deleted, resulting in The asparagine, serine and tyrosine at positions 40, 41 and 42 of the protein encoded by the LOC_Os04g48490 gene are deleted; the sequence of the coding region of the LOC_Os04g48490 gene is shown in SEQ ID NO. 69, and the sequence of the protein encoded by the LOC_Os04g48490 gene is shown in SEQ ID NO. 3 shown.
  • the method includes: making rice express a mutant of male fertility-related protein shown in SEQ ID NO: 8 and not expressing a wild-type plant male fertility-related protein shown in SEQ ID NO: 3.
  • the above mutations can be achieved through gene editing, hybridization, backcrossing, selfing, or asexual reproduction.
  • the present invention has the following beneficial effects: the rice pollen development regulation gene GMS2 provided by the present invention directly participates in the regulation of pollen development. After the gene is knocked out or its expression is inhibited, the pollen is completely sterile, resulting in plant males. Sterile.
  • the present invention uses CRISPR-Cas9 gene editing technology to perform gene editing on the GMS2 gene, and obtains a rice male sterile mutant of the GMS2 gene mutation. Compared with the existing three-line and two-line sterile lines, the sterile rice mutant caused by the GMS2 mutation has stable sterility characteristics and is not affected by environmental conditions.
  • Utilizing the GMS2 gene and its mutants of the present invention can cultivate new nuclear sterile lines by transgene and other methods and provide a method for restoring the fertility of the sterile lines, laying a foundation for the cultivation and propagation of rice nuclear sterile lines, The utilization of heterosis and sterile hybrid seed production will play an important role.
  • the GMS2 mutant of the rice male fertility regulating protein provided by the present invention can make rice pollen completely sterile, resulting in rice male sterility.
  • the nuclear sterile mutant of the present invention can be used to cultivate new nuclear sterile lines, which provides a simple, rapid and effective method for the cultivation of rice nuclear sterile lines.
  • the rice sterile mutant caused by the GMS2 mutant of the present invention has the advantages of stable sterility and high germplasm resource utilization, and can be used for recurrent selection breeding that requires a large number of crosses. , It has great application value in the field of hybrid rice breeding and seed production.
  • Figure 1 shows the plant morphology of wild-type (left) and gms2 mutant (right) at the filling stage in Example 2 of the present invention.
  • Figure 2 shows the spikelet morphology of wild type (left) and gms2 mutant (right) in Example 2 of the present invention.
  • Figure 3 shows the morphology of panicle flowering in wild-type (left) and gms2 mutant (right) in Example 2 of the present invention.
  • Figure 4 shows the floret morphology of wild type (left) and gms2 mutant (right) after dissection in Example 2 of the present invention.
  • Figure 5 shows the anther morphology of wild-type (left) and gms2 mutant (right) in Example 2 of the present invention.
  • Figure 6 shows wild-type (left) and gms2 mutant (right) pollen iodine staining in Example 2 of the present invention.
  • Fig. 7 shows the use of InD48490 marker in Example 4 of the present invention to identify the genotypes of sterile individual plants in the localized population.
  • the size of the upper band is 149bp, and the size of the lower band is 140bp.
  • the DNA templates in the first two lanes on the left are gms2 mutant and Minghui 63, respectively, and the next lane is the sterile single plant in the positioning population.
  • Fig. 8A is a map-based cloning diagram of the GMS2 gene in Example 4 of the present invention.
  • Fig. 8B is a schematic diagram of the mutation site of the gms2 mutant in Example 4 of the present invention.
  • Figure 9 is the nucleotide sequence of the GMS2 gene in Example 4 of the present invention in the materials of 9311 (48490-9311), Minghui 63 (48490-MH63), Nipponbare (48490-Nip) and gms2 mutant (48490-3148) difference. The differences are highlighted with a gray background. The position of the last nucleotide in each line in the entire gene sequence is indicated at the end of the line. The start codon ATG and the stop codon TGA are marked with boxes respectively.
  • Figure 10 shows the difference in amino acid sequence of GMS2 encoded protein in Example 4 of the present invention between 9311 (48490-9311) and gms2 mutant (48490-3148). The differences are highlighted with a light gray background. The position of the last amino acid residue in each line in the entire protein sequence is indicated at the end of the line.
  • Figure 11 is the genotype identification of the progeny of the GMS2 heterozygous strain in Example 4 of the present invention.
  • the size of the upper band is 149bp, and the size of the lower band is 140bp.
  • the arrow points to samples from male sterility.
  • Fig. 12 shows the expression level of GMS2 in young panicles of rice in different tissues and different developmental stages in Example 5 of the present invention.
  • Flowers 1-flower 9 represent the period of spikelet primordium differentiation to pollen maturity in the development of young panicles.
  • FIG 13 is a schematic diagram of the pC9M-GMS2 vector in Example 6 of the present invention.
  • T1 stands for target site 1
  • T2 stands for target site 2.
  • Fig. 14A is a sequence analysis of the target site of some transgenic positive plants after GMS2 gene knockout using the CRISPR/Cas9 system in Example 6 of the present invention.
  • 14B is a sequencing peak diagram of the transgenic plant PC9M-GMS2-Line17 at target site 1 and target site 2 in Example 6 of the present invention. Among them, in the sequencing peak map at target site 1, the arrow points to the deletion site; in the sequencing peak map at target site 2, the arrow points to the insertion site.
  • Figure 15 is the morphology of the GMS2 wild-type (left) and knockout plant PC9M-GMS2-Line17 (right) in Example 6 of the present invention.
  • Figure 16 shows the glume morphology of GMS2 wild-type (left) and knockout plant PC9M-GMS2-Line17 (right) in Example 6 of the present invention.
  • Figure 17 shows the anther morphology of GMS2 wild type (left) and knockout plant PC9M-GMS2-Line17 (right) in Example 6 of the present invention.
  • Figure 18 shows the results of pollen iodine staining of GMS2 wild type (left) and knockout plant PC9M-GMS2-Line17 (right) in Example 6 of the present invention.
  • Figure 19 is a schematic diagram of the pUbi1301-48490-CDS vector in Example 7 of the present invention.
  • Figure 20 shows the RT-PCR expression analysis of GMS2 in overexpression plants in Example 7 of the present invention.
  • the histogram is the result of quantifying the brightness of the bands in the RT-PCR gel image and dividing the brightness value of 48490 by the brightness value of the corresponding GAPDH.
  • Figure 21 is a schematic diagram of the pC1300-48490-genome vector in Example 8 of the present invention.
  • Figure 22 shows the plant morphology of the wild-type plant (left) and the gms2 mutant complementary plant (right) in Example 8 of the present invention.
  • Figure 23 shows the husk morphology of the wild-type plant (left) and the gms2 mutant complementary plant (right) in Example 8 of the present invention.
  • Figure 24 shows the anther morphology of the wild-type plant (left) and the gms2 mutant complementary plant (right) in Example 8 of the present invention.
  • Figure 25 shows the results of pollen iodine staining of wild-type plants (left) and gms2 mutant complementary plants (right) in Example 8 of the present invention.
  • Figure 26 is a sequence alignment diagram of the protein encoded by the rice GMS2 gene in Example 9 of the present invention and homologous proteins in the genomes of other species. Including Arabidopsis lyrata protein AT3G60900.1, Musa acuminata protein GSMUA_Achr11P03090_001, African cultivated rice (Oryza glaberrima) protein ORGLA04G0194100.1, short drug wild rice (Oryza brachyantha) protein OB04G29380.1, barley Hordeum vulgare protein MLOC_7985.1, sorghum (Sorghum bicolor) protein Sb06g026030.1, corn (Zea mays) protein GRMZM2G003752_P01, millet (Setaria italica) protein Si010135m.
  • the NxYL conserved sequence is boxed.
  • Figure 27 is a phylogenetic tree analysis of the protein encoded by the rice GMS2 gene in Example 9 of the present invention.
  • the male sterility of the present invention specifically refers to the abnormal development of plant male reproductive organs (inability to produce normal stamens, anthers or normal male gametophytes) and loss of fertility due to functional changes in plant nuclear genes, which is commonly referred to as Genic male sterility instead of Cytoplasmic male sterility.
  • the abnormality and restoration of the fertility of male reproductive organs are controlled by genes in the nucleus.
  • the present invention also includes the use of the sequences described in the sequence listing to regulate the male gamete fertility of plants, that is, the use of the gene sequence provided by the present invention to affect the same or the same in other plants at the genome, and/or transcriptome, and/or proteome level
  • the function of the source gene thus achieves the purpose of controlling the fertility of male reproductive organs.
  • the gene expression inhibition or the loss of protein function caused by the mutation of the natural sequence, the antisense sequence of the gene or the hairpin structure is introduced into the plant, or the gene is combined with other Sequences (DNA or RNA) combine to produce new functionally active DNA or RNA strands to influence or change the function of plant genes.
  • the present invention includes the rice GMS2 gene, the dominant allele of which has a key effect on the male fertility of plants, and the recessive allele with loss of function can lead to male sterility.
  • the gene is located on rice chromosome 4, and the specific location of the gene is shown in Figure 8A and Figure 8B.
  • the gene sequence and its homologous sequence can be obtained from various plants, including but not limited to Selaginella moellendorffii, populus trichocarpa, Brassica rapa, Arabidopsis lyrata ), Arabidopsis thaliana, soybean (Glycine max), potato (Solanum tuberosum), grape (Vitis vinifera), small fruit wild banana (Musa acuminata), millet (Setaria italica), sorghum (Sorghum bicolor), corn (Zea mays), Brachypodium distachyon, Barley (Hordeum vulgare), Oryza brachyantha, African cultivated rice (Oryza glaberrima), Indica (Oryza sativa Indica Group), Japonica (Oryza sativa) Japonica Group), Physcomitrella patens, etc.
  • Obtaining methods include, but are not limited to: using rice GMS2 gene sequence through blastx, blastn or using rice GMS2 amino acid sequence through blastp to retrieve from genome sequence databases, and/or cDNA sequence databases, and/or protein sequence databases of other plants;
  • the DNA or cDNA or RNA sequence of rice GMS2 gene is used as the reference sequence to design primers, which are directly obtained from the genomic DNA or cDNA or RNA of other plants by PCR; the probe is designed with the gene sequence of rice GMS2, and the nucleic acid hybridization method is used to obtain DNA or cDNA or RNA fragments containing homologous gene sequences are isolated from genomic libraries.
  • the homologous sequence of the GMS2 gene refers to the plant gene sequence with Identities greater than or equal to 35% and Positives greater than or equal to 50% after blastp comparison and analysis with the amino acid sequence of SEQ ID NO: 3.
  • All parameters follow the default settings shown in http://blast.ncbi.nlm.nih.gov/.
  • the wild-type pollen grains were large and round and dyed blue-black, while the mutant pollen grains Shrink and cannot be dyed.
  • the wild-type plants of the same family normally bear fruit after bagging and selfing, but the gms2 mutant does not bear fruit. But with rice variety 93-11 as the male parent to pollinate the gms2 mutant, the fruit can be produced. This indicates that the mutant is a male sterile mutant.
  • Map-based cloning was used to map the GMS2 gene.
  • a F 2 population containing 66 mutant plants was constructed by crossing Minghui 63 as the male parent with gms2 mutant.
  • GMS2 was located in the range of 6861.252Kb between SSR markers RM17332 and RM280 on chromosome 4, and was closely linked to SSR marker RM303 and Indel marker 4826.
  • the number of exchanges between the GMS2 gene and the above four markers is 8, 1, 1, 32, respectively.
  • the use of linkage markers to select gms2 heterozygous individuals in the F 2 population developed an F 3 population containing 1937 mutant individuals.
  • the nucleotide sequence of the LOC_Os04g48490 genome is 1582 bp (denoted as 48490-Nip, the sequence is as SEQ ID NO: 1), and the CDS nucleotide sequence is 1296 bp (the sequence is as SEQ ID NO: 2), including 1 exon ( Figure 8A and Figure 8B), encoding a protein with a length of 432 amino acid residues (sequence such as SEQ ID NO: 3).
  • the sequence of the marker primer pair used to locate the GMS2 gene is shown in Table 1 (SEQ ID NO. 39-68).
  • Table 1 The sequence of the marker primer pair used to locate the GMS2 gene
  • the primers were designed according to the 48490-Nip sequence to amplify and sequence the alleles of the LOC_Os04g48490 gene in 93-11, Minghui 63 and gms2 mutants.
  • the primer sequences are shown in Table 2. All PCR amplifications use KOD FX DNA Polymerase (TOYOBO CO., LTD.
  • Thermo Scientific Arktik thermal cycler in accordance with the reaction system and conditions specified in the product.
  • the PCR products were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing.
  • the sequencing results were assembled with DNAman 6.0.
  • the LOC_Os04g48490 genes in 93-11, Minghui 63 and gms2 mutants are recorded as 48490-9311 (sequence such as SEQ ID NO: 4), 48490-MH63 (sequence such as SEQ ID NO: 5), and 48490-3148 (sequence such as SEQ ID NO: 6).
  • LOC_Os04g48490 gene is highly conserved in rice, and its nucleotide sequence only differs by 4 bases even between the indica and japonica subspecies, but there is no difference in the protein sequence.
  • the CDS nucleotide sequence of LOC_Os04g48490 is shown in SEQ ID NO: 69, and the coding protein sequence is shown in SEQ ID NO: 3.
  • the CDS nucleotide sequence and amino acid sequence of LOC_Os04g48490 in the gms2 mutant are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • InD48490_F and InD48490_R primers 41 gms2 isolates of M6 were genotyped. As shown in Figure 11, the wild type either amplified two bands of 149bp and 140bp, or amplified a band of 149bp, while the sterile mutants could only amplify a band of 140bp. This indicates that the mutant genotype and the sterile phenotype are co-segregated, and LOC_Os04g48490 is the GMS2 gene.
  • RNA was extracted from the tissues of each period of 93-11, and reverse transcribed into cDNA.
  • Real-time quantitative PCR method was adopted for expression analysis.
  • the expression level of GMS2 gene in roots and stems was significantly lower than other tissues, and in seeds, it was significantly higher than other tissues.
  • stem nodes, leaves, leaf sheaths and ears the expression level of GMS2 gene is moderate, but not the same.
  • flower 1 flower length 1mm
  • flower 2 flower length 2mm
  • flower 3 flower length 3mm
  • flower 4 flower length 4mm
  • flower 5 flower length 5mm
  • flower 6 flower length 5.5mm
  • the CRISPR-Cas9 system was used for targeted knockout of the GMS2 gene.
  • two target sites were selected for knockout at the same time.
  • the target site 1 is located on the negative strand of the exon, and the sequence is GCGGTCGGTGGCGGCCATGG (SEQ ID NO: 17, located at the 45th and 64th positions of the sequence SEQ ID NO: 1), and the target site 2 is located on the exon.
  • the sequence is CGCCTCCCTCGCCGTCGCGG (SEQ ID NO: 18, located at positions 85 to 104 in the sequence of SEQ ID NO: 1). According to the method of Ma et al. (Ma X, et al.
  • Recombinant Agrobacterium Ab-pC9M-GMS2 was used to infect the callus of japonica rice Zhonghua 11 (ZH11). After hygromycin resistance screening, differentiation and rooting, 25 regenerated transgenic lines were obtained. After refining seedlings and transplanting, 22 surviving plants were obtained. The total DNA of the leaves of the plants was extracted, and the primers SP1: CCCGACATAGATGCAATAACTTC (SEQ ID NO: 29) and SP2: GGCCGGTGTCATCTATGTTACT (SEQ ID NO: 30) were used for positive detection. Positive strain.
  • Target 1-F AAACCCACGCCCAGAAA (SEQ ID NO: 31) and target 1-R: GCCAGGAGGAAGAGCAG (SEQ ID NO: 32) and the primers on both sides of target site 2
  • Target 2-F GCCTGCTCTTCCTCCTG (SEQ ID NO: 33) and target 2-R: GTGCTCCGGCTGTTGAT (SEQ ID NO: 34) amplify genomic DNA, and compare the amplified product with the genome after sequencing. The results showed that 14 T0 plants had gene editing, one of which had a homozygous mutation, and 8 T0 seedlings were not edited.
  • the genomic DNA of plant PC9M-GMS2-Line17 has homozygous mutations at both target site 1 and target site 2, and TG base deletion (SEQ ID NO: 27) occurred at target site 1, which is in the target site.
  • the genomic DNA of PC9M-GMS2-Line1 has a biallelic mutation at target site 1, in which allele 1 has an A base insertion, and allele 2 has a T base deletion;
  • PC9M-GMS2-Line1 Genomic DNA also has a biallelic mutation at the target site 2, a G/T base SNP occurred in allele 1, and a G/C base SNP occurred in allele 2.
  • PC9M-GMS2-Line3 has a biallelic mutation at target site 1, in which allele 1 has a TG base deletion, and allele 2 has an A base insertion;
  • PC9M-GMS2-Line3 Genomic DNA also has a biallelic mutation at the target site 2, a G/T base SNP occurred in allele 1, and a C base deletion occurred in allele 2.
  • the genotypes of PC9M-GMS2-Line2, PC9M-GMS2-Line5, and PC9M-GMS2-Line7 were not changed in the transgene-negative single plants (Figure 14A).
  • primers 3148OX-F CggggtaccATGGCCGCCACCGAC: (SEQ ID NO: 35) and 3148OX-R: CGCggatccTCACAAGAACGACGC (SEQ ID NO: 36) were used to amplify the complete nucleotide sequence with GMS2 encoding (SEQ ID NO: 2) DNA fragment. This fragment was digested with Kpn I and BamH I and then ligated into pBLU5 to obtain plasmid pUbi1301-48490-CDS ( Figure 19). The Escherichia coli with pUbi1301-48490-CDS was named E.
  • the pUbi1301-48490-CDS was transformed into Agrobacterium strain EHA105 by electric shock, and the resulting strain was named Ab-pUbi1301-48490-CDS.
  • Recombinant Agrobacterium Ab-pUbi1301-48490-CDS was used to infect the callus of japonica rice Zhonghua 11, and 6 transgenic positive plants were obtained after hygromycin resistance screening, differentiation and rooting.
  • InD48490-F GCTCCGGCTGTTGATCT (SEQ ID NO: 19)
  • InD48490-R GCTGCTCTTCCTCCTG (SEQ ID NO: 20) in Example 5
  • GAPDH-RTF GAATGGCTTTCCGTGTT (SEQ ID NO: 25)
  • GAPDH-RTR CAAGGTCCTCCTCAACG (SEQ ID NO: 26) to analyze the expression level of GMS2 in transgenic positive plants.
  • the expression level of GMS2 in the overexpression plants 20 and 28 increased by 9 times and 45 times, respectively, but the overexpression plants did not co-segregate with the expression level.
  • the obvious phenotype indicates that the overexpression of GMS2 gene has no obvious effect on the phenotype of rice.
  • the Escherichia coli with pC1300-48490-genome was named E.coli-pC1300-48490-genome.
  • the pC1300-48490-genome was transformed into Agrobacterium strain EHA105 by electric shock, and the resulting strain was named Ab-pC1300-48490-genome.
  • Recombinant Agrobacterium Ab-pC1300-48490-genome was used to infect gms2 mutant callus. After resistance screening, differentiation, and rooting, a total of 4 transgenic positive plants were obtained, and the fertility of the 4 plants returned to normal ( Figure 22, Figure 23, Figure 24 and Figure 25). This further proves that the GMS2 gene regulates pollen development, and mutations in this gene can cause pollen abortion.
  • the amino acid sequence of the fertility gene in Arabidopsis lyrata is shown in SEQ ID NO: 9; the amino acid sequence of the fertility gene in banana (Musa acuminata) is shown in SEQ ID NO: 10; Africa The amino acid sequence of the fertility gene in Oryza glaberrima is shown in SEQ ID NO: 11; the amino acid sequence of the fertility gene in Oryza brachyantha is shown in SEQ ID NO: 12; Barley ( The amino acid sequence of the fertility gene in Hordeum vulgare is shown in SEQ ID NO: 13: the amino acid sequence of the fertility gene in sorghum (Sorghum bicolor) is shown in SEQ ID NO: 14; the fertility gene in Zea mays The amino acid sequence of the sex gene is shown in SEQ ID NO: 15; the amino acid sequence of the fertility gene in Setaria italica is shown in SEQ ID NO: 16.
  • gms2 mutants and normal fertility receptors such as H28B
  • H28B normal fertility receptors
  • molecular markers are used for gms2 gene and genetic background selection, and finally H28B background with homozygous GMS2 Recessive Genic Male Sterile Line of Mutant Gene.
  • F 1 as the parent and recipient parent, such as H28B, backcross to obtain BC 1 F 1 .
  • Plant BC 1 F 1 and use primers InD48490_F: GCTCCGGCTGTTGATCT (SEQ ID NO: 19) and InD48490_R: GCCTGCTCTTCCTCCTG (SEQ ID NO: 20) to detect gms2 genotype.
  • Select gms2 heterozygous genotype that is, plants with 149bp and 140bp bands can be amplified at the same time.
  • genotypes Use a set of (for example, 100, or 200, etc.) genotypes to have polymorphisms between the gms2 mutant and the recurrent parent genome, and evenly distribute molecular markers (which can be but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR and other types of markers), the genetic background of the single plant selected in step 3 is identified, and the genotype of the recurrent parent is highly similar (such as greater than 88% similarity, or 2% selection rate) Etc.) of the plant.
  • molecular markers which can be but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR and other types of markers
  • step 4 Use the plant selected in step 4 and the recipient parent, such as H28B, to backcross to obtain BC 2 F 1 .
  • Plant BC 2 F 1 repeat steps 3 and 4, and select plants with gms2 genotype heterozygous and high genetic background recovery rate (such as greater than 98%, or 2% selection rate, etc.), and harvest them from bred BC 2 F 2 .
  • Plant BC 2 F 2 repeat steps 3 and 4, select the plants with the gms2 genotype heterozygous and the highest genetic background homozygous rate, and harvest them from the cross BC 2 F 3 .
  • the gms2 homozygous strain isolated from the progeny of BC 2 F 3 is the gms2 recessive nuclear sterile line.
  • BC 2 F 3 is used to preserve the germplasm resources of the gms2 recessive nuclear sterile line.
  • the invention provides a rice male fertility regulating gene, its application, and a method for regulating rice fertility by using CRISPR-Cas9; and a rice male fertility regulating gene mutant and its molecular markers and applications.
  • the present invention provides a rice gene GMS2 with functions of regulating the development of rice male germ cells and pollen fertility. Its nucleotide sequence is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and its amino acid sequence is shown in SEQ ID. NO: shown in 3.
  • the GMS2 mutant of rice male fertility regulatory protein provided by the present invention can make rice pollen completely sterile, resulting in rice male sterility.
  • the genomic nucleotide sequence of the GMS2 mutant is shown in SEQ ID NO: 6, and the CDS sequence is shown in SEQ ID.
  • the amino acid sequence is shown in NO:7, and the amino acid sequence is shown in SEQ ID NO:8.
  • the nuclear sterile mutant of the present invention can be used to cultivate new nuclear sterile lines, which provides a simple, rapid and effective method for the cultivation of rice nuclear sterile lines.
  • the nuclear sterile rice material of the present invention can be used to replace artificial emasculation during rice hybridization, save labor, and can be especially used for recurrent selection breeding that requires a large number of crosses, and has an important role in expanding the germplasm base of rice.
  • the rice gene GMS2 and its mutants provided by the invention can be used for sterile seed production and production of rice hybrids, and have great application value and economic value.

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Abstract

本发明涉及生物技术领域，具体涉及水稻雄性育性调控基因、其应用以及利用CRISPR-Cas9调控水稻育性的方法；以及一种水稻雄性育性调控基因突变体及其分子标记和应用。本发明提供具有调控水稻雄性生殖细胞发育和花粉育性功能的水稻基因GMS2。本发明提供的水稻雄性育性调控蛋白GMS2突变体可使水稻花粉完全不育，导致水稻雄性不育。本发明提供的水稻基因GMS2及其突变体可用于水稻杂交种的不育化制种和生产，具有巨大的应用价值和经济价值。

Description

水稻雄性育性调控基因、水稻雄性育性调控基因突变体、其应用以及调控水稻育性的方法
交叉引用
本申请要求2020年6月2日提交的专利名称为“一种水稻雄性育性调控基因突变体及其分子标记和应用”的第202010491115.0号中国专利申请以及2020年6月2日提交的专利名称为“水稻雄性育性调控基因、其应用以及利用CRISPR-Cas9调控水稻育性的方法”的第202010491100.4号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物技术领域，具体地涉及一个植物育性调控基因GMS2、GMS2编码蛋白、GMS2的基因敲除突变体，GMS2基因、蛋白和突变体在杂交育种中的应用；以及植物育性相关蛋白GMS2突变体，该突变体的编码基因、其分子标记及其在杂交育种中的应用。
背景技术
水稻是世界上最重要的粮食作物之一。随着人口的增长和生活品质的提升，据预计到2050年水稻的年产量要提高1-2倍才能满足人类发展的需求。杂交水稻是父母本杂交后获得的子一代，其产量往往比常规稻亲本提高15％以上，抗性和适应性也远胜于亲本。因此，应用和推广杂交水稻是提高水稻产量的一个重要途径。
雄性不育系是杂交水稻育制种技术的关键节点。雄性不育系是指雄配子发育异常而丧失生育能力，雌配子发育正常的植物株系。它只能作为母本接受父本的花粉，自交不能结实。目前杂交水稻生产上应用的雄性不育系有核质互作型和光温敏型两种。核质互作型雄性不育系的不育基因在细胞质中，细胞核中没有育性恢复基因。当细胞核中有育性恢复基因的恢复系与其配组杂交时可以生产可育的子一代杂交种，当细胞核中没有育性恢复基因而细胞质中也没有不育基因的保持系与其杂交时可以繁殖不育系种子。由于需要不育系、保持系和恢复系三系配套，这种杂交水稻育制种技术常被称为“三系法”。一些控制核质互作型不育及相应育性恢复的基因已经被克隆(Chen and Liu，2014，Male sterility and fertility restoration in crops，Annu Rev Plant Biol，65：579-606)。核质互作型不育系是杂交水稻育制种中第一种大规模应用的不育系，为杂交水稻产业的建立和发展奠定了材料基础。然而由于核质互作型不育系的组配受到恢复系基因型的限制，导致只有约5％的种质资源能被利用。而细胞质的不育基因有导致米质差、特定病虫害流行的潜在风险。
光温敏型雄性不育系是一种育性受光温环境调控的不育系。在一定的光温条件下这种不育系保持不育，可用于组配杂交。当条件改变时不育系恢复育性，可用于不育系繁殖。由于光温敏雄性不育系实现了不育系和保持系的合二为一，只需要父本与其配组生产子一代杂交种，因此相应的育制种技术常被称为“两系法”。调控光温敏雄性不育的基因在细胞核中，目前已经克隆的基因包括PMS3、TMS5、CSA和TMS10(Chen and Liu，2014，Male sterility and fertility restoration in crops，Annu Rev Plant Biol，65:579-606；Zhou H，et al，2014，RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice，Nature Communications，5:4884-4892)。与核质互作型不育系相比，光温敏型不育系繁殖程序简单，配组因恢复基因广泛存在而更自由。光温敏不育系的大规模应用极大地巩固和推动了杂交水稻产业发展。然而，由于该型不育系的育性受光温环境影响，也导致制种风险高，制种地域受到限制。
为了克服目前杂交水稻育制种技术中存在的关键性缺陷，创造和利用新类型的不育系将是重要的突破口。细胞核雄性不育由细胞核基因突变产生，有显性突变和隐性突变，有孢子体基因突变和配子体基因突变。显性突变和配子体基因突变只能通过雌配子遗传，隐性突变既可通过雌配子也可通过雄配子进行遗传，而且遵循孟德尔定律。本发明提供了一种植物育性相关调控基因及基于该基因突变所产生的隐性核不育类型的雄性 不育系。该不育系育性稳定，只受核编码的单基因调控，不受光温环境的影响。该不育系的育性恢复基因广泛存在于水稻种质资源中，也可以通过转野生型基因恢复育性。该基因和该基因突变产生的不育系为研发水稻新型杂交育制种技术提供了元件，为解决现有技术存在的问题奠定了基础。
发明内容
本发明的一个目的是提供一种植物育性相关蛋白、其编码基因以及通过操作该基因在调控植物雄性生育力中的应用。非限制性地举例而言，下文描述的任何方法都可与本发明所提供的植物育性相关蛋白的相应核苷酸序列一起使用，例如，使植株中所述植物育性相关蛋白的内源编码序列突变、向植株中引入该序列的反义序列、使用发卡形式、或将其与其它核苷酸序列连接起来调控植株的表型，或者是本领域技术人员己知的可用于影响植株的雄性生育力的多种方法中的任一方法。
本发明在水稻中发现了一个具有雄性育性调控功能的花粉发育调控基因GMS2。GMS2位于水稻第4号染色体上，其在粳稻品种日本晴中的基因组核苷酸序列如SEQ ID NO:1所示，CDS序列如SEQ ID NO:2所示，氨基酸序列如SEQ ID NO:3所示。在籼稻品种9311中其基因组核苷酸序列如SEQ ID NO:4所示，其CDS序列如SEQ ID NO:69所示，其氨基酸序列与粳稻品种日本晴相同。在琴叶拟南芥(Arabidopsis lyrata)中该育性基因的氨基酸序列如SEQ ID NO:9所示；在香蕉(Musa acuminata)中该育性基因的氨基酸序列如SEQ ID NO:10所示；非洲栽培稻(Oryza glaberrima)中该育性基因的氨基酸序列如SEQ ID NO:11所示；在短药野生稻(Oryza brachyantha)中该育性基因的氨基酸序列如SEQ ID NO:12所示；大麦(Hordeum vulgare)中该育性基因的氨基酸序列如SEQ ID NO:13所示：高粱(Sorghum bicolor)中该育性基因的氨基酸序列如SEQ ID NO:14所示；玉米(Zea mays)中该育性基因的氨基酸序列如SEQ ID NO:15所示；小米(Setaria italica)中该育性基因的氨基酸序列如SEQ ID NO:16所示。
上述所述育性基因，可从各种植物中分离获得。本领域技术人员应该理解，本发明所述的育性基因包括与GMS2基因高度同源，并且具有同样的育性调控功能的高度同源的功能等价序列。所述高度同源的功能等价序列包括在严谨条件下能够与本发明所公开的GMS2基因的核苷酸序列杂交的DNA序列。本发明中所使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和l mM EDTA的杂交液中于60℃杂交12-16小时，然后在65℃下用含0.1％SDS、和0.1×SSC的洗涤液洗涤15-60分钟。
功能等价序列还包括与本发明所公开的GMS2基因的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、或99％序列相似性，且具有育性调控功能的DNA序列，可以从任何植物中分离获得。其中，序列相似性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法(Bioinformatics，4(1)：1117，1988)、Needleman-Wunsch全局比对法(J Mol Biol，48(3)：443-453，1970)、Smith-Waterman局部比对法(J Mol Biol，147：195-197，1981)、Pearson和Lipman相似性搜索法(PNAS，85(8)：2444-2448，1988)、Karlin和Altschul的算法(Altschul等，J Mol Biol，215(3)：403-410，1990；PNAS，90：5873-5877，1993)。这对于本领域技术人员来说是熟悉的。
基于本发明的上述发现，本发明的第一个方面是提供一种植物雄性育性相关蛋白，所述植物雄性育性相关蛋白为如下(1)或(2)所述的蛋白：
(1)具有SEQ ID NO:3、9、10、11、12、13、14、15或16所示氨基酸序列的蛋白；
(2)将SEQ ID NO:3、9、10、11、12、13、14、15或16经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有调控植物雄性育性活性的蛋白。
本发明提供编码所述植物雄性育性相关蛋白的核酸。
本发明所述的核酸可以从任何植物中分离获得，包括但不限于芸苔属、玉米、小麦、高梁、短药野生稻、非洲栽培稻、短柄草属、两节荠属、白芥、草麻子、芝麻、棉籽、亚麻子、大豆、拟南芥属、菜豆属、花生、茵宿、燕麦、油菜籽、大麦、燕麦、黑麦(Rye)、小米、蜀黍、小黑麦、单粒小麦、斯佩尔特小麦(Spelt)、 双粒小麦、亚麻、格兰马草(Gramma grass)、摩擦禾、假蜀黍、羊茅、多年生麦草、甘廉、红莓苔子、番木瓜、香蕉、红花、油棕、香瓜、苹果、黄瓜、石角斗、剑兰、菊花、百合科、棉花、校、向日葵、芸苔、甜菜、咖啡、观赏植物和松类等。优选地，植物包括玉米、小米、拟南芥、二穗短柄草、大豆、红花、芥菜、小麦、大麦、黑麦、短药野生稻、非洲栽培稻、棉花和高粱。
以水稻为例，所述核酸的序列为以下任一：
(1)具有SEQ ID NO:1或2所示的核苷酸序列的核酸；
(2)具有SEQ ID NO:4或69所示的核苷酸序列的核酸；
(3)在严格条件下能够与(1)、(2)之任一所述序列的DNA杂交的DNA片段；
(4)与(1)、(2)之任一所述序列互补的DNA片段；
(5)在(1)、(2)之任一所述序列的基础之上，经过一至数个碱基的替换和/或一至数个碱基的插入和/或缺失，或经过大片段的核苷酸序列插入/缺失/易位/倒位所形成能够影响植物花粉生育能力的DNA片段；
(6)与(1)、(2)之任一所述序列的DNA片段具有85％、90％、95％、96％、97％、98％、或99％以上的同一性且编码水稻雄性育性相关蛋白的DNA片段。
本发明提供编码所述植物雄性育性相关蛋白的核酸的抑制因子，所述抑制因子导入植物中能够使编码所述植物雄性育性相关蛋白的核酸的表达量降低、不表达或发生突变而失活。所述抑制因子可为蛋白或核酸(包括但不限于反义基因、siRNA及其DNA、dsRNA及其DNA、sgRNA及其DNA等)。
本发明提供一种生物材料，其含有编码所述植物雄性育性相关蛋白的核酸，或含有编码所述植物雄性育性相关蛋白的核酸的抑制因子，所述生物材料为表达盒、载体、宿主细胞、转基因细胞系或转基因植物。
本发明提供一种植物、植物组织或植物细胞，其表现为雄性不育性状，由编码所述植物雄性育性相关蛋白的核酸的突变所造成，所述突变为一个或多个核苷酸的缺失、插入或取代突变，或为通过反义基因的转入、共抑制或发夹结构的引入所产生的突变；所述突变导致所述的植物雄性育性相关蛋白的表达量降低、不表达或失活。
所述植物、植物组织或植物细胞可通过自然突变或人工诱变获得，可以是转基因植物、植物组织或植物细胞也可以是非转基因植物、植物组织或植物细胞。
所述人工诱变包括理化诱变、插入突变、基因打靶敲除、反义基因的转入、共抑制或发夹结构的引入等。
所述植物包括但不限于芸苔属、玉米、小麦、高梁、短药野生稻、非洲栽培稻、短柄草属、两节荠属、白芥、芝麻、大豆、拟南芥属、菜豆属、花生、茵宿、燕麦、油菜籽、大麦、燕麦、黑麦(Rye)、小米、蜀黍、小黑麦、单粒小麦、斯佩尔特小麦(Spelt)、双粒小麦、亚麻、格兰马草(Gramma grass)、摩擦禾、假蜀黍、羊茅、多年生麦草、甘廉、红莓苔子、番木瓜、香蕉、红花、油棕、香瓜、苹果、黄瓜、石角斗、剑兰、菊花、百合科、棉花、向日葵、芸苔、甜菜、咖啡、观赏植物和松类等。优选地，包括玉米、小米、拟南芥、二穗短柄草、芥菜、小麦、大麦、黑麦、短药野生稻、非洲栽培稻、棉花和高粱。
可选地，所述植物、植物组织或植物细胞为采用CRISPR-Cas9方法获得，所述CRISPR-Cas9方法使用的靶序列位于编码所述植物雄性育性相关蛋白的核酸的序列中，靶序列的反向互补序列具有5’-(N)X-NGG-3’结构，其中，N表示A，T，C和G中的任意一个，X为19或20nt的任意核苷酸序列。
具体地，所述植物、植物组织或植物细胞衍生系为采用CRISPR-Cas9方法以GCGGTCGGTGGCGGCCATGG(SEQ ID NO:17)和CGCCTCCCTCGCCGTCGCG G(SEQ ID NO:18)为靶位点得到的在靶位点或靶位点相邻区域发生突变的植物。
本发明的第二个方面提供所述植物雄性育性相关蛋白或编码所述植物雄性育性相关蛋白的核酸或所述核酸的抑制因子或所述生物材料或所述植物、植物组织或植物细胞的如下任一种应用：
(1)在调控植物雄性育性中的应用；
(2)在制备雄性不育植物中的应用；
(3)在恢复由编码所述植物雄性育性相关蛋白的核酸突变导致的隐性核不育的雄性育性中的应用；
(4)在植物杂交育种中的应用；
(5)在植物种质资源改良中的应用。
上述(1)中，调控植物雄性育性可为使植物的雄性育性降低或丧失。具体可通过调控植物雄性生殖细胞、花粉的发育实现。其中，使植物的雄性育性降低或丧失可通过将植物中所述植物雄性育性相关蛋白的编码基因突变，使其表达量降低或不表达，或者通过将编码所述植物雄性育性相关蛋白的核酸的抑制因子引入植物中实现。
上述(2)中，所述雄性不育植物为隐性核不育系，带有编码所述植物雄性育性相关蛋白的核酸的纯合突变。
上述(3)中，通过将编码所述植物雄性育性相关蛋白的核酸引入植株来恢复由所述植物雄性育性相关蛋白突变或失活导致的植株隐性核不育的雄性育性，以便导入外源基因从而获得优质的转基因作物。
上述(4)中，使用带有编码所述植物雄性育性相关蛋白的核酸的纯合突变的隐性核不育系进行杂交育、制种。
上述(5)中，所述改良包括产量提高、品质提高、抗病虫害、抗逆、抗倒伏等。
以上所述植物是自花授粉或异花授粉作物，包括但不限于水稻、玉米、小麦、高梁。
本发明的第三个方面是提供通过影响所述植物雄性育性相关蛋白或编码该蛋白的核酸的序列，或者通过影响该核酸的转录、翻译从而影响植物育性的方法。所述影响植物育性是指使所述植物的育性发生改变，如导致植物雄性不育。具体地，取决于实际应用需求，可以通过多种方法来影响所述植物雄性育性相关蛋白或编码该蛋白的核酸的序列或其在植物体内的表达和翻译，从而达到调控植物雄性育性的效果。更具体地，影响所述植物雄性育性相关蛋白或编码该蛋白的核酸的序列或其在植物体内的表达和翻译可以使用许多本领域普通技术人员可获得的工具进行，例如，通过理化诱变、插入突变、基因打靶敲除、反义基因的转入、共抑制或发夹结构的引入等，都可以用于破坏所述植物雄性育性相关蛋白的正常表达，从而获得雄性不育的植株。
本发明的第四个方面是提供所述植物雄性育性相关蛋白的突变体，其为在所述植物雄性育性相关蛋白的编码基因中插入、和/或缺失、和/或取代若干个核苷酸得到，该突变体能够导致水稻雄性不育。
本发明提供一种适用于CRISPR-Cas9方法对编码所述植物育性相关蛋白的核酸进行定向敲除的靶位点，其为靶位点1：GCGGTCGGTGGCGGCCATGG(SEQ ID NO:17)和/或靶位点2：CGCCTCCCTCGCCGTCGCGG(SEQ ID NO:18)。
本发明还提供特异性靶向上述靶位点1和靶位点2的sgRNA。
含有上述sgRNA的DNA序列的CRISPR-Cas9打靶载体也属于本发明的保护范围。
本发明的第五个方面是提供所述靶位点或靶向该靶位点的sgRNA或含有该sgRNA的DNA的CRISPR-Cas9打靶载体的如下任一种应用：
(1)在调控植物雄性育性中的应用；
(2)在制备雄性不育植物中的应用；
(3)在植物杂交育种中的应用；
(4)在植物种质资源改良中的应用。
上述(1)中，调控植物雄性育性可为使植物的雄性育性降低或丧失。具体可通过调控植物雄性生殖细胞、花粉的发育实现。其中，使植物的雄性育性降低或丧失可通过将植物中所述植物雄性育性相关蛋白的编码基因突变，使其表达量降低或不表达，或者通过将编码所述植物雄性育性相关蛋白的核酸的抑制因子引入植物中实现。
上述(2)中，所述雄性不育植物为隐性核不育系，带有编码所述植物雄性育性相关蛋白的核酸的纯合突变。
上述(3)中，使用所述核苷酸的抑制子是所述育性调控蛋白失活，从而创制出隐性核雄性不育的植株，以便用于杂交育、制种。
上述(4)中，所述改良包括产量提高、品质提高、抗病虫害、抗逆、抗倒伏等。
本发明还提供一种制备雄性不育植物的方法，其为使植物中的所述植物雄性育性相关蛋白的表达量降低、不表达或失活。
作为本发明的一种优选方案，本发明提供利用CRISPR-Cas9技术制备雄性不育水稻的方法，其为利用CRISPR-Cas9技术敲除或突变水稻中编码所述植物育性相关蛋白的核酸。
具体地，利用CRISPR-Cas9技术以靶位点GCGGTCGGTGGCGGCCATGG(SEQ ID NO:17)和/或靶位点CGCCTCCCTCGCCGTCGCGG(SEQ ID NO:18)为靶位点，使得靶位点或靶位点及相邻核苷酸序列突变。
本发明还提供了一种获得GMS2基因在植物中的直系同源基因片段的方法，以及利用该方法获得拟南芥、香蕉、非洲栽培稻、短药野生稻、大麦、高粱、玉米、小米同源GMS2的氨基酸序列及其应用。
本发明提供的一种获得GMS2基因在植物中的直系同源基因片段的方法包括：使用前述GMS2基因的DNA片段在核苷酸数据库中进行blastx搜索；获得的所有Identities大于或等于35％、Positives大于或等于50％即为GMS2基因的直系同源的基因片段。
本发明的另一个目的是提供植物育性相关蛋白GMS2突变体，该突变体的编码基因、其分子标记及其在杂交育种中的应用。
本发明发现了一个水稻育性相关蛋白GMS2突变体，该突变体相对于野生型GMS2蛋白在第40～42位缺失天冬酰胺、丝氨酸和酪氨酸3个氨基酸。
GMS2位于水稻第4号染色体上，其在粳稻品种日本晴中的基因组核苷酸序列如SEQ ID NO:1所示，CDS序列如SEQ ID NO:2所示，氨基酸序列如SEQ ID NO:3所示。在籼稻品种9311中其基因组核苷酸序列如SEQ ID NO:4所示，其CDS序列如SEQ ID NO:69所示，其氨基酸序列与粳稻品种日本晴。在琴叶拟南芥(Arabidopsis lyrata)中该育性基因的氨基酸序列如SEQ ID NO:9所示；在香蕉(Musa acuminata)中该育性基因的氨基酸序列如SEQ ID NO:10所示；非洲栽培稻(Oryza glaberrima)中该育性基因的氨基酸序列如SEQ ID NO:11所示；在短药野生稻(Oryza brachyantha)中该育性基因的氨基酸序列如SEQ ID NO:12所示；大麦(Hordeum vulgare)中该育性基因的氨基酸序列如SEQ ID NO:13所示：高粱(Sorghum bicolor)中该育性基因的氨基酸序列如SEQ ID NO:14所示；玉米(Zea mays)中该育性基因的氨基酸序列如SEQ ID NO:15所示；小米(Setaria italica)中该育性基因的氨基酸序列如SEQ ID NO:16所示。
上述所述育性基因，可从各种植物中分离获得。本领域技术人员应该理解，本发明所述的育性基因包括与GMS2基因高度同源，并且具有同样的育性调控功能的高度同源的功能等价序列。所述高度同源的功能等价序列包括在严谨条件下能够与本发明所公开的GMS2基因的核苷酸序列杂交的DNA序列。本发明中所使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和l mM EDTA的杂交液中于60℃杂交12-16小时，然后在65℃下用含0.1％SDS、和0.1×SSC的洗涤液洗涤15-60分钟。
功能等价序列还包括与本发明所公开的GMS2基因的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、或99％序列相似性，且具有育性调控功能的DNA序列，可以从任何植物中分离获得。其中，序列相似性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法(Bioinformatics，4(1)：1117，1988)、Needleman-Wunsch全局比对法(J Mol Biol，48(3)：443-453，1970)、Smith-Waterman局部比对法(J Mol Biol，147：195-197，1981)、Pearson和Lipman相似性搜索法(PNAS，85(8)：2444-2448，1988)、Karlin和Altschul 的算法(Altschul等，J Mol Biol，215(3)：403-410，1990；PNAS，90：5873-5877，1993)。这对于本领域技术人员来说是熟悉的。
基于本发明的上述发现，本发明的又一个方面是提供植物雄性育性相关蛋白突变体，所述突变体相对于野生型植物雄性育性相关蛋白包含如下氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸中的至少一个的缺失；
其中，x为S或N；
所述野生型植物雄性育性相关蛋白为如下(1)或(2)所述的蛋白：
(1)具有SEQ ID NO:3、9、10、11、12、13、14、15或16所示的氨基酸序列的蛋白；
(2)将SEQ ID NO:3、9、10、11、12、13、14、15或16经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有调控植物雄性育性活性的蛋白。
优选地，所述突变体相对于野生型植物雄性育性相关蛋白包含如下氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸的缺失；其中，x为S或N。
对于水稻而言，所述突变体相对于其野生型雄性育性相关蛋白包含如下氨基酸突变：第40、41和42位氨基酸缺失，其野生型雄性育性相关蛋白具有SEQ ID NO:3所示序列或将SEQ ID NO:3经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有调控植物雄性育性活性的蛋白。
本发明提供水稻GMS2蛋白突变体，所述突变体具有如SEQ ID NO.8所示的氨基酸序列。
本发明还提供了编码所述植物雄性育性相关蛋白突变体的核酸。
本发明所述植物雄性育性相关蛋白突变体的核酸可以从任何植物中分离获得，包括但不限于芸苔属、玉米、小麦、高梁、短药野生稻、非洲栽培稻、短柄草属、两节荠属、白芥、芝麻、大豆、拟南芥属、菜豆属、花生、茵宿、燕麦、大麦、燕麦、黑麦(Rye)、小米、蜀黍、小黑麦、单粒小麦、斯佩尔特小麦(Spelt)、双粒小麦、亚麻、格兰马草(Gramma grass)、摩擦禾、假蜀黍、羊茅、多年生麦草、甘廉、红莓苔子、番木瓜、香蕉、红花、油棕、香瓜、苹果、黄瓜、石角斗、剑兰、菊花、百合科、棉花、向日葵、甜菜、咖啡、观赏植物和松类等。优选地，植物包括玉米、小米、拟南芥、二穗短柄草、大豆、红花、芥菜、小麦、大麦、黑麦、短药野生稻、非洲栽培稻、棉花和高粱。
具体地，以水稻为例，编码所述突变体的核酸相对于编码所述野生型植物雄性育性相关蛋白的核酸包含如下核苷酸突变：对应于LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失。
对于水稻GMS2蛋白突变体，突变后的GMS2的基因组核苷酸序列如SEQ ID NO:6所示，CDS序列如SEQ ID NO:7所示。
本发明还提供一种生物材料，其为含有编码所述植物雄性育性相关蛋白突变体的核酸的表达盒、载体、宿主细胞、转基因细胞系或转基因植物。
本发明的再一个方面是提供所述突变体或所述核酸或所述生物材料的如下任一种应用：
(1)在调控植物雄性育性中的应用；
(2)在制备雄性不育植物中的应用；
(3)在植物杂交育种中的应用；
(4)在植物种质资源改良中的应用。
上述(1)中，调控植物雄性育性可为使植物的雄性育性降低或丧失。具体可通过调控植物雄性生殖细胞、花粉的发育实现。其中，使植物的雄性育性降低或丧失可通过将植物中所述植物雄性育性相关蛋白的编码基因突变，使其表达量降低或不表达，或者通过将编码所述植物雄性育性相关蛋白的核酸的抑制因子引入植物中实现。
上述(2)中，所述雄性不育植物为隐性核不育系，带有编码所述植物雄性育性相关蛋白的核酸的纯合突 变。
上述(3)中，使用带有编码所述植物雄性育性相关蛋白的核酸的纯合突变的隐性核不育系进行杂交育、制种。
上述(4)中，所述改良包括产量提高、品质提高、抗病虫害、抗逆、抗倒伏等。
以上所述植物是自花授粉或异花授粉作物，包括但不限于玉米、小麦、高梁、水稻。
本发明的再一个方面是提供一种植物、植物组织或植物细胞，其具有雄性不育性状，其基因组序列中野生型植物雄性育性相关蛋白的基因编码的蛋白发生包含如下的氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸中至少一个的缺失，其中野生型植物雄性育性相关蛋白如前所述。
优选地，所述植物、植物组织或植物细胞的基因组序列中野生型植物雄性育性相关蛋白的基因编码的蛋白发生包含如下的氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸的缺失。
优选地，所述植物为水稻，所述植物组织为水稻组织，所述植物细胞为水稻细胞。
本发明提供一种水稻，所述水稻的基因组或转录组包含如下突变：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失；LOC_Os04g48490基因编码区的序列如SEQ ID NO.69所示，LOC_Os04g48490基因编码蛋白的序列如SEQ ID NO.3所示。
本发明还提供一种水稻组织，所述水稻组织的基因组或转录组包含如下突变：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失。
本发明还提供一种水稻细胞，所述水稻细胞的基因组或转录组包含如下突变：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失。
本发明进一步提供基因组序列中LOC_Os04g48490基因的序列突变为如SEQ ID NO:6所示的序列，导致LOC_Os04g48490基因的CDS突变为如SEQ ID NO:7所示序列，编码蛋白突变为如SEQ ID NO:8所示序列的水稻、水稻组织或水稻细胞。
本发明提供水稻突变体材料gms2，gms2表现为雄性不育，其基因组序列中LOC_Os04g48490(GMS2)基因的序列突变为如SEQ ID NO:6所示的序列，LOC_Os04g48490基因的CDS突变为如SEQ ID NO:7所示序列，编码蛋白的氨基酸序列突变为如SEQ ID NO:8所示序列；所述LOC_Os04g48490基因的基因组序列如SEQ ID NO:4所示。
具体地，水稻突变体材料gms2的基因组序列在LOC_Os04g48490基因(GMS2)编码区(序列如SEQ ID NO:69)第118～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白(序列如SEQ ID NO:3)中第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失。
上述GMS2的第40、40和第42位的天冬酰胺、丝氨酸和酪氨酸缺失，导致GMS2蛋白丧失功能，进而导致水稻雄性不育。
本领域技术人员应该知晓，可以将如SEQ ID NO:6所示的核苷酸序列通过杂交、回交或转基因的方法导入受体植物中，从而获得新的雄性不育突变体材料。
本发明的再一个方面是提供用于检测所述突变体或所述突变体材料的分子标记，所述分子标记通过核苷酸序列如SEQ ID NO.19-20所示的引物扩增得到。
本发明提供用于扩增所述分子标记的特异性引物，其核苷酸序列如SEQ ID NO:19-20所示。
本发明提供含有核苷酸序列如SEQ ID NO:19-20所示的引物的检测试剂或试剂盒。
本发明还提供所述分子标记或所述检测试剂或试剂盒的如下任一种应用：
(1)在检测所述植物雄性育性相关蛋白突变体或所述突变体材料中的应用；
(2)在筛选或培育雄性不育水稻突变体中的应用。
具体地，当选用SEQ ID NO:19-20所示引物扩增水稻基因组DNA时，若只能扩增出140bp一条带，则水稻表达所述植物雄性育性相关蛋白突变体、具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的纯合基因型且表现出雄性不育性状；若只能扩增出149bp一条带，则水稻不表达植物雄性育性相关蛋白突变体、不具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的基因型且表现出雄性可育性状；若能同时扩增出140bp和149bp两条带，则水稻表达植物雄性育性相关蛋白突变体、具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的杂合基因型且表现出雄性可育性状。
本发明的再一个方面是提供一种制备雄性不育植物的方法，包括：使野生型植物雄性育性相关蛋白发生突变，所述突变包含如下氨基酸的突变：在NxYL保守序列中发生N、x、Y三个氨基酸中的至少一个的缺失；所述野生型植物雄性育性相关蛋白同上所述。
优选地，所述植物为水稻，所述方法包括：使水稻的基因组或转录组发生突变，所述突变包含如下突变：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失；LOC_Os04g48490基因编码区的序列如SEQ ID NO.69所示，LOC_Os04g48490基因编码蛋白的序列如SEQ ID NO.3所示。
更优选地，所述方法包括：使水稻表达序列如SEQ ID NO:8所示的雄性育性相关蛋白突变体，且不表达SEQ ID NO:3所示的野生型植物雄性育性相关蛋白。
上述突变可通过基因编辑、杂交、回交、自交或无性繁殖的方法实现。
与现有技术相比，本发明具有如下的有益效果：本发明提供的水稻花粉发育调控基因GMS2直接参与花粉发育调控，该基因被敲除或表达受到抑制后，花粉完全不育，导致植物雄性不育。本发明利用CRISPR-Cas9基因编辑技术对GMS2基因进行了基因编辑，获得了GMS2基因突变的水稻雄性不育突变体。由GMS2突变导致的水稻不育突变体与现有三系和两系不育系相比，其不育性状稳定，不受环境条件影响。利用本发明的GMS2基因及其突变体可通过转基因等方法培育新的核不育系并提供恢复所述不育系育性的方法，为水稻核不育系培育和繁殖奠定了基础，在农作物的杂种优势利用和不育化杂交种制种生产中都将发挥重要作用。本发明提供的水稻雄性育性调控蛋白GMS2突变体可使水稻花粉完全不育，导致水稻雄性不育。利用本发明的核不育突变体可培育新的核不育系，为水稻核不育系培育提供了一个简单、迅速、有效的方法。由本发明的GMS2突变体导致的水稻不育突变体与现有三系和两系不育系相比，具有不育性状稳定，种质资源利用率高等优势，可用于需要进行大量杂交的轮回选择育种，在杂交水稻育制种领域具有极大的应用价值。
附图说明
图1为本发明实施例2中灌浆期野生型(左)和gms2突变体(右)的植株形态。
图2为本发明实施例2中野生型(左)和gms2突变体(右)的小穗形态。
图3为本发明实施例2中野生型(左)和gms2突变体(右)穗开花的形态。
图4为本发明实施例2中解剖后野生型(左)和gms2突变体(右)的小花形态。
图5为本发明实施例2中野生型(左)和gms2突变体(右)的花药形态。
图6为本发明实施例2中野生型(左)和gms2突变体(右)花粉碘染。
图7为本发明实施例4中利用InD48490标记鉴定定位群体中不育单株的基因型。上带大小149bp，下带大小140bp。左边前2个泳道的DNA模板分别为gms2突变体和明恢63，后面泳道为定位群体中的不育单株。
图8A为本发明实施例4中GMS2基因图位克隆图。
图8B为本发明实施例4中gms2突变体的突变位点示意图。
图9为本发明实施例4中GMS2基因在9311(48490-9311)，明恢63(48490-MH63)，日本晴(48490-Nip)和gms2突变体(48490-3148)材料中的核苷酸序列差异。有差异的地方用灰色背景突出。每行最后一个核苷酸在整个基因序列中的位置标示在行末。起始密码子ATG和终止密码子TGA分别用方框标出。
图10为本发明实施例4中GMS2编码蛋白在9311(48490-9311)和gms2突变体(48490-3148)中的氨基酸序列差异。有差异的地方用浅灰色背景突出。每行最后一个氨基酸残基在整个蛋白序列中的位置标示在行末。
图11为本发明实施例4中GMS2杂合株后代基因型鉴定。上带大小149bp，下带大小140bp。箭头所指为来自雄性不育的样品。
图12为本发明实施例5中GMS2在水稻不同组织和不同发育时期幼穗中的表达量。花1-花9代表幼穗发育的颖花原基分化期至花粉成熟时期。
图13为本发明实施例6中pC9M-GMS2载体示意图。T1代表靶位点1，T2代表靶位点2。
图14A为本发明实施例6中利用CRISPR/Cas9系统对GMS2基因敲除后部分转基因阳性植株的靶位点序列分析。
图14B为本发明实施例6中转基因植株PC9M-GMS2-Line17在靶位点1和靶位点2处的测序峰图。其中，在靶位点1处的测序峰图中，箭头指向缺失位点；在靶位点2处的测序峰图中，箭头指向插入位点。
图15为本发明实施例6中GMS2野生型(左)、敲除植株PC9M-GMS2-Line17(右)的整株形态。
图16为本发明实施例6中GMS2野生型(左)、敲除植株PC9M-GMS2-Line17(右)的颖壳形态。
图17为本发明实施例6中GMS2野生型(左)、敲除植株PC9M-GMS2-Line17(右)的花药形态。
图18为本发明实施例6中GMS2野生型(左)、敲除植株PC9M-GMS2-Line17(右)的花粉碘染结果。
图19为本发明实施例7中pUbi1301-48490-CDS载体示意图。
图20为本发明实施例7中GMS2在超表达植株中的RT-PCR表达量分析。柱状图为对RT-PCR胶图中的条带亮度进行量化后，用48490的亮度值分别除以对应GAPDH的亮度值得到的结果。
图21为本发明实施例8中pC1300-48490-genome载体示意图。
图22为本发明实施例8中野生型植株(左)和gms2突变体互补植株(右)的植株形态。
图23为本发明实施例8中野生型植株(左)和gms2突变体互补植株(右)的颖壳形态。
图24为本发明实施例8中野生型植株(左)和gms2突变体互补植株(右)的花药形态。
图25为本发明实施例8中野生型植株(左)和gms2突变体互补植株(右)的花粉碘染结果。
图26为本发明实施例9中水稻GMS2基因编码蛋白与其他物种基因组中的同源蛋白的序列比对图。包括琴叶拟南芥(Arabidopsis lyrata)蛋白AT3G60900.1、香蕉(Musa acuminata)蛋白GSMUA_Achr11P03090_001、非洲栽培稻(Oryza glaberrima)蛋白ORGLA04G0194100.1、短药野生稻(Oryza brachyantha)蛋白OB04G29380.1、大麦(Hordeum vulgare)蛋白MLOC_7985.1、高粱(Sorghum bicolor)蛋白Sb06g026030.1、玉米(Zea mays)蛋白GRMZM2G003752_P01、小米(Setaria italica)蛋白Si010135m。NxYL保守序列用方框标出。
图27为本发明实施例9中水稻GMS2基因编码蛋白系统进化树分析。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明的应用范围。下述实施例中的所有技术和科学术语，如无特殊说明，均为本发明所属领域普通技术人员通常所理解的相同含义。除非有相反指明，本发明所使用或提及的技术均为本领域普通技术人员公认的标准技术。所述试验材料，如无特别注明，均为本发明领域通用的试验材料。下述实施例中所用的试验试剂，如无特殊说明，均为自常规生化试剂商店购买得到的。
本发明所述的雄性不育，特指由植物细胞核基因发生功能变化导致植物雄性生殖器官发育出现异常(无 法产生正常雄蕊、花药或者正常的雄性配子体)并出现育性的丧失，即通常所说的雄性核不育(Genic male sterility)而非细胞质核不育(Cytoplasmic male sterility)。雄性生殖器官育性的异常和恢复均由细胞核内的基因加以控制。
因此，本发明也包括利用序列表所述序列调控植株的雄配子生育能力，即利用本发明提供的基因序列在基因组、和/或转录组、和/或蛋白质组水平影响其它植物中相同或同源基因的功能从而达到控制雄性生殖器官育性的目的。例如，包括但不限于下述方法：通过天然序列的变异导致基因表达抑制或蛋白质功能的丧失、通过向植物中转入所述基因的反义序列或引入发卡结构、或将所述基因与其它序列(DNA或RNA)相结合产生新的具有功能活性的DNA或RNA链，来影响或改变植物基因的功能。或其它本领域技术人员己知的可用于影响植物雄性育性的技术方法中的任何一种技术方法。
本发明包括水稻GMS2基因，其显性等位基因对植物雄性育性具有关键作用，功能缺失性的隐性等位基因会导致雄性不育。该基因位于水稻4号染色体，其基因具体位置如图8A、图8B所示。
该基因序列及其同源序列可从各种植物中获得，包括但不限于卷柏(Selaginella moellendorffii)、毛果杨(populus trichocarpa)、芜菁(Brassica rapa)、琴叶拟南芥(Arabidopsis lyrata)、拟南芥(Arabidopsis thaliana)、大豆(Glycine max)、马铃薯(Solanum tuberosum)、葡萄(Vitis vinifera)、小果野芭蕉(Musa acuminata)、小米(Setaria italica)、高粱(Sorghum bicolor)、玉米(Zea mays)、二穗短柄草(Brachypodium distachyon)、大麦(Hordeum vulgare)、短药野生稻(Oryza brachyantha)、非洲栽培稻(Oryza glaberrima)、籼稻(Oryza sativa Indica Group)、粳稻(Oryza sativa Japonica Group)、小立碗藓(Physcomitrella patens)等。获得方法包括但不限于：使用水稻GMS2基因序列通过blastx、blastn或使用水稻GMS2氨基酸序列通过blastp从其它植物的基因组序列数据库、和/或cDNA序列数据库、和/或蛋白质序列数据库中调取；以水稻GMS2基因的DNA或cDNA或RNA序列为参考序列设计引物，从其它植物的基因组DNA或cDNA或RNA中利用PCR的方法直接获得；以水稻GMS2的基因序列设计探针，利用核酸杂交的方法从基因组文库中分离含有同源基因序列的DNA或cDNA或RNA片段。
GMS2基因同源序列指在与SEQ ID NO:3的氨基酸序列进行blastp比较分析后，Identities大于或等于35％、Positives大于或等于50％的植物基因序列。进行blastp时，所有参数均遵照http://blast.ncbi.nlm.nih.gov/所示的默认设置进行。
下文通过说明和阐述提供了更为详细的描述，但这并非意欲对本发明的范围加以限制。
实施例1 水稻雄性不育突变体gms2的筛选
2013年6月用钴60辐射93-11种子10公斤得到M 0代。辐射后的种子种植于海南省临高县试验田，成熟后分单株收种，共获得M 1代材料约6500份。2014年春，选种子量较多的3617个M 1代材料种植成株系，每个株系种50个单株。分别在分蘖期、孕穗期、抽穗期、开花期、灌浆期筛选株型、穗型、育性、产量等各种类型突变体，并收种保存。其中在编号为3148的株系中发现一个不育突变体，命名为gms2。
实施例2 水稻雄性不育突变体gms2的表型分析
与野生型相比，gms2突变体植株(图1)和小穗(图2)形态正常，花期稍迟。内、外稃大小、小花开张尺寸、开张时间与野生型没有明显差异(图3)。体式显微镜下观察突变体小花形态，发现子房，花柱，柱头均比野生型略大(图4)，但花药比野生型瘦小，颜色较浅(图5)。用碘-碘化钾溶液(0.6％KI，0.3％I 2，w/w)溶液对花粉进行染色，如图6所示，野生型花粉粒大而圆并且被染成蓝黑色，而突变体花粉粒皱缩并且不能被染色。同一家系野生型植株套袋自交后正常结实，而gms2突变体不结实。而以水稻品种93-11为父本给gms2突变体授粉则可以结实。这表明该突变体为雄性不育突变体。
实施例3 水稻雄性不育突变体gms2的遗传分析
在M5代种植gms2的分离群体80株，其中64株育性正常，16株不育，可育与不育株分离比符合3:1(χ 2＝0.57，P>0.05)。用gms2与93-11回交，F1代植株全部可育。在F2代种植gms2的分离群体70株，其中57株育性正常，13株不育，可育与不育株分离比符合3:1(χ 2＝0.85，P>0.05)。上述结果表明gms2的不育性状是由隐性单基因控制。
实施例4 水稻雄性不育基因GMS2的克隆
使用图位克隆的方法对GMS2基因进行定位。以明恢63为父本与gms2突变体杂交构建了一个包含66棵突变植株的F 2群体。利用该群体将GMS2定位于4号染色体SSR标记RM17332和RM280之间6861.252Kb范围内，与SSR标记RM303及Indel标记4826紧密连锁。GMS2基因与上述四个标记之间的交换单株分别为8个，1个，1个，32个。利用连锁标记挑选F 2群体中的gms2杂合单株发展了一个F 3群体，包含1937个突变体单株。在F 3群体中RM303、4826、S10与GMS2基因之间的交换单株分别为10个，7个，8个。在RM303和S10之间通过分析和比较93-11和明恢63基因组的序列，开发并实验证实了5个新的单核苷酸多态性标记S4b，S3b，S2，S1，S8。在F3群体中，上述标记的交换单株分别为6个，6个，1个，4个，8个，(图7)。以S2上下游77kb为候选区段，发现在该区段内共有11个注释基因，其中LOC_Os04g48490预测编码一个成束蛋白样阿拉伯半乳聚糖蛋白，推测是GMS2基因。在日本晴中，LOC_Os04g48490基因组核苷酸序列长1582bp(记为48490-Nip，序列如SEQ ID NO:1)，CDS核苷酸序列长1296bp(序列如SEQ ID NO:2)，包含1个外显子(图8A和图8B)，编码一个长432个氨基酸残基的蛋白(序列如SEQ ID NO:3)。用于定位GMS2基因的标记引物对序列如表1所示(SEQ ID NO.39-68)。
表1 用于定位GMS2基因的标记引物对序列
Figure PCTCN2020137145-appb-000001
根据48490-Nip序列设计引物对LOC_Os04g48490基因在93-11、明恢63和gms2突变体中的等位基因进行扩增和测序，引物序列如表2所示。所有PCR扩增均使用KOD FX DNA Polymerase(TOYOBO CO.,LTD.
Life Science Department,Osaka,Japan)，并按照产品说明的反应体系和条件，在Thermo scientific Arktik thermal cycler上进行PCR扩增。PCR产物送往南京金斯瑞生物科技有限公司进行测序。测序结果用DNAman 6.0进行拼接。93-11、明恢63和gms2突变体中的LOC_Os04g48490基因分别记为48490-9311(序列如SEQ ID NO:4)，48490-MH63(序列如SEQ ID NO:5)，48490-3148(序列如SEQ ID NO:6)。
表2 用于扩增LOC_Os04g48490的引物对序列
Figure PCTCN2020137145-appb-000002
对48490-9311、48490-3148、48490-MH63和48490-Nip进行多序列比对，结果如图9所示。48490-9311和48490-3148相比，48490-3148只在ATG后第118个碱基开始存在一个AACAGCTAC的缺失(图8B和图9)。氨基酸序列分析显示，该突变将导致LOC_Os04g48490基因编码蛋白中第40到第42位的天冬酰胺、丝氨酸和酪氨酸残基缺失(图10)。48490-MH63和48490-Nip与48490-3148也在ATG后118个碱基处存在上述相同差异(图9)。这表明ATG后118位碱基开始的AACAGCTAC的缺失突变是造成gms2突变体雄性不育的原因。此外48490-9311和48490-MH63的序列完全一致，而与48490-Nip相比，在第8位有一个A变C的SNP，在第109位有一个C变G的SNP，在1288位有一个T变C的SNP，在第1515位有一个G碱基的插入(图9)。两处核苷酸差异分别落在5’UTR和3’UTR中，另外两处核苷酸差异虽然落在外显子中，但并不影响编码蛋白。这表明LOC_Os04g48490基因在水稻中高度保守，其核苷酸序列即使在籼、粳亚种间也只存在4个碱基的差异，而蛋白质序列则没有差异。在93-11中LOC_Os04g48490的CDS核苷酸序列如SEQ ID NO:69所示，编码蛋白序列如SEQ ID NO:3。在gms2突变体中LOC_Os04g48490的CDS核苷酸序列和氨基酸序列分别如SEQ ID NO:7和SEQ ID NO:8所示。
基于LOC_Os04g48490基因突变位点的测序结果，在突变位点两侧设计特异引物InD48490_F：GCTCCGGCTGTTGATCT(SEQ ID NO:19)和InD48490_R：GCCTGCTCTTCCTCCTG(SEQ ID NO:20)。当InD48490_F和InD48490_R配对扩增野生型LOC_Os04g48490基因时将产生149bp条带，扩增突变型LOC_Os04g48490基因时将产生140bp条带。使用InD48490_F和InD48490_R引物对对41株gms2的M6分离群体进行基因型检测。如图11所示，野生型要么扩增出149bp和140bp两条带，要么扩增出149bp一条带，而不育突变体均只能扩增出140bp一条带。这说明突变基因型与不育表型共分离，LOC_Os04g48490就是GMS2基因。
实施例5 GMS2基因的表达分析
取93-11各时期组织提取总RNA，反转录成cDNA。使用引物InD48490_F：GCTCCGGCTGTTGATCT(SEQ ID NO:19)和InD48490_R：GCCTGCTCTTCCTCCTG(SEQ ID NO:20)检测GMS2基因的表达量，使用引物GAPDH-RTF：GAATGGCTTTCCGTGTT(SEQ ID NO:25)和GAPDH-RTR：CAAGGTCCTCCTCAACG(SEQ ID NO:26)检测内参基因GAPDH的表达量。采取实时定量PCR方法进行表达量分析。如图12所示，GMS2基因的表达量在根和茎中明显低于其它组织，在种子中明显高于其它组织。在茎节、叶、叶鞘和穗中，GMS2基因的表达量处于中等，但并不相同。在花1(花长1mm)、花2(花长2mm)、花3(花长3mm)、花4(花长4mm)、花5(花长5mm)、花6(花长5.5mm)、花7(花长6mm)、花8(花长7mm)、花9(花长8mm)到即颖花原基分化期至花粉成熟时期的穗中，GMS2的表达量呈现先降低后升高最后再降低的波动。
实施例6 GMS2基因敲除株系的获得及表型分析
利用CRISPR-Cas9系统对GMS2基因进行定向敲除。为了提高敲除效率，选择两个靶位点同时进行敲除。靶位点1位于外显子的负链上，序列为GCGGTCGGTGGCGGCCATGG(SEQ ID NO:17，位于序列SEQ ID NO:1的第45位第64位)，靶位点2位于外显子的上，序列为CGCCTCCCTCGCCGTCGCGG(SEQ  ID NO:18，位于SEQ ID NO:1序列的第85位至第104位)。根据Ma等(Ma X,et al.A Robust CRISPR-Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant,2015,8:1274-84)的方法将靶位点1和靶位点2连入载体pC9M中，获得载体pC9M-GMS2(图13)。有pC9M-GMS2的大肠杆菌被命名为E.coli-pC9M-GMS2。将pC9M-GMS2通过电击转入农杆菌菌株EHA105中，得到的菌株命名为Ab-pC9M-GMS2。
利用重组农杆菌Ab-pC9M-GMS2侵染粳稻中花11(ZH11)愈伤组织，经潮霉素抗性筛选、分化、生根获得再生转基因株系25株。炼苗移栽后得到存活植株22株，提取所述植株叶片的总DNA，利用引物SP1：CCCGACATAGATGCAATAACTTC(SEQ ID NO:29)和SP2：GCGCGGTGTCATCTATGTTACT(SEQ ID NO:30)做阳性检测，全部为株阳性株。用靶位点1两侧的引物靶1-F：AAACCCACGCCCAGAAA(SEQ ID NO:31)和靶1-R：GCCAGGAGGAAGAGCAG(SEQ ID NO:32)以及靶位点2两侧的引物靶2-F：GCCTGCTCTTCCTCCTG(SEQ ID NO:33)和靶2-R：GTGCTCCGGCTGTTGAT(SEQ ID NO:34)扩增基因组DNA，扩增产物测序后与基因组进行比对。结果显示14株T0植株发生了基因编辑，其中一株发生纯合突变，8株T0代苗未被编辑。
植株PC9M-GMS2-Line17的基因组DNA在靶位点1和靶位点2处均发生了纯合突变，其中在靶位点1处发生了TG碱基缺失(SEQ ID NO:27)，在靶位点2处发生了T碱插入(SEQ ID NO:28)(图14B)。PC9M-GMS2-Line1的基因组DNA在靶位点1处发生了双等位突变，其中等位基因1发生了A碱基插入，等位基因2发生了T碱基缺失；PC9M-GMS2-Line1的基因组DNA在靶位点2处也发生了双等位突变，在等位基因1发生了G/T碱基SNP，等位基因2发生了G/C碱基SNP。PC9M-GMS2-Line3的基因组DNA在靶位点1处发生了双等位突变，其中等位基因1发生了TG碱基缺失，等位基因2发生了A碱基插入；PC9M-GMS2-Line3的基因组DNA在靶位点2处也发生了双等位突变，在等位基因1发生了G/T碱基SNP，等位基因2发生了C碱基缺失。而转基因阴性单株PC9M-GMS2-Line2，PC9M-GMS2-Line5，PC9M-GMS2-Line7基因型并未改变(图14A)。
开花后对上述阳性株进行表型分析。与野生型ZH11相比，GMS2敲除植株PC9M-GMS2-Line17在株叶和小穗形态上并无明显差别(图15和图16)。但GMS2敲除植株的花药明显更加瘦小(图17)。花粉碘染结果表明，野生型ZH11的花粉大而圆，可以被染色，而GMS2敲除植株的花粉小而皱缩，不能被染色(图18)。其它GMS2双等位突变植株也表现出雄性不育的性状。
实施例7 GMS2基因超表达株系的获得及表型分析
以9311的RNA反转录产物为模板，用引物3148OX-F CggggtaccATGGCCGCCACCGAC：(SEQ ID NO:35)和3148OX-R：CGCggatccTCACAAGAACGACGC(SEQ ID NO:36)扩增获得带有GMS2完整编码核苷酸序列(SEQ ID NO:2)的DNA片段。将该片段用Kpn I和BamH I双酶切后连入pBLU5获得质粒pUbi1301-48490-CDS(图19)。有pUbi1301-48490-CDS的大肠杆菌被命名为E.coli-pUbi1301-48490-CDS。将pUbi1301-48490-CDS通过电击转入农杆菌菌株EHA105中，得到的菌株命名为Ab-pUbi1301-48490-CDS。
利用重组农杆菌Ab-pUbi1301-48490-CDS侵染粳稻中花11愈伤组织，经潮霉素抗性筛选、分化、生根获得转基因阳性植株6株。使用实时定量PCR方法，利用实施例5中的引物InD48490-F：GCTCCGGCTGTTGATCT(SEQ ID NO:19)和InD48490-R：GCCTGCTCTTCCTCCTG(SEQ ID NO:20)，GAPDH-RTF:GAATGGCTTTCCGTGTT(SEQ ID NO:25)和GAPDH-RTR:CAAGGTCCTCCTCAACG(SEQ ID NO:26)对转基因阳性植株中GMS2的表达量进行分析。如图20所示，和转基因阴性单株2和8相比，超表达植株20和28中GMS2的表达量分别上升了9倍和45倍以上，但超表达植株并未出现与表达量共分离的明显表型，说明GMS2基因超表达对水稻表型并没有明显影响。
实施例8 gms2突变体转基因互补株系的获得及表型分析
以9311的基因组DNA为模板，用引物3148HB-F：CgcgtttcgaaatttTGATTTCTTCATCGCACT(SEQ ID NO:37)和3148HB-R：GtcgcgatcgcatgcACAACATGGTGCAACAGTG(SEQ ID NO:38)扩增获得带有GMS2起始密码子ATG上游2kb和终止密码子TGA下游515bp的基因全长片段。将该片段用Kpn I和BamH I双酶切后连入pC1300获得质粒pC1300-48490-genome(图21)。有pC1300-48490-genome的大肠杆菌被命名为E.coli-pC1300-48490-genome。将pC1300-48490-genome通过电击转入农杆菌菌株EHA105中，得到的菌株命名为Ab-pC1300-48490-genome。利用重组农杆菌Ab-pC1300-48490-genome侵染gms2突变体愈伤组织，经抗性筛选、分化、生根共获得转基因阳性植株4株，4株育性均恢复正常(图22、图23、图24和图25)。这进一步证明GMS2基因调控花粉发育，该基因突变会导致花粉败育。
实施例9 GMS2编码蛋白与其同源蛋白的序列比对及进化树分析
利用blastp工具在NCBI的Genbank数据库中对水稻GMS2基因编码蛋白的氨基酸序列进行同源性搜索，得到了琴叶拟南芥(Arabidopsis lyrata)、香蕉(Musa acuminata)、非洲栽培稻(Oryza glaberrima)、短药野生稻(Oryza brachyantha)、大麦(Hordeum vulgare)、高粱(Sorghum bicolor)、玉米(Zea mays)、小米(Setaria italica)基因组中预测的同源蛋白，将这些蛋白序列进行比对分析，结果显示来自不同植物的同源蛋白都具有非常相似的保守序列，彼此之间同源性很高(图26和图27)，表明该蛋白在植物花的雄性器官发育过程中生物学功能保守，起着非常重要的作用。
在琴叶拟南芥(Arabidopsis lyrata)中该育性基因的氨基酸序列如SEQ ID NO:9所示；香蕉(Musa acuminata)中该育性基因的氨基酸序列如SEQ ID NO:10所示；非洲栽培稻(Oryza glaberrima)中该育性基因的氨基酸序列如SEQ ID NO:11所示；短药野生稻(Oryza brachyantha)中该育性基因的氨基酸序列如SEQ ID NO:12所示；大麦(Hordeum vulgare)中该育性基因的氨基酸序列如SEQ ID NO:13所示：高粱(Sorghum bicolor)中该育性基因的氨基酸序列如SEQ ID NO:14所示；玉米(Zea mays)中该育性基因的氨基酸序列如SEQ ID NO:15所示；小米(Setaria italica)中该育性基因的氨基酸序列如SEQ ID NO:16所示。
实施例10 转育带有GMS2基因的隐性核不育系
用gms2突变体与育性正常的受体，如H28B，进行杂交、回交和自交，并在此过程中用分子标记进行gms2基因和遗传背景选择，最终获得H28B背景下带有纯合GMS2突变基因的隐性核不育系。具体实施步骤如下：
1、以受体亲本，如H28B，为父本与gms2杂交获得F 1。
2、以F 1为母本与受体亲本，如H28B，回交获得BC 1F 1。
3、种植BC 1F 1，使用引物InD48490_F：GCTCCGGCTGTTGATCT(SEQ ID NO:19)和InD48490_R：GCCTGCTCTTCCTCCTG(SEQ ID NO:20)检测gms2基因型。选择gms2杂合基因型，即同时能扩增出149bp和140bp条带的植株。
4、使用一组(例如100个，或200个等)基因型在gms2突变体和轮回亲本基因组之间存在多态性，且分布均匀的分子标记(可以是但不限于SSR、SNP、INDEL、EST、RFLP、AFLP、RAPD、SCAR等类型标记)，对步骤3中选出的单株进行遗传背景鉴定，选取与轮回亲本基因型相似度高(如大于88％相似度，或2％中选率等)的植株。
5、用步骤4中选出的植株与受体亲本，如H28B，回交获得BC 2F 1。
6、种植BC 2F 1，重复步骤3和步骤4，选出gms2基因型杂合，遗传背景回复率高(如大于98％，或2％中选率等)的植株，收自交种BC 2F 2。
7、种植BC 2F 2，重复步骤3和步骤4，选出gms2基因型杂合，遗传背景纯合率最高的植株，收自交种BC 2F 3。BC 2F 3后代中分离的gms2纯合株即gms2隐性核不育系，BC 2F 3用于保存gms2隐性核不育系种质资 源。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种水稻雄性育性调控基因、其应用以及利用CRISPR-Cas9调控水稻育性的方法；以及一种水稻雄性育性调控基因突变体及其分子标记和应用。本发明提供具有调控水稻雄性生殖细胞发育和花粉育性功能的水稻基因GMS2，其核苷酸序列如SEQ ID NO:1所示，CDS序列如SEQ ID NO:2所示，氨基酸序列如SEQ ID NO:3所示。本发明提供的水稻雄性育性调控蛋白GMS2突变体可使水稻花粉完全不育，导致水稻雄性不育，GMS2突变体的基因组核苷酸序列如SEQ ID NO:6所示，CDS序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:8所示。利用本发明的核不育突变体可培育新的核不育系，为水稻核不育系培育提供了一个简单、迅速、有效的方法。本发明的核不育水稻材料可用于在水稻杂交时取代人工去雄，节约劳力，尤其可用于需要进行大量杂交的轮回选择育种，对扩大水稻的种质基础具有重要作用。本发明提供的水稻基因GMS2及其突变体可用于水稻杂交种的不育化制种和生产，具有巨大的应用价值和经济价值。

Claims (20)

  1. 植物雄性育性相关蛋白，其特征在于，所述植物雄性育性相关蛋白为如下(1)或(2)所述的蛋白：
    (1)具有SEQ ID NO:3、9、10、11、12、13、14、15或16所示氨基酸序列的蛋白；
    (2)将SEQ ID NO:3、9、10、11、12、13、14、15或16经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有调控植物雄性育性活性的蛋白。
  2. 一种核酸，其特征在于，所述核酸编码权利要求1所述植物雄性育性相关蛋白；
    优选地，当所述植物雄性育性相关蛋白来源于水稻时，所述核酸为以下任一：
    (1)具有SEQ ID NO:1或2所示的核苷酸序列的核酸；
    (2)具有SEQ ID NO:4或69所示的核苷酸序列的核酸；
    (3)在严格条件下能够与(1)、(2)之任一所述序列的DNA杂交的DNA片段；
    (4)与(1)、(2)之任一所述序列互补的DNA片段；
    (5)在(1)、(2)之任一所述序列的基础之上，经过一至数个碱基的替换和/或一至数个碱基的插入和/或缺失，或经过大片段的核苷酸序列插入/缺失/易位/倒位所形成能够影响植物花粉生育能力的DNA片段；
    (6)与(1)、(2)之任一所述序列的DNA片段具有85％、90％、95％、96％、97％、98％或99％以上的同一性且编码水稻雄性育性相关蛋白的DNA片段。
  3. 植物雄性育性相关蛋白突变体，其特征在于，所述野生型植物雄性育性相关蛋白同权利要求1中所述；所述突变体相对于野生型植物雄性育性相关蛋白包含如下氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸中的至少一个的缺失；
    其中，x为S或N。
  4. 根据权利要求3所述的突变体，其特征在于，所述突变体相对于野生型植物雄性育性相关蛋白包含如下氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸的缺失；其中，x为S或N；
    优选地，当所述野生型植物雄性育性相关蛋白来源于水稻时，所述突变体相对于所述野生型植物雄性育性相关蛋白包含如下氨基酸突变：第40、41和42位氨基酸缺失；
    更优选地，当所述野生型植物雄性育性相关蛋白来源于水稻时，所述突变体具有如SEQ ID NO.8所示的氨基酸序列。
  5. 编码权利要求3或4所述突变体的核酸；
    优选地，当所述野生型植物雄性育性相关蛋白来源于水稻时，编码所述突变体的核酸相对于编码所述野生型植物雄性育性相关蛋白的核酸包含如下核苷酸突变：对应于LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失；
    更优选地，当所述野生型植物雄性育性相关蛋白来源于水稻时，编码所述突变体的核酸的基因组序列如SEQ ID NO:6所示，CDS序列如SEQ ID NO:7所示。
  6. 一种生物材料，其特征在于，所述生物材料包含权利要求2或2所述核酸或权利要求2或5所述核酸的抑制因子，所述生物材料为表达盒、载体、宿主细胞、转基因细胞系或转基因植物。
  7. 一种植物、植物组织或植物细胞，其特征在于，所述植物、植物组织或植物细胞表现为雄性不育性状，由权利要求2所述核酸的突变所造成，所述突变为一个或多个核苷酸的缺失、插入或取代突变，或为通过反义基因的转入、共抑制或发夹结构的引入所产生的突变；所述突变导致权利要求1所述的植物雄性育性相关蛋白的表达量降低、不表达或失活；
    优选地，所述植物、植物组织或植物细胞为采用CRISPR-Cas9方法获得，所述CRISPR-Cas9方法使用的靶序列位于权利要求2所述核酸的序列中，靶序列的反向互补序列具有5’-(N)X-NGG-3’结构，其中，N表示 A，T，C和G中的任意一个，X为19或20nt的任意核苷酸序列。
  8. 根据权利要求7所述的植物、植物组织或植物细胞，其特征在于，其为采用CRISPR-Cas9方法以GCGGTCGGTGGCGGCCATGG和CGCCTCCCTCGCCGTCGCGG为靶位点得到的在靶位点或靶位点相邻区域发生突变的植物、植物组织或植物细胞。
  9. 一种植物、植物组织或植物细胞，其特征在于，其具有雄性不育性状，其基因组序列中野生型植物雄性育性相关蛋白的基因编码的蛋白发生包含如下的氨基酸突变：在NxYL保守序列中发生N、x、Y三个氨基酸中的至少一个的缺失；所述野生型植物雄性育性相关蛋白同权利要求1中所述；
    优选地，所述植物为水稻，所述植物组织为水稻组织，所述植物细胞为水稻细胞，所述水稻、水稻组织或水稻细胞的基因组或转录组包含如下突变：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失；LOC_Os04g48490基因编码区的序列如SEQ ID NO.69所示，LOC_Os04g48490基因编码蛋白的序列如SEQ ID NO.3所示；
    更优选地，所述水稻、水稻组织或水稻细胞的基因组序列中LOC_Os04g48490基因的序列突变为如SEQ ID NO:6所示的序列，导致LOC_Os04g48490基因的CDS突变为如SEQ ID NO:7所示序列，编码蛋白突变为如SEQ ID NO:8所示序列。
  10. 权利要求1所述植物雄性育性相关蛋白或权利要求3或4所述突变体或权利要求2或5所述核酸或权利要求2或5所述核酸的抑制因子或权利要求6所述生物材料或权利要求7-9中任一项所述的植物、植物组织或植物细胞的如下任一种应用：
    (1)在调控植物雄性育性中的应用；
    (2)在制备雄性不育植物中的应用；
    (3)在植物杂交育种中的应用；
    (4)在植物种质资源改良中的应用。
  11. 一种适用于CRISPR-Cas9方法对权利要求2所述核酸进行定向敲除的靶位点，其特征在于，其为GCGGTCGGTGGCGGCCATGG和/或CGCCTCCCTCGCCGTCGCGG。
  12. 特异性靶向权利要求11所述靶位点的sgRNA。
  13. 含有权利要求12所述sgRNA的DNA序列的CRISPR-Cas9打靶载体。
  14. 权利要求11所述靶位点或权利要求12所述sgRNA或权利要求13所述CRISPR-Cas9打靶载体的如下任一种应用：
    (1)在调控植物雄性育性中的应用；
    (2)在制备雄性不育植物中的应用；
    (3)在植物杂交育种中的应用；
    (4)在植物种质资源改良中的应用。
  15. 一种制备雄性不育植物的方法，其特征在于，使植物中权利要求1所述的植物雄性育性相关蛋白的表达量降低、不表达或失活。
  16. 一种获得权利要求2所述核酸在植物中的直系同源基因片段的方法，其特征在于，包括：使用权利要求2所述核酸在核苷酸数据库中进行blastx搜索，所有Identities大于或等于35％、Positives大于或等于50％的核苷酸序列即为与所述核酸直系同源的基因片段。
  17. 用于检测权利要求3或4所述突变体或权利要求9所述植物、植物组织或植物细胞的分子标记，其特征在于，通过核苷酸序列如SEQ ID NO:19-20所示的引物扩增得到。
  18. 含有核苷酸序列如SEQ ID NO:19-20所示的引物的检测试剂或试剂盒。
  19. 权利要求17所述分子标记或权利要求18所述检测试剂或试剂盒的如下任一种应用：
    (1)在检测权利要求3或4所述突变体或权利要求9所述植物、植物组织或植物细胞中的应用；
    (2)在筛选或培育雄性不育水稻中的应用；
    优选地，当选用SEQ ID NO:19-20所示引物扩增水稻基因组DNA时，若只能扩增出140bp一条带，则水稻表达权利要求4所述的突变体、具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的纯合基因型且表现出雄性不育性状；若只能扩增出149bp一条带，则水稻不表达权利要求4所述的突变体、不具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的基因型且表现出雄性可育性状；若能同时扩增出140bp和149bp两条带，则水稻表达权利要求4所述的突变体、具有LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失的杂合基因型且表现出雄性可育性状。
  20. 一种制备雄性不育植物的方法，其特征在于，包括：使野生型植物雄性育性相关蛋白发生突变，所述突变包含如下氨基酸的突变：在NxYL保守序列中发生N、x、Y三个氨基酸中的至少一个的缺失；所述野生型植物雄性育性相关蛋白同权利要求1中所述；
    优选地，所述植物为水稻，所述方法包括：使水稻的基因组或转录组发生突变，所述突变包含：LOC_Os04g48490基因编码区的第118位～126位的AACAGCTAC碱基发生缺失，导致LOC_Os04g48490基因编码蛋白的第40、41和42位的天冬酰胺、丝氨酸和酪氨酸缺失；LOC_Os04g48490基因编码区的序列如SEQ ID NO.69所示，LOC_Os04g48490基因编码蛋白的序列如SEQ ID NO.3所示；
    更优选地，所述方法包括：使水稻表达序列如SEQ ID NO:8所示的雄性育性相关蛋白突变体，且不表达SEQ ID NO:3所示的野生型植物雄性育性相关蛋白。
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