WO2020042412A1 - 一种水稻育性调控基因及其突变体与应用 - Google Patents

一种水稻育性调控基因及其突变体与应用 Download PDF

Info

Publication number
WO2020042412A1
WO2020042412A1 PCT/CN2018/119270 CN2018119270W WO2020042412A1 WO 2020042412 A1 WO2020042412 A1 WO 2020042412A1 CN 2018119270 W CN2018119270 W CN 2018119270W WO 2020042412 A1 WO2020042412 A1 WO 2020042412A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
gene
gms1
sequence
nucleotide sequence
Prior art date
Application number
PCT/CN2018/119270
Other languages
English (en)
French (fr)
Inventor
黄培劲
龙湍
唐杰
刘昊
李佳林
李新鹏
安保光
曾翔
吴永忠
Original Assignee
海南波莲水稻基因科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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
Publication of WO2020042412A1 publication Critical patent/WO2020042412A1/zh

Links

Images

Classifications

    • 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
    • A01H5/02—Flowers
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a plant fertility regulation gene GMS1, a protein encoded by GMS1, a radiation mutagenesis and gene knockout mutant of GMS1, and application of the GMS1 gene, protein, and mutant in hybrid breeding.
  • Hybrid rice is the offspring obtained after parental crosses, and its yield is often more than 15% higher than that of conventional rice parents, and the resistance and adaptability are far better than the parents. Therefore, the application and promotion of hybrid rice is an important way to increase rice yield.
  • Male sterile lines are the key nodes of hybrid rice breeding technology.
  • Male sterile lines refer to plant lines in which the male gamete develops abnormally and loses fertility, and the female gamete develops normally. It can only accept the pollen of the father as the mother, and it cannot be strong.
  • nucleoplasm interaction type there are two types of male sterility lines used in hybrid rice production: nucleoplasm interaction type and light temperature sensitive type.
  • Sterile genes of cytoplasmic male-sterile lines are in the cytoplasm, and there is no fertility restoration gene in the nucleus.
  • the nuclear-plasma-interacting sterile line is the first large-scale sterile line used in hybrid rice breeding, laying a material foundation for the establishment and development of the hybrid rice industry.
  • the karyoplasmic interaction type CMS lines are limited, so only about 5% of the germplasm resources can be used.
  • the cytoplasmic sterility gene has the potential risk of causing poor rice quality and the spread of specific diseases and insect pests.
  • thermo-sensitive male-sterile line is a kind of sterile line whose fertility is regulated by the light-temperature environment. Under certain conditions of light and temperature, this type of sterile line remains sterile and can be used for mating crosses. When conditions change, the sterile line restores fertility and can be used for sterile line reproduction. Since the thermo-sensitive male sterility line realizes the merging of the sterility line and the maintainer line into one, only the male parent and its mating pair are required to produce the offspring hybrid, so the corresponding breeding technology is often called "two-line method" ". Genes that regulate light and temperature-sensitive male sterility are in the nucleus.
  • thermo-sensitive CMS lines have greatly consolidated and promoted the development of hybrid rice industry. However, because the fertility of this type of sterile line is affected by the light and temperature environment, it also leads to high risk of seed production, and the area of seed production is limited.
  • the invention provides a crop fertility gene and a male sterility line of recessive nuclear sterility type generated based on the mutation of the gene.
  • the sterility of this sterility line is stable, it is only regulated by a single gene encoded by the nuclear, and it is not affected by the light and temperature environment.
  • the fertility restoring gene of this sterile line is widely present in rice germplasm resources, and fertility can also be restored by transducing wild type genes.
  • the gene and the sterile line produced by the gene mutation provide elements for the development of new hybrid rice breeding technology and lay the foundation for solving the problems existing in the existing technology.
  • the invention provides a fertility-related gene and its nucleotide and protein sequences, and also includes the application of manipulating the gene in regulating male fertility of a plant.
  • any of the methods described below can be used with the corresponding nucleotide sequence provided by the present invention, for example, introducing a mutant sequence of the fertility gene into a plant to cause plant male sterility, Mutating the endogenous sequence of the plant, introducing the antisense sequence of the sequence into the plant, using a hairpin form, or linking it with other nucleotide sequences to regulate the phenotype of the plant, or can be used by those skilled in the art to know Any of a number of methods that affect the male fertility of a plant.
  • a first aspect of the present invention is to provide a pollen development regulating gene GMS1.
  • GMS1 is located on rice chromosome 2. Its genomic nucleotide sequence in japonica is shown as SEQ ID NO: 1, CDS sequence is shown as SEQ ID NO: 2, and its amino acid sequence is shown as SEQ ID NO: 3. The genome nucleotide sequence of indica rice is shown in SEQ ID NO: 4, and its CDS and amino acid sequence are the same as those of japonica rice.
  • the genomic nucleotide sequence of the fertility gene in Arabidopsis thaliana is shown in SEQ ID NO: 26, the CDS sequence is shown in SEQ ID NO: 27, and the amino acid sequence is shown in SEQ ID NO: 28;
  • the genomic nucleotide sequence of the fertility gene in Huawei (Setaria italica) is shown in SEQ ID NO: 29, the CDS sequence is shown in SEQ ID NO: 30, the amino acid sequence is shown in SEQ ID NO: 31;
  • sorghum The genomic nucleotide sequence of the fertility gene in Sorghum (biorg) is shown in SEQ ID NO: 32, the CDS sequence is shown in SEQ ID NO: 33, the amino acid sequence is shown in SEQ ID NO: 34; in Zeamays
  • the genomic nucleotide sequence of the fertility gene is shown in SEQ ID NO: 35, the CDS sequence is shown in SEQ ID NO: 36, the amino acid sequence is shown in SEQ ID NO: 37; Brachypodium distachyon The genomic nucleot
  • the fertility restoration gene according to the present invention includes a highly homologous functional equivalent sequence that is highly homologous to the GMS1 gene and has the same fertility regulatory function.
  • the highly homologous functional equivalent sequence includes a DNA sequence capable of hybridizing to the nucleotide sequence of the GMS1 gene disclosed in the present invention under stringent conditions.
  • the "stringent conditions" used in the present invention are well known and include, for example, hybridization in a hybridization solution 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 functional equivalent sequence also includes at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the sequence shown by the GMS1 gene disclosed in the present invention, and has fertility regulation
  • Functional DNA sequences 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 Mol Biol, 48 ( 3): 443-453, 1970), Smith-Waterman local comparison method (Jol 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.
  • a DNA fragment of the present invention having the function of regulating male fertility of a plant has a sequence of any of the following:
  • the DNA fragment is the GMS1 gene or a specific fragment thereof.
  • the present invention provides a protein encoded by the DNA fragment, which is the protein described in 1) or 2) below:
  • SEQ ID NO: 3, 28, 31, 34, 37, 40, 43, 46, or 49 is substituted and / or deleted and / or added by one or several amino acid residues and has the ability to regulate plant male fertility Of protein.
  • the present invention provides a biological material containing the above-mentioned DNA sequence.
  • the biological material is an expression cassette, an expression vector, an engineered bacterium, a transgenic plant, or a transgenic cell line.
  • the DNA fragment according to the present invention can be isolated from any plant, including but not limited to Brassica, corn, wheat, sorghum, short-medicine wild rice, African cultivated rice, Brachypodium, Two-segment genus, White mustard , Grass seed, sesame, cottonseed, flaxseed, soybean, Arabidopsis, kidney bean, peanut, indica, oat, rapeseed, barley, oat, rye (Rye), millet, milo, triticale, single Grain wheat, Spelt, double grain wheat, flax, Gramma grass, friction grass, false mimosa, fescue, perennial wheatgrass, ganlian, cranberry moss, papaya, Banana, safflower, oil palm, cantaloupe, apple, cucumber, stone gladiator, gladiolus, chrysanthemum, lily family, cotton, school, sunflower, brassica, beet, coffee, ornamental plant and pine.
  • the plant includes corn, millet, Arabidopsis thaliana, Brachypodium spp., Soybean, safflower, mustard, wheat, barley, rye, short-medicine wild rice, African cultivated rice, cotton and sorghum.
  • a second aspect of the present invention is to provide a method for affecting plant fertility by affecting the nucleotide sequence of GMS1 or by regulating the transcriptional expression of the GMS1 gene.
  • the influencing plant fertility refers to changing the fertility of the plant by regulating the expression of the GMS1 gene, such as causing male sterility of the plant.
  • GMS1 gene in plants, thereby achieving the effect of regulating male fertility of plants.
  • regulating the expression of the GMS1 gene can be performed using many tools available to one of ordinary skill in the art, for example, by physicochemical mutagenesis, insertion mutations, gene knockout, transfer of antisense genes, co-suppression, or hairpins
  • the introduction of structures can be used to disrupt the normal expression of the GMS1 gene, thereby obtaining male-sterile plants.
  • the present invention also includes restoring male fertility of a plant in which GMS1 expression is disrupted by introducing a nucleotide sequence of wild-type GMS1 into a plant.
  • a third aspect of the present invention provides a DNA molecule containing the DNA fragment, and inserting, and / or deleting, and / or replacing several nucleotides in the DNA fragment, thereby causing rice male fertility to occur change.
  • the present invention provides a sterile mutant sequence of the GMS1 gene and its male sterile mutant material.
  • the male sterility mutant material is caused by a mutation of a nucleotide sequence, and a plant containing the nucleotide sequence after the mutation exhibits male sterility, and the nucleotide sequence is as shown in SEQ ID NO: 1,2, As shown in 4, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, or 48, the mutations are point mutations, DNA deletions, insertions Or substitution mutations, transfer of antisense genes, co-suppression or introduction of hairpin structures or mutations generated by gene silencing methods.
  • the male sterility mutant material is a process in which the plant body loses male fertility by mutating the endogenous GMS1 gene of rice, or mutating the nucleotide sequence of a gene that is highly homologous to the gene.
  • the "mutation” includes, but is not limited to, a method such as a genetic mutation caused by a physical or chemical method, and the mutation may be a point mutation, or a DNA deletion or insertion mutation. Mutations can also be generated by gene silencing methods such as RNAi, CRSPR-Cas9, artificial nucleases, and site-directed mutations.
  • the mutant material is a CRISPR-Gas9 method, with the sequence CTTCGTGATCGGCGACTCCA (target site 1) and / or the sequence GAGAGAATGCCAGCTGCCG (target site 2) as target sites, so that A target site or a plant obtained by mutating a target site and an adjacent nucleotide sequence.
  • the plants include, but are not limited to, Brassica, corn, wheat, sorghum, Brassica rapa, African cultivated rice, Brassica spp., Two knots, white mustard, grass seed, sesame, cottonseed, flax, soybean , Arabidopsis, Phaseolus, Peanuts, Indica, Oats, Rapeseed, Barley, Oats, Rye, Millet, Shuyu, Triticale, Single Wheat, Spelt, Double-grain wheat, flax, Gramma grass, friction grass, false mimosa, fescue, perennial wheatgrass, ganlian, cranberry moss, papaya, banana, safflower, oil palm, cantaloupe, apple, Cucumber, Gladiator, Gladiolus, Chrysanthemum, Liliaceae, Cotton, School, Sunflower, Brassica, Beet, Coffee, Ornamental Plant and Pine.
  • it includes corn, millet, Arabidopsis thaliana, Brachypodium spp., Soybean, safflower, mustard, wheat, barley, rye, short-medicine wild rice, African cultivated rice, cotton and sorghum.
  • the mutant material is a rice mutant material, and has the following 1) in the target site 1 and target site 2 or the target site and adjacent regions of the rice genome, and / Or 1 or more mutations in 2), such as 2, 3, and 4 mutations:
  • the present invention provides a target site suitable for directional knockdown of a plant GMS1 gene by the CRISPR / Cas9 system, which is CTTCGTGATCGGCGACTCCA (target site 1) and / or GAGAGAATGCCAGCTGCCG (target site 2).
  • the invention also provides an sgRNA that specifically targets the above-mentioned target site 1 or target site 2.
  • a CRISPR / Cas9 targeting vector containing the DNA sequence of the sgRNA belongs to the protection scope of the present invention.
  • the present invention provides a rice male sterility mutant, which contains a mutated male sterility gene, and the genomic nucleotide sequence of the mutated male sterility gene is shown in SEQ ID NO: 5
  • the CDS sequence is shown in SEQ ID NO: 6, and the amino acid sequence is shown in SEQ ID NO: 7.
  • Alignment of the wild type and mutant sequences revealed that the TTGT at base 3600 of exon 3 of the coding region of the genome sequence of the LOC_Os02g18870 gene was replaced with A, resulting in the 230th and Leucine and valine at position 231 are mutated to a histidine, which causes the protein to lose function.
  • the nucleotide sequence SEQ ID NO: 5 can be introduced into the recipient plant by crossing, backcrossing or transgenic methods, thereby obtaining new male sterile mutant material.
  • the invention provides an application of the GMS1 gene or a protein encoded by the GMS1 gene or a biological material containing the GMS1 gene or a mutant material thereof in regulating male fertility of a plant.
  • the biological material is an expression cassette, an expression vector, an engineered bacterium, a transgenic plant, or a transgenic cell line containing the GMS1 gene.
  • the GMS1 gene is used for restoring male sterility in crop plants in order to introduce foreign genes to obtain high-quality transgenic crops.
  • the improvement includes improvement of growth traits such as increased yield, improved quality, resistance to diseases and insect pests, stress resistance, lodging resistance and the like.
  • the crop is a self-pollinated or cross-pollinated crop.
  • the crop includes, but is not limited to, corn, wheat, sorghum, rice.
  • the present invention also provides the GMS1 gene or its encoded protein or biological material or DNA molecule containing the same or any of the mutant materials described above in the transmission of a recessive nuclear sterility line with a homozygous GMS1 mutant gene. application.
  • the invention also provides a method for obtaining an orthologous gene of the GMS1 gene in other plants, and an amino acid sequence of the sorghum, millet, brachypodium, and rice homologous GMS1 by using the method.
  • the invention provides a method for obtaining an orthologous gene of the GMS1 gene in other plants, including:
  • the present invention provides a molecular marker closely linked to the GMS1 gene, which is obtained by amplifying a nucleotide sequence such as the primers described in SEQ ID NO: 8-9.
  • the present invention provides a molecular marker for detecting GMS1 gene mutant material, which is obtained by amplifying a nucleotide sequence such as the primers described in SEQ ID NO: 10-11.
  • the rice to be tested was a GMS1 gene mutant.
  • a detection reagent or a kit containing a primer having a nucleotide sequence such as SEQ ID NO: 8-9 or SEQ ID ID NO: 10-11 belongs to the protection scope of the present invention.
  • the present invention provides an application of the above-mentioned two kinds of molecular markers or the above-mentioned detection reagent or kit in detecting a rice GMS1 gene mutant.
  • the invention provides the application of the above two molecular markers or the above detection reagents or kits in screening or breeding male sterile rice mutants.
  • the rice pollen development regulation gene GMS1 provided by the present invention directly participates in the regulation of pollen development. After the gene is knocked out or the expression is suppressed, the pollen is completely sterile. Compared with the existing three-line and two-line sterile lines, the sterile traits of gms1 sterile mutants are stable and are not affected by environmental conditions. Through the plant biotechnology approach, the present invention will play an important role in the utilization of heterosis in crops and in the production of sterile hybrid seed production.
  • Figure 1 Plant morphology of wild-type (left) and gms1 mutants (right) at the filling stage.
  • Figure 3 Morphology of flowering of spikes of wild type (left) and gms1 mutant (right).
  • Figure 4 Floret morphology of wild type (left) and gms1 mutant (right) after dissection.
  • Figure 6 Pollen iodine staining of wild type (left) and gms1 mutants (right).
  • Figure 7 uses Indel3 markers to identify genotypes of sterile plants in a localized population.
  • the upper band is 124bp and the lower band is 120bp.
  • the DNA templates for lanes 1-4 are the gms1 mutant, Minghui 63, which locates the sterile plants 2.5 and 2.6 in the population.
  • FIG. 8A is a clone map of the GMS1 gene map
  • FIG. 8B is a schematic diagram of the mutation site of the gms1 mutant.
  • Figure 9 Nucleotide sequence differences of the GMS1 gene in the materials of 9311 (9311-870), Minghui 63 (MH63-870), Nippon Harumi (Nip-870) and gms1 mutant (gms1-870). Differences are highlighted with a black background. The position of the last nucleotide in each line in the entire gene sequence is indicated at the end of the line.
  • FIG. 10 Difference in amino acid sequence of GMS1 encoded protein in 9311 (9311-870) and gms1 mutant (gms1-870). Differences are highlighted with a black background. The position of the last amino acid residue in each line throughout the protein sequence is indicated at the end of the line.
  • FIG. 11 Genotype identification of GMS1 heterozygous offspring.
  • the upper band is 111bp and the lower band is 108bp.
  • the arrow indicates the sterile DNA.
  • FIG. 12 GMS1 expression in different tissues of rice.
  • FIG. 13 Schematic diagram of pC9M-GMS1 vector.
  • T1 represents target site 1 and T2 represents target site 2.
  • Figure 14 Sequencing peak map of transgenic plant L138-1-1 at target site 1. The arrow points to the missing site.
  • Figure 15 Sequencing peak map of transgenic plant L138-3-1 at target site 2. The arrow points to the missing site.
  • FIG. 16 Spikelet morphology of GMS1 wild type (left), knockout plants L138-1-1 (middle) and L138-3-1 (right).
  • FIG. Anther morphology of GMS1 wild type (left), knockout plants L138-1-1 (middle) and L138-3-1 (right).
  • FIG. 1 Pollen iodine staining of GMS1 wild type (left), knockout plants L138-1-1 (middle) and L138-3-1 (right).
  • Figure 19 Schematic diagram of pBLU5-870-CDS vector.
  • Figure 21 Schematic diagram of pC2300-870-genome vector.
  • Figure 22 Plant morphology of gms1 mutant plants (left) and complementary plants (right).
  • FIG. 23 Sequence comparison of the protein encoded by the rice GMS1 gene and homologous proteins in the genomes of other species. Includes Arabidopsis thaliana protein AT4G10950.1, Setaria italica protein Si019557m, Sorghum bicolor protein Sb04g011320.1, Zea mays protein GRMZM2G166330_P01, Brachypodium distachyon1 BRA protein .1. Barley (Hordeum vulgare) protein MLOC_10139.2, short-dried wild rice (Oryza brachyantha) protein OB02G21480.1, African cultivated rice (Oryza gliberrima) protein ORGLA02G0115000.1.
  • test materials are test materials commonly used in the field of the present invention.
  • test reagents used in the following examples were purchased from conventional biochemical reagent stores.
  • the male sterility described in the present invention refers to the abnormal development of the male reproductive organs of the plant due to functional changes in plant cell nuclear genes (the inability to produce normal stamens, anthers, or normal male gametophytes) and the loss of fertility, which is commonly known as Gento male sterility rather than Cytoplasmic male sterility.
  • the abnormality and recovery of fertility in male reproductive organs are controlled by genes in the nucleus.
  • the present invention also includes the use of 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 proteomic level.
  • the function of the source gene thus achieves the purpose of controlling the fertility of male reproductive organs.
  • the following methods are not limited to the following methods: Inhibition of gene expression or loss of protein function by mutation of a natural sequence, transfer of an antisense sequence of the gene into a plant or introduction of a hairpin structure, or the gene Combined with other sequences (DNA or RNA) to generate new functionally active DNA or RNA strands to affect or change the function of plant genes.
  • the invention includes the rice GMS1 gene, the dominant allele of which has a key role in male flower fertility of the plant, and the recessive allele with loss of function will cause male sterility.
  • This gene is located on rice chromosome 2 and the specific location of the gene is shown in Figures 8A and 8B.
  • the gene sequence and its homologous sequences can be obtained from various plants, including but not limited to Selaginella moellendorffii, populus trichocarpa, Brassica rapa, Arabidopsis lyrata ), Arabidopsis thaliana, Glycine max, Solanum tuberosum, Vitis vinifera, Musaacuminata, Millet (Setaria italica), Sorghum bicolor , Zea mays, Brachypodium distachyon, Hordeum vulgare, Oryza brachyantha, Oryza glaberrima, Oryza sativa, Indica group, Japonica rice Oryza (sativa, Japonica, Group), Physcomitrella (pahys) and others.
  • Obtaining methods include, but are not limited to: using blastx, blastn, or amino acid sequence using blastp from rice GMS1 gene sequence to retrieve from the genome sequence database, and / or cDNA sequence database, and / or protein sequence database of other plants; using rice GMS1
  • the DNA or cDNA or RNA sequence of the gene is used as a reference sequence to design primers, which can be directly obtained from the genomic DNA or cDNA or RNA of other plants by PCR: design probes based on the rice GMS1 gene sequence and use nucleic acid hybridization from the genomic library Isolate DNA or cDNA or RNA fragments containing homologous gene sequences.
  • the homologous sequence of the GMS1 gene refers to the DNA sequence of a plant gene having Identities greater than or equal to 35% and Positives greater than or equal to 50% after performing a blastx comparison analysis with the amino acid sequence of SEQ ID NO: 3.
  • All parameters follow the default settings shown at http://blast.ncbi.nlm.nih.gov/.
  • the gms1 mutant plants (Fig. 1) and spikelets (Fig. 2) were normal in morphology and slightly later in flowering. There were no significant differences between the size of the inner and outer ridges, the opening size of the floret, and the opening time compared with the wild type ( Figure 3). Observing the morphology of mutant florets under a stereo microscope, it was found that the ovary, style, and stigma were slightly larger than the wild type ( Figure 4), but the anthers were thinner and lighter than the wild type ( Figure 5). Pollen was stained with iodine-potassium iodide solution (0.6% KI, 0.3% I 2 , w / w) solution. As shown in FIG.
  • mutant pollen grains were large and round and stained blue-black, while mutant pollen grains were wrinkled. Shrink and cannot be stained. Wild-type plants from the same family were bagging normally after self-bagging, but the 3013 mutant was not. Pollination of the 3013 mutant with the rice cultivar 93-11 was firm. This indicates that the mutant is a male-sterile mutant.
  • Map-based cloning was used to map the GMS1 gene.
  • An F 2 population consisting of 350 plants was constructed by crossing Minghui 63 as the male parent and the gms1 mutant. This population was used to locate GMS1 between chromosome 2 SSR markers RM13004 and RM13018, and was co-segregated from SSR marker RM13011. GMS1 gene and the above three markers are one, zero and one.
  • a gms1 heterozygous single plant in the F 2 population was selected using linkage markers to develop an F 3 population containing 7,832 individual plants. In the F 3 population, there were 9, 3, and 13 exchanged individual plants between the RM13004, RM13011, RM13018, and GMS1 genes, respectively.
  • Indel3 By analyzing and comparing the sequences of the 93-11 and Nipponbare genomes between RM13004 and RM13018, a new insertion and deletion marker Indel3 was developed and experimentally confirmed.
  • the primers used to detect Indel3 were Indel3-F: GGGAAGAAACAGAGGTGCC (SEQ ID NO: 8) and Indel3-R: GCTTGGAGGTATTTGGGACA (SEQ ID NO: 9).
  • Indel3-F and Indel3-R one 120bp (sequence such as SEQ ID NO: 62) and one 124bp (sequence such as SEQ ID NO: 63) can be amplified in the gms1 mutant and Minghui 63 genomic DNA, respectively.
  • the nucleotide sequence of the LOC_Os02g18870 genome is 5604bp in length (denoted as Nip-870, the sequence is as SEQ ID NO: 1), and the CDS nucleotide sequence is 1,200bp in length (the sequence is as SEQ ID NO: 2).
  • Figure 8B 4 introns ( Figure 8B), encoding a protein containing 399 amino acid residues (sequence such as SEQ ID NO: 3).
  • the primers were designed based on the Nip-870 sequence to amplify and sequence the alleles of the LOC_Os02g18870 gene in the 93-11, Minghui 63, and gms1 mutants.
  • the primer sequences are shown in Table 1. All PCR amplifications were performed using KODFX DNA DNA Polymerase (TOYOBO CO., LTD. Life Science Department, Osaka, Japan), and PCR amplification was performed on a Thermo Scientific Arktik thermal thermal cycler in accordance with the reaction system and conditions described in the product description.
  • the PCR products were sent to Nanjing Kingsray Biotechnology Co., Ltd. for sequencing.
  • the sequencing results were spliced with DNAman 6.0.
  • the LOC_Os02g18870 gene in the 93-11, Minghui 63, and gms1 mutants were recorded as 9311-870 (sequence such as SEQ ID NO: 4), MH63-870, and gms1-870 (sequence such as SEQ ID NO: 5).
  • the CDS nucleotide sequence of LOC_Os02g18870 is shown in SEQ ID NO: 2, and the coding protein sequence is shown in SEQ ID NO: 3.
  • the CDS nucleotide sequence and amino acid sequence of LOC_Os02g18870 in the gms1 mutant are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
  • the wild type either amplified two bands of 111bp and 108bp or one band of 111bp, and the sterile mutant could only amplify one band of 108bp.
  • LOC_Os02g18870 is the GMS1 gene.
  • Primers 3013_RTF AATTCATCCGCCAGCACC (SEQ ID NO: 12) and 3013_RTR: GCCCACAGCAGGCATCAG (SEQ ID NO: 13) were designed based on the nucleotide sequence of GMS1 genome and coding region, and primers were designed using rice Actin gene as internal control Actin-RTF: GATGCTTATGTCGGTGAT (SEQ ID NO: 14) and Actin-RTR: ATGCTCTTCTGGTGCTAC (SEQ ID NO: 15). Real-time quantitative PCR was used to analyze the expression level. As shown in Fig.
  • the GMS1 gene was expressed in rice roots, stems, leaves, seeds, lemmas, and lemmas, but the expression level was low.
  • the expression of GMS1 increased rapidly in the young ears of the first to third stages, and then gradually decreased until the young ears of the eighth stage. Among them, the expression of GMS1 in the young ears of the seventh stage increased slightly. In addition, GMS1 expression was significantly increased in the anthers of the eighth-phase panicles.
  • Target site 1 is located on the positive strand of the first exon
  • the sequence is CTTCGTGATCGGCGACTCCA (positions 168 to 187 of the SEQ ID NO: 1 sequence)
  • target site 2 is located on the negative strand of the second exon.
  • the sequence is GAGAGAATGCCAGCTGCCG (positions 1629 to 1647 of the SEQ ID NO: 1 sequence).
  • E. coli with pC9M-GMS1 was named E. coli-pC9M-GMS1.
  • PC9M-GMS1 was transformed into human Agrobacterium strain EH105 by electric shock, and the obtained strain was named Ab-pC9M-GMS1.
  • Recombinant Agrobacterium tumefaciens Ab-pC9M-GMS1 was used to infect japonica rice 11 callus, and 33 hygromycin-resistant screened, differentiated and rooted 33 transgenic lines were obtained.
  • Total DNA was extracted from the leaves of the above plants, and primers SP1: CCCGACATAGATGCAATAACTTC (SEQ ID NO: 16) and SP2: GCGCGGTGTCATCTATGTTACT (SEQ ID NO: 17) were used for positive detection, and 30 transgenic positive plants were identified.
  • target 1-F TTCGCCCTGCTCTTCCCG (SEQ ID NO: 18) and target 1-R: AGATCAGATGCCCCAAATTCATA (SEQ ID NO: 19) and target site 2 on both sides
  • the primer target 2-F TGGAGCCTCTTGGTTAGAGTC (SEQ ID NO: 20) and the target 2-R: CCTCCTTCTGATTCGTCCT (SEQ ID NO: 21).
  • plant L138-1-1 had a homozygous deletion mutation of the CGGCGACTCCA sequence at target site 1 ( Figure 14).
  • Plant L138-3-1 had a homozygous mutation for G deletion at target site 2 ( Figure 15).
  • Mutations that occur at target site 1 or target site 1 and adjacent sequences also include CTCCACGGCG, TCGGCGACTCCA, TCGGCGACTCCA, CCACGGCG, ATCGGCGAC, C, CGGCGGCGGCGGCGGCGCCGCGCCGCACACGCCGCTCGTCCCGGCGCTCTTCTCGTGATCGGCGACT, CTCTTCGTGATCGGCGACAC base deletion or mutation Table 2).
  • Mutations occurring at target site 2 or within target site 2 and adjacent sequences also include AGCTGC, G, GCTG, TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGC
  • the primers 3013OXS: tttggtaccATGGCGCTCCCCTTCCTC (SEQ ID ID NO: 22) and 3013OXAS: tttggatccCTACTTGAGTTTTACCATCTGCTGCA (SEQ ID ID NO: 23) were used to amplify the nucleotide sequence with GMS1 (SEQ ID ID NO). : 2) DNA fragment. This fragment was double digested with Kpn I and BamH I and ligated into pBLU5 to obtain plasmid pBLU5-870-CDS ( Figure 19). E. coli with pBLU5-870-CDS was named E. coli-pBLU5-870-CDS. PBLU5-870-CDS was transformed into human Agrobacterium strain EH105 by electric shock, and the obtained strain was named Ab-pBLU5-870-CDS.
  • Recombinant Agrobacterium Ab-pBLU5-870-CDS was used to infect 11 callus in japonica rice, and 42 transgenic positive plants were obtained through hygromycin resistance selection, differentiation and rooting.
  • the primers 3013_RTF: AATTCATCCGCCAGCACC (SEQ ID NO: 12) and 3013_RTR: GCCCACAGCAGGCATCAG (SEQ ID ID: 13), Actin-RTF: GATGCTTATGTCGGTGAT (SEQ ID ID NO: 14), and Actin- RTF: ATGCTCTTCTGGTGCTAC (SEQ ID NO: 15) was used to analyze the expression of GMS1 in transgenic positive plants.
  • the expression level of GMS1 in the over-expressing plants generally increased by more than three times, but the over-expressing plants did not show a clear phenotype that was co-segregated from the expression level. This indicates that GMS1 gene overexpression has no significant effect on rice phenotype.
  • Example 8 Obtaining a gms1 mutant transgenic complementary strain and phenotypic analysis
  • primers 870F GGGGTACCGAGCATAAGGCAAACTGGCG (SEQ ID NO: 24) and 870R: CGGGATCCATGGCCAAACCATGGGAAGA (SEQ ID ID: 25) were amplified to obtain a GMS1 start codon ATG upstream of 1TG and a stop codon TAG downstream 795bp full-length fragment of the gene.
  • This fragment was double digested with Kpn I and BamH I and ligated into pC2300 to obtain plasmid pC2300-870-genome ( Figure 21).
  • E. coli with pC2300-870-genome was named E. coli-pC2300-870-genome.
  • PC2300-870-genome was transformed into human Agrobacterium strain EH105 by electric shock, and the obtained strain was named Ab-pC2300-870-genome.
  • Recombinant Agrobacterium Ab-pC2300-870-genome was used to infect gms1 mutant callus, and 25 transgenic positive plants were obtained through resistance screening, differentiation and rooting, all of which restored the fertility of the gms1 mutant to normal ( Figure 22) . This further proves that GMS1 gene regulates pollen development, and this gene mutation will cause pollen abortion.
  • the genomic nucleotide sequence of the fertility gene in Arabidopsis thaliana is shown in SEQ ID NO: 26, the CDS sequence is shown in SEQ ID NO: 27, and the amino acid sequence is shown in SEQ ID NO: 28;
  • the genomic nucleotide sequence of the fertility gene in Huawei (Setaria italica) is shown in SEQ ID NO: 29, the CDS sequence is shown in SEQ ID NO: 30, the amino acid sequence is shown in SEQ ID NO: 31;
  • sorghum The genomic nucleotide sequence of the fertility gene in Sorghum (biorg) is shown in SEQ ID NO: 32, the CDS sequence is shown in SEQ ID NO: 33, the amino acid sequence is shown in SEQ ID NO: 34; in Zeamays
  • the genomic nucleotide sequence of the fertility gene is shown in SEQ ID NO: 35, the CDS sequence is shown in SEQ ID NO: 36, the amino acid sequence is shown in SEQ ID NO: 37; Brachypodium distachyon The genomic nucleot
  • the gms1 mutant is used to cross, fertilize, and self-cross with a fertile receptor, such as H28B.
  • a fertile receptor such as H28B.
  • molecular markers are used to select the gms1 gene and genetic background.
  • homozygous GMS1 is obtained in the H28B background. Recessive genic male sterile line of mutant gene.
  • F 1 is obtained by hybridizing the recipient parent, such as H28B, with gms1 as the parent.
  • Select gms1 heterozygous genotype that is, plants that can amplify both 111bp and 108bp bands.
  • genotypes e.g., 100, or 200, etc.
  • a uniformly distributed molecular marker which can be, but is not limited to, SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR and other types of markers
  • a high degree of similarity with the recurrent parent genotype such as greater than 88% similarity, or 2% selection rate Etc.
  • step 4 Backcross the plant selected in step 4 with the recipient parent, such as H28B, to obtain BC 2 F 1 .
  • Plant BC 2 F 1 repeat step 3 and step 4 to select gms1 genotype heterozygous plants with high genetic background recovery rate (such as greater than 98%, or 2% selection rate, etc.), and collect from cross BC 2 F 2 .
  • high genetic background recovery rate such as greater than 98%, or 2% selection rate, etc.
  • Plant BC 2 F 2 repeat step 3 and step 4 to select the gms1 genotype heterozygous, the plant with the highest genetic background homozygosity rate, and collect it from cross BC 2 F 3 .
  • the gms1 heterozygous plant isolated from the offspring of BC 2 F 3 is the gms1 recessive nuclear sterile line.
  • BC 2 F 3 is used to save the gms1 recessive nuclear sterile line.
  • the invention provides a rice fertility regulating gene, a mutant thereof and application thereof.
  • the rice gene GMS1 provided by the present invention has the functions of regulating male germ cell development and pollen fertility. Its CDS sequence is shown in SEQ ID NO: 2, and its amino acid sequence is shown in SEQ ID NO: 3.
  • the invention also provides a radiation mutagenesis mutant and a CRISPR knockout mutant of the GMS1 gene, and provides a molecular marker identification method for the mutant.
  • the rice gene GMS1 provided by the invention can be used for sterile breeding and production of rice hybrids, and has great economic value and application prospects.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Botany (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种水稻育性调控基因及其突变体与应用。提供了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GMS1，其CDS序列如SEQ ID NO:2所示，氨基酸序列如SEQ ID NO:3所示。提供了GMS1基因的辐射诱变突变体和CRISPR敲除突变体，并提供了所述突变体的分子标记鉴定方法。提供的水稻基因GMS1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

一种水稻育性调控基因及其突变体与应用
交叉引用
本申请要求2018年8月30日提交的专利名称为“一种水稻育性调控基因及其突变体与应用”的第201811004799.6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
本发明属于生物技术领域，具体地涉及一个植物育性调控基因GMS1、GMS1编码蛋白、GMS1的辐射诱变和基因敲除突变体，以及GMS1基因、蛋白和突变体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 ZS1processes UbL40mRNAs 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。本发明提供了一种作物育性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
本发明提供一种育性相关基因及其核苷酸和蛋白序列，还包括通过操作该基因在调控植株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相应核苷酸序列一起使用，例如，将所述育性基因的突变体序列引入植株以导致植株雄性不育、使植株内源序列突变、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。
本发明第一个方面是提供一个花粉发育调控基因GMS1。GMS1位于水稻第2号染色体上，其在粳稻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。在籼稻中其基因组核苷酸序列如SEQ ID NO:4所示，其CDS和氨基酸序与粳稻相同。在拟南芥(Arabidopsis thaliana)中该育性基因的基因组核苷酸序列如SEQ ID NO:26所示，CDS序列如SEQ ID NO:27所示，氨基酸序列如SEQ ID NO:28所示；在小米(Setaria italica)中该育性基因的基因组核苷酸序列如SEQ ID NO:29所示，CDS序列如SEQ ID NO:30所示，氨基酸序列如SEQ ID NO:31所示；高粱(Sorghum bicolor)中该育性基因的基因组核苷酸序列如SEQ ID NO:32所示，CDS序列如SEQ ID NO:33所示，氨基酸序列如SEQ ID NO:34所示；玉米(Zeamays)中该育性基因的基因组核苷酸序列如SEQ ID NO:35所示，CDS序列如SEQ ID NO:36所示，氨基酸序列如SEQ ID NO:37所示；二穗短柄草(Brachypodium distachyon)中该育性基因的基因组核苷酸序列如SEQ ID NO:38所示，CDS序列如SEQ ID NO:39所示，氨基酸序列如SEQ ID NO:40所示：大麦(Hordeum vulgare)中该育性基因的基因组核苷酸序列如SEQ ID NO:41所示，CDS序列如SEQ ID NO:42所示，氨基酸序列如SEQ ID NO:43所示；短药野生稻(Oryza brachyantha)中该育性基因的基因组核苷酸序列如SEQ ID NO:44所示，CDS序列如SEQ ID NO:45所示，氨基酸序列如SEQ ID NO:46所示；非洲栽培稻(Oryza glaberrima)中该育性基因的基因组核苷酸序列如SEQ ID NO:47所示，CDS序列如SEQ ID NO:48所示，氨基酸序列如SEQ ID NO:49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知晓，本发明所述的育性恢复基因包括与GMS1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GMS1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GMS1基因所示的序列有至少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，本发明一种具有调控植物雄性育性功能的DNA片段，其序列为以下任一：
1)具有SEQ ID NO:1或2所示的核苷酸序列；
2)具有SEQ ID NO:4或2所示的核苷酸序列；
3)具有SEQ ID NO:26或27所示的核苷酸序列；
4)具有SEQ ID NO:29或30所示的核苷酸序列；
5)具有SEQ ID NO:32或33所示的核苷酸序列；
6)具有SEQ ID NO:35或36所示的核苷酸序列；
7)具有SEQ ID NO:38或39所示的核苷酸序列；
8)具有SEQ ID NO:41或42所示的核苷酸序列；
9)具有SEQ ID NO:44或45所示的核苷酸序列；
10)具有SEQ ID NO:47或48所示的核苷酸序列；
11)在严格条件下能够与(1)-(10)之任一序列的DNA杂交的DNA片段；或
12)与(1)-(11)之任一所述序列互补的DNA片段；或
13)在(1)-(11)之任一所述序列的基础之上，经过一至数个碱基替换和/或一至数个碱基的插入和/或缺失以及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；或
14)与(1)-(11)之任一所述序列的DNA片段具有85％以上的同一性且编码水稻雄性育性相关蛋白的DNA片段。
上述DNA片段为GMS1基因或其特异性片段。
本发明提供了所述DNA片段编码的蛋白，为如下1)或2)所述的蛋白：
1)SEQ ID NO:3、28、31、34、37、40、43、46或49所示的氨基酸序列组成的蛋白；
2)将SEQ ID NO:3、28、31、34、37、40、43、46或49经过一个或若干个氨基酸残基的取代和/或缺失和/或添加且具有调控植物雄性育性活性的蛋白。
本发明提供一种生物材料，含有上述的DNA序列，所述生物材料为表达盒，表达载体、工程菌、转基因植物或转基因细胞系。
本发明所述的DNA片段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。
本发明的第二方面是提供了通过影响GMS1的核苷酸序列或者通过调控GMS1基因的转录表达从而影响植株育性的方法。所述影响植株育性是指通过调控GMS1基因的表达，从而使所述植株的育性发生改变，如导致植株雄性不育。具体地，取决于实际应用需求，可以通过多种方法来影响GMS1基因在植物体内的表达，从而达到调控植株雄性育性的效果。更具体地，调控GMS1基因的表达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GMS1基因的正常表达，从而获得雄性不育的植株。另一方面，本发明还包括通过将野生型GMS1的核苷酸序列引入植株来恢复GMS1表达被破坏的植株的雄性生育力。
本发明的第三方面提供了一种DNA分子，其含有所述DNA片段，并在所述DNA片段中插入、和/或缺失、和/或取代若干个核苷酸从而导致水稻雄性育性发生改变。
进而本发明提供一种GMS1基因的不育突变体序列及其雄性不育突变体材料。所述雄性不育突变体材料是由核苷酸序列的突变所造成，含有该突变后核苷酸序列的植株表现为雄性不育，所述核苷酸序列如SEQ ID NO:1、2、4、26、27、29、30、32、33、35、36、38、39、41、42、44、45、47或48任一所示，所述的突变为点突变、DNA缺失、插入或取代突变、反义基因的转入、共抑制或发夹结构的引入或通过基因沉默手段产生的突变。
具体地，所述雄性不育突变体材料是通过突变水稻内源的GMS1基因，或突变与其高度同源的基因的核苷酸序列，使该植物体丧失雄性育性 的过程。所述“突变”包括但不限于以下方法，如用物理或化学的方法导致的基因突变，所述突变可以是点突变，也可以是DNA缺失或插入突变。突变还可以是通过RNAi、CRSPR-Cas9、人工核酸酶、定点突变等基因沉默手段产生。
在本发明的一个优选的实施方案中，所述的突变体材料为采用CRISPR-Gas9方法，以序列CTTCGTGATCGGCGACTCCA(靶位点1)和/或序列GAGAGAATGCCAGCTGCCG(靶位点2)为靶位点，使靶位点或靶位点及相邻核苷酸序列突变后得到的植物。
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、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，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。
在本发明的优选实施例中，所述的突变体材料为水稻突变体材料，在水稻基因组所述靶位点1和靶位点2或靶位点及相邻区域内具有以下1)和/或2)中的1种或多种突变，如2种、3种、4种突变：
1)在靶位点1或靶位点1及相邻序列内发生了CGGCGACTCCA、CTCCACGGCG、TCGGCGACTCCA、TCGGCGACTCCA、CCACGGCG、ATCGGCGAC、C、CGGCGGCGGCGGCGGCCCCGCGCCGCACGCCGCTCGTCCCGGCGCTCTTCGTGATCGGCGACT、CTCTTCGTGATCGGCGACTCCA或TCCACGGCGGAC序列缺失突变，或T碱基插入突变；
2)在靶位点2或靶位点2及相邻序列内发生了AGCTGC、G、GCTG、TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGCTGGAGAGAATGCCAGCTGCCGCGGATGCATAGTTG或TG序列缺失突变，或A、CT、GT或GC碱基插入突变，或从TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGCTGGAGAGAATGCCAGCTGCCGCGGATGC到GTTTGT或从CAGCTG到ATAGTTAAAAAATT序列替换突变。
进一步，本发明提供了一种适用于CRISPR/Cas9系统对植物GMS1基因进行定向敲除的靶位点，其为CTTCGTGATCGGCGACTCCA(靶位点1)和/或GAGAGAATGCCAGCTGCCG(靶位点2)。
本发明还提供了一种特异性靶向上述靶位点1或靶位点2的sgRNA。
含有上述sgRNA的DNA序列的CRISPR/Cas9打靶载体属于本发明的保护范围。
更具体地，本发明提供了一种水稻雄性不育突变体，其含有突变后的雄性不育基因，所述突变后的雄性不育基因的基因组核苷酸序列如SEQ ID NO:5所示，CDS序列如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:7所示。对野生型和突变体序列进行比对发现，在LOC_Os02g18870基因的基因组序列编码区第3外显子的第3600位碱基处的TTGT被替换为A，导致LOC_Os02g18870基因编码蛋白中第230位和第231位的亮氨酸和缬氨酸突变为一个组氨酸，进而导致蛋白丧失功能。本领域技术人员应该知晓，可以将所述核苷酸序列SEQ ID NO:5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。
本发明提供了GMS1基因或其编码蛋白或含有其的生物材料或其突变体材料在调控植物雄性育性活性中的应用。所述生物材料为含有GMS1基因的表达盒、表达载体、工程菌、转基因植物或转基因细胞系。
其中所述GMS1基因用于恢复作物植株雄性不育，以便导入外源基因以获得优质的转基因作物。
提供上述GMS1基因用于转基因改良作物的用途。
所述改良包括产量提高、品质提高、抗病虫害、抗逆、抗倒伏等生长性状的改良。
在另一具体实施方案中，所述作物是自花授粉或异花授粉作物。
在一个更加具体的实施方案中，所述作物包括但不限于玉米、小麦、高梁、水稻。
本发明还提供了所述的GMS1基因或其编码蛋白或含有其的生物材料或DNA分子或上述任一种突变体材料在转育带有纯合GMS1突变基因的隐性核不育系中的应用。
本发明还提供了一种在其它植物中获取GMS1基因的直系同源基因的方法，以及利用该方法获得高粱、小米、短柄草、水稻同源GMS1的氨基酸序列。
本发明提供了一种在其它植物中获取GMS1基因的直系同源基因的方法包括：
1)使用前述GMS1基因的DNA片段在核苷酸数据库中进行blastx搜索；
2)所有Identities大于或等于35％、Positives大于或等于50％即为与所述DNA片段直系同源的基因片段。
本发明提供了一种与GMS1基因紧密连锁的分子标记，通过核苷酸序列如SEQ ID NO:8-9所述的引物扩增得到。
本发明提供了一种用于检测GMS1基因突变体材料的分子标记，通过核苷酸序列如SEQ ID NO:10-11所述的引物扩增得到。
选用SEQ ID NO:10-11所示引物扩增待测水稻基因组DNA时，若只能扩增出108bp一条带，则待测水稻为GMS1基因突变体。
含有核苷酸序列如SEQ ID NO:8-9或SEQ ID NO:10-11所述的引物的检测试剂或试剂盒属于本发明的保护范围。
进一步本发明提供了上述两种分子标记或上述检测试剂或试剂盒在 检测水稻GMS1基因突变体中的应用。
本发明提供了上述两种分子标记或上述检测试剂或试剂盒在筛选或培育雄性不育水稻突变体中的应用。
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GMS1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。gms1不育突变体与现有三系和两系不育系相比不育性状稳定，不受环境条件影响。通过植物生物技术途径，本发明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。
附图说明
图1灌浆期野生型(左)和gms1突变体(右)的植株形态。
图2野生型(左)和gms1突变体(右)的小穗形态。
图3野生型(左)和gms1突变体(右)穗开花的形态。
图4解剖后野生型(左)和gms1突变体(右)的小花形态。
图5野生型(左)和gms1突变体(右)的花药形态。
图6野生型(左)和gms1突变体(右)花粉碘染。
图7利用Indel3标记鉴定定位群体中不育单株的基因型。上带大小124bp，下带大小120bp。泳道1-4的DNA模板分别为gms1突变体，明恢63，定位群体中的不育单株2.5和2.6。
图8A为GMS1基因图位克隆图，图8B为gms1突变体的突变位点示意图。
图9 GMS1基因在9311(9311-870)，明恢63(MH63-870)，日本晴(Nip-870)和gms1突变体(gms1-870)材料中的核苷酸序列差异。有差异的地方用黑色背景突出。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。
图10 GMS1编码蛋白在9311(9311-870)和gms1突变体(gms1-870)中的氨基酸序列差异。有差异的地方用黑色背景突出。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 GMS1杂合株后代基因型鉴定。上带大小111bp，下带大小108bp。箭头所指为不育株DNA。
图12 GMS1在水稻不同组织中的表达量。
图13 pC9M-GMS1载体示意图。T1代表靶位点1，T2代表靶位点2。
图14转基因植株L138-1-1在靶位点1处的测序峰图。箭头指向缺失位点。
图15转基因植株L138-3-1在靶位点2处的测序峰图。箭头指向缺失位点。
图16 GMS1野生型(左)、敲除植株L138-1-1(中)和L138-3-1(右)的小穗形态。
图17 GMS1野生型(左)、敲除植株L138-1-1(中)和L138-3-1(右)的花药形态。
图18 GMS1野生型(左)、敲除植株L138-1-1(中)和L138-3-1(右)的花粉碘染。
图19 pBLU5-870-CDS载体示意图。
图20 GMS1在超表达植株中的表达量。
图21 pC2300-870-genome载体示意图。
图22 gms1突变植株(左)和互补植株(右)的植株形态。
图23水稻GMS1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。包括拟南芥(Arabidopsis thaliana)蛋白AT4G10950.1、小米(Setaria italica)蛋白Si019557m、高粱(Sorghum bicolor)蛋白Sb04g011320.1、玉米(Zea mays)蛋白GRMZM2G166330_P01、二穗短柄草(Brachypodium distachyon)蛋白BRADI3G01797.1、大麦(Hordeum vulgare)蛋白MLOC_10139.2、短药野生稻(Oryza brachyantha)蛋白OB02G21480.1、非洲栽培稻(Oryza glaberrima)蛋白ORGLA02G0115000.1。
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，下述方法但不限于下述方法：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花育性的技术方法中的任何一种技术方法。
本发明包括水稻GMS1基因，其显性等位基因对植物雄花育性具有关键作用，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2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)、茄属马铃薯(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：通过水稻GMS1基因序列利用blastx、blastn或通过氨基酸序列利用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GMS1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GMS1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。
GMS1基因同源序列指在与SEQ ID NO:3的氨基酸序列进行blastx比较分析后，Identities大于或等于35％、Positives大于或等于50％的植物基因的DNA序列。进行blastx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gms1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。其中一个突变体表现为雄性不育，被命名为gms1。
实施例2 水稻雄性不育突变体gms1的表型分析
与野生型相比，gms1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3013突变体不结实。而以水稻品种93-11为父本给3013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gms1的遗传分析
在M4代种植gms1的分离群体436株，其中330株育性正常，106株不育，可育与不育株分离比符合3:1(χ 2＝0.08，P<0.05)。用gms1与明恢63杂交，F1代植株全部可育。在F3代种植gms1的分离群体7832株，其中5834株育性正常，1998株不育，可育与不育株分离比符合3:1(χ 2＝1.06，P<0.05)。上述结果表明gms1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GMS1的克隆
使用图位克隆的方法对GMS1基因进行定位。以明恢63为父本与gms1突变体杂交构建了一个包含350棵植株的F 2群体。利用该群体将GMS1定位于2号染色体SSR标记RM13004和RM13018之间，与SSR标记RM13011共分离。GMS1基因与上述三个标记之间的交换单株分别为1个，0个，1个。利用连锁标记挑选F 2群体中的gms1杂合单株发展了一个F 3群体，包含7832个单株。在F 3群体中RM13004、RM13011、RM13018与GMS1基因之间的交换单株分别为9个，3个，13个。在RM13004和RM13018之间通过分析和比较93-11和日本晴基因组的序列，开发并实验证实了一个新的插入缺失标记Indel3。检测Indel3的引物为Indel3-F：GGGAAGAAACAGAGGTGCC(SEQ ID NO:8)和Indel3-R：GCTTGGAGGTATTTGGGACA(SEQ ID NO:9)。利用Indel3-F和Indel3-R 可以在gms1突变体和明恢63基因组DNA中分别扩增出1条120bp(序列如SEQ ID NO:62)和1条124bp(序列如SEQ ID NO:63)条带(图7)。实验结果显示定位群体中所有不育单株都只扩增出了一条120bp的条带(图7)，表明Indel3与gms1不育表型紧密连锁(图8A)。以Indel3上下游20kb为候选区段，发现在该区段内共有6个注释基因，其中LOC_Os02g18870预测编码一个GDSL类脂肪酶/酰基水解酶，推测是GMS1基因。在日本晴中，LOC_Os02g18870基因组核苷酸序列长5604bp(记为Nip-870，序列如SEQ ID NO:1)，CDS核苷酸序列长1200bp(序列如SEQ ID NO:2)，包含5个外显子和4个内含子(图8B)，编码一个包含399个氨基酸残基的蛋白(序列如SEQ ID NO:3)。
根据Nip-870序列设计引物对LOC_Os02g18870基因在93-11、明恢63和gms1突变体中的等位基因进行扩增和测序，引物序列如表1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gms1突变体中的LOC_Os02g18870基因分别记为9311-870(序列如SEQ ID NO:4)，MH63-870，gms1-870(序列如SEQ ID NO:5)。
表1用于扩增LOC_Os02g18870的引物对序列
Figure PCTCN2018119270-appb-000001
Figure PCTCN2018119270-appb-000002
对9311-870、gms1-870、MH63-870和Nip-870进行多序列比对，结果如图9所示。9311-870和gms1-870只在第3个外显子的第3600位碱基处存在一个TTGT到A的替换(图8和图9)。氨基酸序列分析显示，该突变将导致LOC_Os02g18870基因编码蛋白中第230位和第231位的亮氨酸和缬氨酸突变为一个组氨酸(图10)。MH63-870和Nip-870与gms1-870也在第3600个碱基处存在上述相同差异(图9)。这表明第3600位碱基处TTGT到A的突变是造成gms1突变体雄性不育的原因。此外9311-870和MH63-870的序列完全一致，而与Nip-870相比，在第308位缺失一个C，在第3303位有一个T变C的SNP(图9)。两处核苷酸差异分别落在第1和第2内含子中，并不影响编码区。这表明LOC_Os02g18870基因在水稻中高度保守，其核苷酸序列即使在籼、粳亚种间也只在非编码区存在两个碱基的差异，而蛋白质序列则没有差异。在93-11中LOC_Os02g18870的CDS核苷酸序列如SEQ ID NO:2所示，编码蛋白序列如SEQ ID NO:3。在gms1突变体中LOC_Os02g18870的CDS核苷酸序列和氨基酸序列分别如SEQ ID NO:6和SEQ ID NO:7所示。
基于LOC_Os02g18870基因突变位点的测序结果，在突变位点两侧设计特异引物3013_F：GCAACGACTTCATCCACT(SEQ ID NO:10)和3013_R：CCTTGATTTCCTGCCTCA(SEQ ID NO:11)。当3013_F和3013_R配对扩增野生型LOC_Os02g18870基因时将产生111bp条带，扩增突变型LOC_Os02g18870基因时将产生108bp条带。使用3013_F和3013_R引物对对38株gms1的M5分离群体进行基因型检测。如图11 所示，野生型要么扩增出111bp和108bp两条带，要么扩增出111bp一条带，而不育突变体均只能扩增出108bp一条带。这说明突变基因型与不育表型共分离，LOC_Os02g18870就是GMS1基因。
实施例5 GMS1基因的表达分析
取93-11各时期组织提取总RNA，反转录成cDNA。根据GMS1的基因组和编码区核苷酸序列设计跨内含子的引物3013_RTF：AATTCATCCGCCAGCACC(SEQ ID NO:12)和3013_RTR：GCCCACAGCAGGCATCAG(SEQ ID NO:13)，同时以水稻Actin基因作为内参对照设计引物Actin-RTF：GATGCTTATGTCGGTGAT(SEQ ID NO:14)和Actin-RTR：ATGCTCTTCTGGTGCTAC(SEQ ID NO:15)。采取实时定量PCR方法进行表达量分析。如图12所示，GMS1基因在水稻的根、茎、叶、种子、外稃、内稃中均有表达但表达量较低。在一期到三期幼穗中，GMS1的表达量快速升高，然后逐渐降低，直到八期幼穗。其中七期幼穗中GMS1表达量略有升高。此外，在八期穗的花药中，GMS1的表达明显升高。
实施例6 GMS1基因敲除株系的获得及表型分析
利用CRISPR/Cas9系统对GMS1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位于第一外显子的正链上，序列为CTTCGTGATCGGCGACTCCA(SEQ ID NO:1序列的第168位至第187位)，靶位点2位于第二外显子的负链上，序列为GAGAGAATGCCAGCTGCCG(SEQ ID NO:1序列的第1629位至第1647位)。根据Ma等(Ma X,et al.A Robust CRISPR/Cas9System 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-GMS1(图13)。有pC9M-GMS1的大肠杆菌被命名为E.coli-pC9M-GMS1。将pC9M-GMS1通过电击转人农杆菌菌株EH105中，得到的菌株命名为Ab-pC9M-GMS1。
利用重组农杆菌Ab-pC9M-GMS1侵染粳稻中花11愈伤组织，经潮霉素抗性筛选、分化、生根获得再生转基因株系33株。提取上述植株叶片的总DNA，利用引物SP1：CCCGACATAGATGCAATAACTTC(SEQ ID NO:16)和SP2：GCGCGGTGTCATCTATGTTACT(SEQ ID NO:17)做阳性检测，共鉴定出转基因阳性植株30株。随机选取17株阳性株，用靶位点1两侧的引物靶1-F：TTCGCCCTGCTCTTCCCG(SEQ ID NO:18)和靶1-R：AGATCAGATGCCCCAAATTCATA(SEQ ID NO:19)以及靶位点2两侧的引物靶2-F：TGGAGCCTCTTGGTTAGAGTC(SEQ ID NO:20)和靶2-R：CCTCCTTCTGATTCGTCCT(SEQ ID NO:21)。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17株阳性株的基因组DNA至少在靶位点1和靶位点2中的一处两条等位染色体均发生了突变(表2)。例如植株L138-1-1在靶位点1处发生了CGGCGACTCCA序列的纯合缺失突变(图14)。植株L138-3-1在靶位点2处发生了G缺失的纯合突变(图15)。发生在靶位点1或靶位点1及相邻序列内的突变还包括CTCCACGGCG、TCGGCGACTCCA、TCGGCGACTCCA、CCACGGCG、ATCGGCGAC、C、CGGCGGCGGCGGCGGCCCCGCGCCGCACGCCGCTCGTCCCGGCGCTCTTCGTGATCGGCGACT、CTCTTCGTGATCGGCGACTCCA或TCCACGGCGGAC等序列缺失突变，或T等碱基插入突变(表2)。发生在靶位点2或靶位点2及相邻序列内的突变还包括AGCTGC、G、GCTG、TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGC
TGGAGAGAATGCCAGCTGCCGCGGATGCATAGTTG或TG等序列缺失突变，或A、CT、GT或GC等碱基插入突变，或从TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGCTGGAGAGAATGCCAGCTGCCGCGGATGC到GTTTGT或从CAGCTG到ATAGTTAAAAAATT等序列替换突变。
开花后对上述17株阳性株进行表型分析。与野生型ZH11相比，GMS1 敲除植株L138-1-1和L138-3-1在株叶和小穗形态上并无明显差别(图16)。但GMS1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GMS1敲除植株的花粉小而皱缩，不能被染色(表2和图18)。GMS1敲除植株的表型与gms1突变体的表型一致。
实施例7 GMS1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3013OXS：tttggtaccATGGCGCTCCCCTTCCTC(SEQ ID NO:22)和3013OXAS：tttggatccCTACTTGAGTTTTACCATCTGCTGCA(SEQ ID NO:23)扩增获得带有GMS1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pBLU5-870-CDS(图19)。有pBLU5-870-CDS的大肠杆菌被命名为E.coli-pBLU5-870-CDS。将pBLU5-870-CDS通过电击转人农杆菌菌株EH105中，得到的菌株命名为Ab-pBLU5-870-CDS。
利用重组农杆菌Ab-pBLU5-870-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42株。使用实时定量PCR方法，利用实施例5中的引物3013_RTF：AATTCATCCGCCAGCACC(SEQ ID NO:12)和3013_RTR：GCCCACAGCAGGCATCAG(SEQ ID NO:13)，Actin-RTF:GATGCTTATGTCGGTGAT(SEQ ID NO:14)和Actin-RTF:ATGCTCTTCTGGTGCTAC(SEQ ID NO:15)对转基因阳性植株中GMS1的表达量进行分析。如图20所示，和转基因阴性单株L136-3-1相比，超表达植株中GMS1的表达量普遍上升了3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GMS1基因超表达对水稻表型并没有显著影响。
实施例8 gms1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870F：GGGGTACCGAGCATAAGGCAAACTGGCG(SEQ ID NO:24)和870R： CGGGATCCATGGCCAAACCATGGGAAGA(SEQ ID NO:25)扩增获得带有GMS1起始密码子ATG上游1373bp和终止密码子TAG下游795bp的基因全长片段。将该片段用Kpn I和BamH I双酶切后连入pC2300获得质粒pC2300-870-genome(图21)。有pC2300-870-genome的大肠杆菌被命名为E.coli-pC2300-870-genome。将pC2300-870-genome通过电击转人农杆菌菌株EH105中，得到的菌株命名为Ab-pC2300-870-genome。利用重组农杆菌Ab-pC2300-870-genome侵染gms1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25株，均使gms1突变体的育性恢复正常(图22)。这进一步证明GMS1基因调控花粉发育，该基因突变会导致花粉败育。
实施例9 GMS1基因编码蛋白与植物基因组中预测同源蛋白的序列比对
利用blastx工具在NCBI的Genbank数据库中对水稻GMS1基因编码蛋白的氨基酸序列进行同源性搜索，得到了拟南芥(Arabidopsis thaliana)、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)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23)，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。
在拟南芥(Arabidopsis thaliana)中该育性基因的基因组核苷酸序列如SEQ ID NO:26所示，CDS序列如SEQ ID NO:27所示，氨基酸序列如SEQ ID NO:28所示；在小米(Setaria italica)中该育性基因的基因组核苷酸序列如SEQ ID NO:29所示，CDS序列如SEQ ID NO:30所示，氨基酸序列如SEQ ID NO:31所示；高粱(Sorghum bicolor)中该育性基因的基因组核苷酸序列如SEQ ID NO:32所示，CDS序列如SEQ ID NO: 33所示，氨基酸序列如SEQ ID NO:34所示；玉米(Zeamays)中该育性基因的基因组核苷酸序列如SEQ ID NO:35所示，CDS序列如SEQ ID NO:36所示，氨基酸序列如SEQ ID NO:37所示；二穗短柄草(Brachypodium distachyon)中该育性基因的基因组核苷酸序列如SEQ ID NO:38所示，CDS序列如SEQ ID NO:39所示，氨基酸序列如SEQ ID NO:40所示：大麦(Hordeumvulgare)中该育性基因的基因组核苷酸序列如SEQ ID NO:41所示，CDS序列如SEQ ID NO:42所示，氨基酸序列如SEQ ID NO:43所示；短药野生稻(Oryzabrachyantha)中该育性基因的基因组核苷酸序列如SEQ ID NO:44所示，CDS序列如SEQ ID NO:45所示，氨基酸序列如SEQ ID NO:46所示；非洲栽培稻(Oryzaglaberrima)中该育性基因的基因组核苷酸序列如SEQ ID NO:47所示，CDS序列如SEQ ID NO:48所示，氨基酸序列如SEQ ID NO:49所示。
实施例10 转育带有GMS1基因的隐性核不育系
用gms1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gms1基因和遗传背景选择，最终获得H28B背景下带有纯合GMS1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gms1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3013_F：GCAACGACTTCATCCACT(SEQ ID NO:10)和3013_R：CCTTGATTTCCTGCCTCA(SEQ ID NO:11)检测gms1基因型。选择gms1杂合基因型，即同时能扩增出111bp和108bp条带的植株。
4、使用一组基因型(例如100个，或200个等)在gms1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gms1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gms1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gms1杂合株即gms1隐性核不育系，BC 2F 3用于保存gms1隐性核不育系种质资源。
Figure PCTCN2018119270-appb-000003
Figure PCTCN2018119270-appb-000004
工业实用性
本发明提供一种水稻育性调控基因及其突变体与应用。本发明提供的水稻基因GMS1具有调控水稻雄性生殖细胞发育和花粉育性功能，其CDS序列如SEQ ID NO:2所示，氨基酸序列如SEQ ID NO:3所示。本发明还提供了GMS1基因的辐射诱变突变体和CRISPR敲除突变体，并提供了所述突变体的分子标记鉴定方法。本发明提供的水稻基因GMS1可用于水稻杂交种的不育化制种和生产，具有巨大的经济价值和应用前景。

Claims (22)

  1. 一种DNA片段，具有调控植物雄性育性的功能，其特征在于，所述DNA片段的序列为以下任一：
    1)具有SEQ ID NO:1或2所示的核苷酸序列；
    2)具有SEQ ID NO:4或2所示的核苷酸序列；
    3)具有SEQ ID NO:26或27所示的核苷酸序列；
    4)具有SEQ ID NO:29或30所示的核苷酸序列；
    5)具有SEQ ID NO:32或33所示的核苷酸序列；
    6)具有SEQ ID NO:35或36所示的核苷酸序列；
    7)具有SEQ ID NO:38或39所示的核苷酸序列；
    8)具有SEQ ID NO:41或42所示的核苷酸序列；
    9)具有SEQ ID NO:44或45所示的核苷酸序列；
    10)具有SEQ ID NO:47或48所示的核苷酸序列；
    11)在严格条件下能够与(1)-(10)之任一序列的DNA杂交的DNA片段；或
    12)与(1)-(11)之任一所述序列互补的DNA片段；或
    13)在(1)-(11)之任一所述序列的基础之上，经过一至数个碱基替换和/或一至数个碱基的插入和/或缺失以及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；或
    14)与(1)-(11)之任一所述序列的DNA片段具有85％以上的同一性且编码水稻雄性育性相关蛋白的DNA片段。
  2. 权利要求1所述DNA片段编码的蛋白，其特征在于，为如下1)或2)所述的蛋白：
    1)SEQ ID NO:3、28、31、34、37、40、43、46或49所示的氨基酸序列组成的蛋白；
    2)将SEQ ID NO:3、28、31、34、37、40、43、46或49经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有调控植物雄性育性 活性的蛋白。
  3. 一种生物材料，其特征在于，该生物材料含有权利要求1所述的DNA序列，所述生物材料为表达盒，表达载体、工程菌、转基因植物或转基因细胞系。
  4. 一种突变体材料，所述突变材料是由核苷酸序列的突变所造成，含有该突变后核苷酸序列的植株表现为雄性不育，其特征在于，所述核苷酸序列如SEQ ID NO:1、2、4、26、27、29、30、32、33、35、36、38、39、41、42、44、45、47或48任一所示，所述的突变为点突变、DNA缺失、插入或取代突变、反义基因的转入、共抑制或发夹结构的引入或通过基因沉默手段产生的突变。
  5. 根据权利要求4所述的突变体材料，其特征在于，所述的突变体材料为采用CRISPR-Gas9方法，以靶位点1的序列CTTCGTGATCGGCGACTCCA和/或靶位点2的序列GAGAGAATGCCAGCTGCCG为靶位点，造成靶位点或靶位点及相邻核苷酸序列突变后得到的植物。
  6. 根据权利要求5所述的突变体材料，所述的突变体材料为水稻突变体材料，其特征在于，在如权利要求5所述的靶位点1和靶位点2或靶位点及相邻区域内具有以下1)和/或2)中的1种或多种突变：
    1)在靶位点1或靶位点1及相邻序列内发生了CGGCGACTCCA、CTCCACGGCG、TCGGCGACTCCA、TCGGCGACTCCA、CCACGGCG、ATCGGCGAC、C、CGGCGGCGGCGGCGGCCCCGCGCCGCACGCCGCTCGTCCCGGCGCTCTTCGTGATCGGCGACT、CTCTTCGTGATCGGCGACTCCA或TCCACGGCGGAC序列缺失突变，或T碱基插入突变；
    2)在靶位点2或靶位点2及相邻序列内发生了AGCTGC、G、GCTG、TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACT
    GCTGGAGAGAATGCCAGCTGCCGCGGATGCATAGTTG或TG序 列缺失突变，或A、CT、GT或GC碱基插入突变，或从TTGGGAAGTTAGGAGGACAAACCAGCTCAGAACCACTGCTGGAGAGAATGCCAGCTGCCGCGGATGC到GTTTGT或从CAGCTG到ATAGTTAAAAAATT序列替换突变。
  7. 根据权利要求4所述的突变体材料，其特征在于，其为水稻突变体材料gms1，其基因组序列在LOC_Os02g18870基因的基因组序列编码区第3外显子的第3600位碱基处的TTGT被替换为A，导致LOC_Os02g18870基因编码蛋白中第230位和第231位的亮氨酸和缬氨酸突变为一个组氨酸。
  8. 根据权利要求7所述的突变体材料，其特征在于，其含有权利要求1所述DNA片段突变后的DNA片段，该突变后的DNA片段为水稻雄性不育基因，所述突变后的水稻雄性不育基因的基因组核苷酸序列如SEQ ID NO:5所示，CDS序列如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:7所示。
  9. 一种适用于CRISPR/Cas9系统对植物GMS1基因进行定向敲除的靶位点，其为CTTCGTGATCGGCGACTCCA和/或GAGAGAATGCCAGCTGCCG。
  10. 特异性靶向权利要求9所述靶位点的sgRNA。
  11. 含有权利要求10所述sgRNA的DNA序列的CRISPR/Cas9打靶载体。
  12. 权利要求1所述的DNA片段或其编码蛋白或含有其的生物材料或DNA分子或权利要求4-8任一所述的突变体材料在调控植物雄性育性性状中的应用。
  13. 根据权利要求12所述的应用，其中所述DNA片段用于诱导作物植株雄性不育，以便导入外源基因以获得优质的转基因作物。
  14. 权利要求1所述的DNA片段或其编码蛋白或含有其的生物材料或DNA分子或权利要求4-8任一所述的突变体材料在作物种质资源改良 中的应用，所述改良包括研发新杂交育制种技术，提高作物产量、提高作物品质、抗病虫害、抗逆、抗倒伏的改良。
  15. 权利要求1所述的DNA片段或其编码蛋白或含有其的生物材料或DNA分子或权利要求4-8任一所述的突变体材料在转育带有纯合GMS1突变基因的隐性核不育系中的应用。
  16. 一种获取权利要求1所述DNA片段在植物中的直系同源基因片段的方法，包括：
    1)使用权利要求1所述DNA片段在核苷酸数据库中进行blastx搜索；
    2)所有Identities大于或等于35％、Positives大于或等于50％即为与权利要求1所述DNA片段直系同源的基因片段。
  17. 一种与权利要求1所述DNA片段紧密连锁的分子标记，其特征在于，通过核苷酸序列如SEQ ID NO:8-9所述的引物扩增得到。
  18. 一种用于检测权利要求7所述突变体材料的分子标记，其特征在于，通过核苷酸序列如SEQ ID NO:10-11所述的引物扩增得到。
  19. 含有核苷酸序列如SEQ ID NO:8-9或SEQ ID NO:10-11所述的引物的检测试剂或试剂盒。
  20. 权利要求17或18所述的分子标记或权利要求19所述的检测试剂或试剂盒在检测水稻GMS1基因突变体中的应用。
  21. 根据权利要求20所述的应用，其特征在于，当选用SEQ ID NO:10-11所示引物扩增待测水稻基因组DNA时，若只能扩增出108bp一条带，则待测水稻为GMS1基因突变体。
  22. 权利要求17或18所述的分子标记或权利要求19所述的检测试剂在筛选或培育雄性不育水稻突变体中的应用。
PCT/CN2018/119270 2018-08-30 2018-12-05 一种水稻育性调控基因及其突变体与应用 WO2020042412A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811004799.6A CN110511945B (zh) 2018-08-30 2018-08-30 一种水稻育性调控基因及其突变体与应用
CN201811004799.6 2018-08-30

Publications (1)

Publication Number Publication Date
WO2020042412A1 true WO2020042412A1 (zh) 2020-03-05

Family

ID=68621953

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/119270 WO2020042412A1 (zh) 2018-08-30 2018-12-05 一种水稻育性调控基因及其突变体与应用

Country Status (2)

Country Link
CN (1) CN110511945B (zh)
WO (1) WO2020042412A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112195269A (zh) * 2020-11-06 2021-01-08 海南波莲水稻基因科技有限公司 与水稻核雄性不育表型相关的分子标记和应用
US11177389B2 (en) 2019-07-30 2021-11-16 Micron Technology, Inc. Integrated transistors having gate material passing through a pillar of semiconductor material, and methods of forming integrated transistors
CN116286871A (zh) * 2023-04-04 2023-06-23 安徽省农业科学院水稻研究所 一种水稻显性雄性不育基因sdgms及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110800606B (zh) * 2019-12-10 2021-07-02 安徽省农业科学院水稻研究所 一种水稻可繁殖杂合雄性不育系分子选育方法及其应用
CN113046359B (zh) * 2019-12-28 2022-09-09 湖南杂交水稻研究中心 调控水稻雌性发育的突变型基因及其编码的蛋白、应用以及引物
CN113754746B (zh) * 2020-06-02 2024-02-23 海南波莲水稻基因科技有限公司 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
WO2021244007A1 (zh) * 2020-06-02 2021-12-09 海南波莲水稻基因科技有限公司 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
CN113754747B (zh) * 2020-06-02 2024-02-23 海南波莲水稻基因科技有限公司 一种水稻雄性育性调控基因突变体及其分子标记和应用
CN111690047A (zh) * 2020-07-13 2020-09-22 中国科学院遗传与发育生物学研究所 一个玉米细胞核雄性育性基因ipe2的克隆与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105695501A (zh) * 2014-11-28 2016-06-22 上海师范大学 创制光温敏不育系的方法及其在植物育种中的应用
CN106834316A (zh) * 2017-03-31 2017-06-13 西南大学 水稻花粉萌发孔发育及花粉育性基因OsAOM、突变基因及其重组表达载体和应用
CN106834294A (zh) * 2017-03-31 2017-06-13 西南大学 水稻花药及种子高效启动子POsAOM及其重组表达载体和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634522B (zh) * 2012-03-07 2013-12-04 四川农业大学 控制水稻育性的基因及其编码蛋白和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105695501A (zh) * 2014-11-28 2016-06-22 上海师范大学 创制光温敏不育系的方法及其在植物育种中的应用
CN106834316A (zh) * 2017-03-31 2017-06-13 西南大学 水稻花粉萌发孔发育及花粉育性基因OsAOM、突变基因及其重组表达载体和应用
CN106834294A (zh) * 2017-03-31 2017-06-13 西南大学 水稻花药及种子高效启动子POsAOM及其重组表达载体和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONG, XIANGSHU ET AL.: "GDSL Esterase/lipase Genes in Brassica Rapa L.: Genome-wide Identification and Expression Analysis", MGG - MOLECULAR GENETICS AND GENOMICS, vol. 291, no. 2, 30 September 2015 (2015-09-30), pages 531 - 542, XP035882078 *
HU , JIHONG ET AL.: "Comparative Transcript Profiling of Alloplasmic Male-sterile Lines Revealed Altered Gene Expression Related to Pollen Development in Rice (Oryza Sativa L.", BMC PLANT BIOLOGY, vol. 16, no. 175, 5 August 2016 (2016-08-05), pages - 16, XP055696880 *
WU, SUOWEI ET AL.: "Construction of Male-sterility System Using Biotechnology and Application in Crop Breeding and Hybrid Seed Production", CHINA BIOTECHNOLOGY, vol. 38, no. 1, 31 January 2018 (2018-01-31), pages 78 - 87, XP055696896 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11177389B2 (en) 2019-07-30 2021-11-16 Micron Technology, Inc. Integrated transistors having gate material passing through a pillar of semiconductor material, and methods of forming integrated transistors
CN112195269A (zh) * 2020-11-06 2021-01-08 海南波莲水稻基因科技有限公司 与水稻核雄性不育表型相关的分子标记和应用
CN112195269B (zh) * 2020-11-06 2024-05-03 海南波莲水稻基因科技有限公司 与水稻核雄性不育表型相关的分子标记和应用
CN116286871A (zh) * 2023-04-04 2023-06-23 安徽省农业科学院水稻研究所 一种水稻显性雄性不育基因sdgms及其应用
CN116286871B (zh) * 2023-04-04 2023-12-05 安徽省农业科学院水稻研究所 一种水稻显性雄性不育基因sdgms及其应用

Also Published As

Publication number Publication date
CN110511945B (zh) 2021-07-27
CN110511945A (zh) 2019-11-29

Similar Documents

Publication Publication Date Title
WO2020042412A1 (zh) 一种水稻育性调控基因及其突变体与应用
US10619168B2 (en) Fertility gene and use thereof
WO2021244007A1 (zh) 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
US20220186238A1 (en) Diplospory gene
Sun et al. CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata
US20210269822A1 (en) Plants comprising wheat g-type cytoplasmic male sterility restorer genes, molecular markers and uses thereof
WO2018019193A1 (zh) 小麦育性恢复基因及其应用
WO2015035951A1 (zh) 雄性核不育基因及其突变体在杂交育种上的应用
CN111868075A (zh) 包含雄性育性恢复等位基因的小麦
WO2018119225A1 (en) Genome editing-based crop engineering and production of brachytic plants
CA3138988A1 (en) Gene for parthenogenesis
US20230383308A1 (en) Modified promoter of a parthenogenesis gene
Farinati et al. Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications
Li et al. Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker
Hu et al. Transposable elements cause the loss of self‐incompatibility in citrus
WO2010115297A1 (zh) 油菜隐性核不育恢复基因BnCYP704B1及应用
US20220275383A1 (en) Sterile genes and related constructs and applications thereof
Tan et al. Molecular characterization and application of a novel cytoplasmic male sterility-associated mitochondrial sequence in rice
CN113754746B (zh) 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
US20220145312A1 (en) Compositions and methods for driving t1 event diversity
CN113754747B (zh) 一种水稻雄性育性调控基因突变体及其分子标记和应用
CN117402887B (zh) 一种玉米雄性育性调控基因ZmMS2085及其突变体与应用
WO2018205521A1 (zh) 小麦育性相关基因TaMS7及其应用方法
Tang et al. Fertility restorer gene CaRf and PepperSNP50K provide a promising breeding system for hybrid pepper

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18931856

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18931856

Country of ref document: EP

Kind code of ref document: A1