WO2018205521A1 - 小麦育性相关基因TaMS7及其应用方法 - Google Patents

小麦育性相关基因TaMS7及其应用方法 Download PDF

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WO2018205521A1
WO2018205521A1 PCT/CN2017/109813 CN2017109813W WO2018205521A1 WO 2018205521 A1 WO2018205521 A1 WO 2018205521A1 CN 2017109813 W CN2017109813 W CN 2017109813W WO 2018205521 A1 WO2018205521 A1 WO 2018205521A1
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gene
sequence
fertility
tams7
pollen
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PCT/CN2017/109813
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English (en)
French (fr)
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李健
王峥
马力耕
邓兴旺
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未名兴旺系统作物设计前沿实验室（北京）有限公司
北京大学
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Priority to US16/611,863 priority Critical patent/US11753650B2/en
Priority to CN201780001491.3A priority patent/CN108064297B/zh
Priority to RU2019139843A priority patent/RU2763468C2/ru
Priority to EP17909493.3A priority patent/EP3623475A4/en
Publication of WO2018205521A1 publication Critical patent/WO2018205521A1/zh

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    • 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
    • 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
    • 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
    • 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)
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a hybrid breeding method for wheat, including wheat genic male sterile line breeding and hybrid seed preparation, and more particularly to a wheat male sterility gene and mutant thereof and application thereof in cross breeding .
  • Wheat is a self-pollinated crop, and the core of its heterosis utilization is to establish a technical system for efficient production of wheat hybrids.
  • the core of its heterosis utilization is to establish a technical system for efficient production of wheat hybrids.
  • there are three main ways to utilize heterosis in wheat production one is the three-line method, which uses the nuclear-matrix interaction male sterile line to produce hybrids through the three lines of sterile line, maintain line and restorer line; It is a chemical killing method, that is, the application of chemical male-killing agent induces male male sterility to carry out hybrid seed production; the third is the two-line method, that is, the light-temperature-sensitive male sterile line is used for hybrid seed production with changes in light temperature.
  • the three-line method has been studied since the 1950s.
  • the killing method was once considered to be a promising hybrid seeding new technology.
  • chemical male-killing agents have problems such as poor stability, easy residue, easy toxic side effects, and heavy environmental pollution. Therefore, in order to apply to a large area for production, it is necessary to develop an ideal chemical male-killing agent.
  • the second-line hybrid wheat has developed rapidly and gradually becomes the main development trend of wheat heterosis research and utilization in the future.
  • the core of the second-line method is the photo-thermophilic male sterile line, which is a male-sterile type caused by genotype-environment interaction.
  • the core of wheat heterosis utilization is to establish a high-efficiency technical system for the production of wheat hybrids, and the key to establishing efficient wheat hybrid seed production techniques is to have a suitable male sterile line.
  • Male sterility refers to the inability of plants to produce anthers, pollen or male gametes with normal functions during sexual reproduction.
  • the discovery of the mechanism of male sterility is the basis for improving the yield and quality of wheat by using heterosis.
  • the wheat genome is large and complex, so on wheat
  • the mechanism of male sterility research has so far accumulated very limited data. Therefore, the application of modern molecular biology and cell biology methods to the study of wheat male sterility mechanism has important theoretical and practical significance for strengthening wheat heterosis research and utilization.
  • the present invention provides a fertility related gene TaMS7, the nucleotide sequence of the fertility-related gene being selected from one of the following group of sequences:
  • the fertility-related genes of the present invention also include homologous gene sequences which are highly homologous to the nucleotide sequence or protein sequence of the TaMS7 gene and which have the same fertility regulation or recovery function.
  • the highly homologous homologous gene having a fertility regulating function includes a DNA sequence capable of hybridizing to a DNA having the sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6 under stringent conditions. Or a nucleotide sequence whose encoded amino acid sequence has 85% or more similarity to the amino acid sequence of the protein represented by SEQ ID NO: 7, 8, or 9.
  • stringent conditions include, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA, preferably at a hybridization temperature of 53 ° C to 60 ° C and a hybridization time of 12 - 16 hours, then washed with a washing solution containing 0.5 x SSC, and 0.1% SDS at 62 ° C - 68 ° C for 15-60 minutes.
  • the above homologous gene further comprises at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to the full length of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
  • a DNA sequence having sexual and regulatory functions can be isolated from any plant.
  • the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm. This is well known to those skilled in the art.
  • the present invention also provides an expression cassette comprising the DNA sequence of the fertility-related gene disclosed in the present invention,
  • the nucleotide sequence of the fertility-related gene is selected from one of the following group of sequences:
  • the fertility-related gene in the above expression cassette is operably linked to a promoter capable of driving expression thereof, including but not limited to a constitutive expression promoter, an inducible promoter, and tissue-specific expression. Promoters, or spatiotemporal specific expression promoters. More specifically, the promoter is an anther-specific expression promoter.
  • the nucleotide sequence of the anther-specific expression promoter is as set forth in SEQ ID NO: 16, 17, or 18.
  • the above expression cassette of the present invention further comprises a pollen inactivating gene which can interfere with the function or formation of a male gamete containing the pollen inactivating gene in the plant.
  • the pollen inactivating gene includes, but is not limited to, a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivating gene is a maize alpha amylase gene, preferably the nucleotide sequence thereof is shown in SEQ ID NO: 25.
  • the above expression cassette of the present invention further comprises a screening gene which can be used for screening plants, plant tissue cells or vectors containing the expression cassette.
  • the screening gene includes, but is not limited to, an antibiotic resistance gene, or a herbicide resistance gene, or a fluorescent protein gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene. Sex gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.
  • the invention also discloses a method for regulating plant fertility, wherein the male fertility of the ms7 male sterile mutant is restored by transferring the fertility-related gene into the ms7 male sterile mutant, wherein the The nucleotide sequence of the fertility-related gene is selected from one of the following group of sequences:
  • the present invention also provides a method for regulating plant fertility, which regulates the fertility of a plant by affecting the expression of the fertility gene TaMS7.
  • a method for regulating plant fertility which regulates the fertility of a plant by affecting the expression of the fertility gene TaMS7.
  • the mutation comprises substituting, deleting or adding one or more nucleotides on the nucleotide sequence of the fertility regulating gene.
  • Methods for obtaining mutations include, but are not limited to, physical mutagenesis, chemical mutagenesis, or gene editing methods such as RNAi, TALEN, CRISPR-Cas9, and the like.
  • the present invention also encompasses a method for obtaining a male sterile mutant of ms7 by mutating a plant endogenous fertility regulating gene TaMS7, or mutating a nucleotide sequence of a gene highly homologous thereto to cause the plant to be lost The process of male fertility.
  • the amino acid sequence of the fertility regulatory gene TaMS7 is set forth in SEQ ID NO: 7, 8, or 9.
  • the nucleotide sequence of the fertility regulatory gene TaMS7 is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
  • the "mutation” includes, but is not limited to, a method such as a mutation caused by a physical or chemical method, and the chemical method includes mutagenesis caused by treatment with a mutagen such as EMS, and the mutation may also be a point mutation. It may also be a DNA deletion or insertion mutation, or may be a method by gene silencing means such as RNAi or by site-directed mutagenesis, and the method of site-directed mutagenesis includes, but is not limited to, ZFN site-directed mutagenesis method, TALEN site-directed mutagenesis method, and/or Gene editing method such as CRISPR/Cas9.
  • the present invention also provides a method for applying a ms7 mutant material, characterized in that the mutant material is caused by a mutation of a nucleotide sequence, and the plant containing the nucleotide sequence of the mutation has a male sterile table.
  • a form wherein the nucleotide sequence is a nucleotide sequence of the TaMS7 gene, preferably as set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
  • the use of the mutant material includes, but is not limited to, the application in cross breeding, and more specifically, the ms7 mutant plant is used as a female parent of the sterile line to hybridize with the restorer line to produce hybrid seeds.
  • the invention also discloses a method for maintaining a male sterile line, wherein the ms7 male sterile mutant is used as a transforming receptor material, and the three closely related target genes are transformed into the sterile mutant recipient plant.
  • the three target genes are the fertility related gene TaMS7, the pollen inactivating gene and the screening marker gene, respectively.
  • the fertility-related gene TaMS7 can restore the transformation of infertility by sports.
  • the pollen inactivating gene can inactivate the pollen containing the transformed foreign gene, that is, lose the ability to inseign, and the screening gene can be used for transgenic seeds or tissues and Sorting of non-transgenic seeds or tissues, sorted non-transgenic seeds are used as hybrid lines for the production of sterile lines, and transgenic seeds are used as a source of maintenance to continuously and stably produce sterile lines.
  • the ms7 male sterile mutant in a wheat plant refers to a male sterile mutant caused by a TaMS7 mutation.
  • the ms7 male sterile mutant of the present invention may also be referred to as an ms7 male sterile line or an ms7 male sterile line.
  • the pollen-inactivated gene includes, but is not limited to, a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivating gene is a maize alpha amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 25.
  • the screening gene can be used to screen a plant or vector containing the expression cassette.
  • the screening gene includes, but is not limited to, an antibiotic resistance gene, or a herbicide resistance gene, or a fluorescent protein gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene.
  • the present invention also discloses a method for breeding a male sterile line, the method comprising the steps of:
  • the pollen inactivating gene includes, but is not limited to, a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivating gene is a maize alpha amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 25.
  • the pollen inactivating gene is linked to a promoter that is preferred for male gamete expression. More specifically, the promoter that favors expression of male gametes includes, but is not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene can be used to screen a plant or vector containing the expression cassette.
  • the screening gene includes, but is not limited to, an antibiotic resistance gene, or a herbicide resistance gene, or a fluorescent protein gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene.
  • the invention also discloses a production method of a retention system, the method comprising the following steps:
  • the pollen-inactivated gene includes, but is not limited to, a barnase gene, an amylase gene, a DAM methylase, and the like. More specifically, the pollen inactivating gene is a maize alpha amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 25.
  • the pollen inactivating gene is linked to a promoter that is preferred for male gamete expression. More specifically, the promoter that favors expression of male gametes includes, but is not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene can be used to screen a plant or vector containing the expression cassette.
  • the screening gene includes, but is not limited to, an antibiotic resistance gene, or a herbicide resistance gene, or a fluorescent protein gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene.
  • the invention also discloses a method for breeding a maintainer, the method comprising the following steps:
  • the invention also discloses a method for producing a seed, the method comprising:
  • the step (a) may also be a method of introducing a fertility-related gene TaMS7 into a common plant. a pollen-inactivated gene and a vector for screening the gene, obtaining a transgenic plant containing the vector, and then crossing with a male sterile line of ms7, and directionally selecting to obtain a male sterile line with a background of ms7 and containing the vector. Keep the plants.
  • nucleotide sequence of the fertility-related gene is selected from the following group of sequences One:
  • the above-mentioned fertility-related gene TaMS7 is also operably linked to an anther-specific promoter, which can drive the expression of TaMS7 gene in plant pollen.
  • the anther-specific promoter is selected from the group consisting of MS26, NP1, MSP1, PAIR1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, MS22, DPW, MADS3, OSC6,
  • One of the groups consisting of promoters of fertility regulatory genes such as RIP1, CSA, AID1, 5126 or Ms45. More specifically, the nucleotide sequence of the anther-specific expression promoter is shown in SEQ ID NO: 16, 17, or 18.
  • the above fertility related gene TaMS7 is also operably linked to a terminator which may be a terminator of any of the disclosed genes. Specifically, the nucleotide sequence of the terminator is SEQ ID NO: 22, 23 or 24 is shown
  • the pollen-inactivated gene includes, but is not limited to, a barnase gene, an amylase gene, and DAM A. Base enzymes, etc. More specifically, the pollen inactivating gene is a maize alpha amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 25.
  • the pollen inactivating gene is linked to a promoter that is preferred for male gamete expression. More specifically, the promoter that favors expression of male gametes includes, but is not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene includes, but not limited to, an antibiotic resistance gene and a herbicide resistance gene. Or fluorescent gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene. Sex gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.
  • the present invention also provides an anther-specific expression promoter having a nucleotide sequence as shown in SEQ ID NO: 16, 17 or 18.
  • SEQ ID NO: 16, 17 or 18 was ligated to the reporter gene GUS, and the vector was constructed to transform rice and wheat, and the GUS expression activity and expression pattern in the transgenic plants were detected and analyzed, and the GUS was carried out on the roots, stems, leaves and flowers of the transgenic plants. As a result of staining analysis, it was found that the promoter-driven GUS gene provided by the present invention was expressed in plant pollen.
  • the SEQ ID NO: 16, 17 or 18 provided by the present invention is an anther-specific expression promoter.
  • the plant anther-specific expression promoter provided by the present invention comprises the nucleotide sequence shown in SEQ ID NO: 16, 17 or 18 in the sequence listing, or comprises the core listed in SEQ ID NO: 16, 17 or 18. a nucleotide sequence having a nucleotide sequence greater than 90% similarity, or comprising 500 and more than 500 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 16, 17 or 18, and which can be driven with the promoter Expression of an operably linked nucleotide sequence in plant pollen. Expression vectors, transgenic cell lines, host bacteria and the like containing the above sequences are all within the scope of the present invention. Primer pairs that amplify any of the nucleotide fragments of the SEQ ID NO: 16, 17 or 18 promoter disclosed herein are also within the scope of the invention.
  • a “promoter” as used herein refers to a DNA regulatory region that typically comprises a TATA box that directs RNA polymerase II to initiate RNA synthesis at a suitable transcription initiation site for a particular coding sequence. Promoters may also contain additional recognition sequences, which are typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences for the promoter regions disclosed herein have been identified, other regulatory elements for isolating and identifying upstream regions of the TATA box of a particular promoter region identified in the present invention are also Within the scope of the invention.
  • the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, for regulating tissue expression and temporal expression functions of the coding sequences.
  • upstream regulatory elements such as those elements, enhancers, and the like
  • promoter elements that enable expression in a target tissue can be identified and isolated for use with other core promoters to verify the preferential expression of male tissues.
  • the core promoter refers to the minimal sequence required for initiation of transcription, such as the sequence known as the TATA box, which is commonly found in the promoters of genes encoding proteins.
  • the upstream promoter of the TaMS7 gene can be used in association with its own or a core promoter from other sources.
  • the core promoter may be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (U.S. Patent No. 5,352,605), the ubiquitin promoter (U.S. Patent No. 5,510,474), the IN2 core promoter ( U.S. Patent No. 5,364,780) or the Scrophularia mosaic virus promoter.
  • the function of the gene promoter can be analyzed by operably linking the promoter sequence to the reporter gene to form a transformable vector, and then transferring the vector into the plant, and observing the report in obtaining the transgenic progeny.
  • the expression of the gene in various tissues and organs of the plant is confirmed to confirm its expression characteristics; or the above vector is subcloned into an expression vector for transient expression experiments, and the function of the promoter or its regulatory region is detected by a transient expression experiment.
  • the choice of appropriate expression vector for testing the function of the promoter or regulatory region will depend on the host and the method by which the expression vector is introduced into the host, such methods being well known to those of ordinary skill in the art.
  • the regions in the vector include regions that control transcription initiation and control processing. These regions are operably linked to a reporter gene, including the YFP, UidA, GUS gene or luciferase.
  • An expression vector comprising a putative regulatory region located in a genomic fragment To be introduced into a complete tissue, such as staged pollen, or to introduce callus for functional verification.
  • the promoter of the present invention may also be ligated to a nucleotide sequence other than the TaMS7 gene to express other heterologous nucleotide sequences.
  • the promoter nucleotide sequences of the present invention, and fragments and variants thereof, can be assembled together with a heterologous nucleotide sequence in an expression cassette for expression in a plant of interest, more specifically, in a male organ of the plant. expression.
  • the expression cassette has suitable restriction sites for insertion of the promoter and heterologous nucleotide sequences.
  • the anther-specific expression promoter disclosed herein can be used to drive expression of a heterologous nucleotide sequence such that a transformed plant obtains a male sterile phenotype, which can encode a carbohydrate Degraded enzymes or modified enzymes, amylases, debranching enzymes, and pectinases, more specifically such as barnase gene, maize alpha amylase gene, auxin gene, rot B, cytotoxin gene, diphtheria toxin, DAM methylase Or a dominant male sterility gene.
  • a male sterile phenotype which can encode a carbohydrate Degraded enzymes or modified enzymes, amylases, debranching enzymes, and pectinases, more specifically such as barnase gene, maize alpha amylase gene, auxin gene, rot B, cytotoxin gene, diphtheria toxin, DAM methylase Or a dominant male sterility gene
  • nucleotide sequences referred to in the present invention are operably linked downstream of a promoter of the present invention, wherein said "nucleotide sequence" can be operably linked as disclosed herein.
  • the present invention also provides a transcription terminator sequence, the nucleotide sequence of which is represented by SEQ ID NO: 22, 23 or 24, and has a function of terminating transcriptional expression of a gene.
  • the invention also provides an expression cassette, vector or engineered strain comprising the anther-specific expression promoter SEQ ID NO: 16, 17 or 18 provided by the invention.
  • the nucleotide sequence of the fertility-related gene TaMS7 provided by the present invention can be constructed downstream of the promoter SEQ ID NO: 16, 17 or 18 provided by the present invention, thereby driving the fertility gene in transformation. Expression in body plants.
  • the anther-specific expression promoter provided by the invention can be used for the specific expression of a foreign gene in pollen, thereby avoiding the adverse effects of the sustained expression of the foreign gene in other tissues of the plant, and can also be used for plant pollen.
  • Functional analysis and identification of genes related to growth and development can be used for the creation of male sterile lines and maintainer lines; and can be applied to pollen abortion experiments to avoid biosafety problems caused by plant transgenic drift or pollen escape, The creation of plant male sterile lines and maintainer lines is of great significance.
  • the nucleotide sequence and promoter sequence or expression cassette of the TaMS7 gene provided by the present invention can be inserted into a vector, a plasmid, a yeast artificial chromosome, a bacterial artificial chromosome or any other vector suitable for transformation into a host cell.
  • Preferred host cells are bacterial cells, especially bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as Escherichia coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • the expression cassette or vector can be inserted into the genome of the transformed plant cell. Insertions can be either positioned or randomly inserted.
  • Transferring a nucleotide sequence, vector or expression cassette into a plant or introducing a plant or transforming a plant according to the present invention Refers to the transfer of a nucleotide sequence, vector or expression cassette into a recipient cell or recipient plant by conventional transgenic methods. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes Agrobacterium-mediated plant transformation methods and the like.
  • the present invention provides a fertility related gene TaMS7 and a promoter thereof, and a method for using the gene for propagation and maintenance of the ms7 male sterile line.
  • the fertility-related genes provided by the present invention, the fertility conservation of the male and male sterile lines, and the breeding methods of the sterile lines have great production and promotion value and application value for the cross breeding breeding of crops.
  • the fertility gene provided by the invention and the sterile line produced by the mutation of the gene provide resources for wheat cross breeding, and also provide necessary components for constructing the third generation hybrid breeding system, and the male sterile line produced by the mutation is used.
  • the production of hybrid seeds is of great significance for breaking through and improving the existing "three-line” and "two-line” hybrid technology.
  • Figure 1 is a graph showing the expression level of comp155942_c0_seq4 in anthers of pollen in meiosis (WT-0), mononuclear (WT-1), binuclear (WT-2) and trinuclear (WT-3),
  • the abscissa is the different developmental stages of pollen, and the ordinate is FPKM, reflecting the expression level of the gene.
  • Figure 2 is an RT-PCR analysis of three homologous genes of TaMS7 in anthers of different tissues and organs of wheat at different developmental stages; 1 indicates roots, 2 indicates stems, 3 indicates leaves, and 4 indicates tassels with pollen at meiosis.
  • 5 indicates the anther of the pollen in the mononuclear stage
  • 6 indicates the anther of the pollen in the binuclear phase
  • 7 indicates the anther of the pollen in the trinuclear phase
  • 8 indicates the flower organ of the pollen in the mononuclear stage except for the anther
  • 9 indicates Pollen is in the flower of the double-nuclear stage except for the flower organs other than the anther
  • 10 means that the pollen is in the flower of the trinuclear stage except for the flower organs other than the anther.
  • Figure 3 shows the genotype and phenotype of seven wheat nuclear recessive male sterile mutant lines obtained by CRISPR-Cas9 technology: a1 and a2 represent two types of TaMS7-A mutations, respectively, and b1 represents TaMS7-B mutation. One type, d1, d2, respectively represents two types of TaMS7-D mutations.
  • Figure 4 shows the traits of complete male sterility in wheat plants with TaMS7-A, TaMS7-B and TaMS7-D mutations:
  • Figure A shows the wild-type and mutant plants, and
  • Figure B shows the wild-type and The anthers and pistils of the mutant plants,
  • C is a photograph of I 2 -KI staining of wild type and mutant plant pollen.
  • Example 1 Whole genome expression profiling of wheat anthers at different developmental stages and acquisition of anther expression contig in early pollen development
  • the results of high-throughput sequencing of wheat transcriptome were first sequenced by Trinity software, and the resulting splicing sequence further removed redundancy and similarity clustering.
  • the high-throughput sequenced sequences in each sample were first aligned with the results of transcript splicing by TopHat (http://tophat.cbcb.umd.edu/) software.
  • TopHat http://tophat.cbcb.umd.edu/
  • the Cufflink software is then able to calculate the homogenous expression of the transcript contigs on the alignment, using the "fragments per kilobase of exon model per million mapped fragments (FPKM)" .
  • comp155942_c0_seq4 is highly expressed in the anthers of pollen in the meiotic and mononuclear phases and not in the anthers in which the pollen is in the dinuclear and trinuclear phases.
  • the gene corresponding to comp155942_c0_seq4 was named TaMS7.
  • the sequencing information of (Aegilops tauschii, D genome donor) was electronically cloned, and three TaMS7 genes were obtained, which were named TaMS7-A, TaMS7-B and TaMS7-D, respectively.
  • the genomic DNA sequences of the three TaMS7 genes are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the homology between the three is 95%-97%;
  • the CDS sequences are respectively SEQ. ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, the homology between the three is 97% - 98%;
  • the protein sequences are SEQ ID NO: 7, SEQ ID NO: 8 and As shown in SEQ ID NO: 9, the homology between the three is 98% to 99%.
  • TaMS7-A, TaMS7-B and TaMS7-D CDS were designed, and the three genes were used in addition to anthers and anthers in wheat roots, stems, leaves, and different developmental stages by RT-PCR. Expression specificity analysis was performed in various tissue materials such as other floral organs. The results are shown in Figure 2.
  • the TaMS7 gene is specific only in the anthers where the pollen is in the meiotic stage and the pollen is in the mononuclear stage. The expression was not expressed in the anthers of the pollen in the binuclear and trinuclear stages, and was not expressed in the flower organs and roots, stems and leaves except for anthers at various developmental stages, indicating that the TaMS7 gene is anther-specific.
  • a gene that is specifically expressed in anthers in the early stage of pollen development, and a promoter that drives its expression is also an anther-specific promoter.
  • the RT-PCR primers for the TaMS7-A gene are:
  • Primer 1 5'-ATACTGACACAAGTTTATGGGGCTG-3' (SEQ ID NO: 10)
  • Primer 2 5'-AATTACATTCAAATATGGCTCCTTG-3' (SEQ ID NO: 11)
  • the RT-PCR primers for the TaMS7-B gene are:
  • Primer 3 5'-GGCCTCGTGAACTCGTCGTATC-3' (SEQ ID NO: 12)
  • Primer 4 5'-TGAATTACATGCAAATTTGGCTCCG-3' (SEQ ID NO: 13)
  • the RT-PCR primers for the TaMS7-D gene are:
  • Primer 5 5'-ATGTCCAACCAGGAGCACTTCAC-3' (SEQ ID NO: 14)
  • TaMS7-A, TaMS7-B and TaMS7-D genes Based on the genomic DNA sequences of TaMS7-A, TaMS7-B and TaMS7-D genes, sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and wheat ancestral Uraltu wheat published in Nature 2013
  • the sequencing information of (Triticum urartu, A genome donor) and Aegilops tauschii (D genome donor) was electronically cloned, and the promoters of TaMS7-A, TaMS7-B and TaMS7-D genes were obtained and named as TaMS7-A promoter, TaMS7-B promoter and TaMS7-D promoter, in the present invention, the promoters may also be referred to as pTaMS7-A, pTaMS7-B and pTaMS7-D, respectively, and their lengths are 2601 bp and 2635 bp, respectively.
  • the nucleotide sequences thereof are shown in S
  • the present invention performed a functional verification experiment of the TaMS7-D promoter.
  • the plant expression vector pBI121 was digested with restriction endonucleases HindIII and EcoRI, and the 35S:GUS fragment was ligated into the pCAMBIA2300 vector of CAMBIA, which was also digested with HindIII and EcoRI, using T4 DNA ligase.
  • the new vector was named as P2300 35S: GUS.
  • Primer 7 5'-aagcttCTGACATAGTACATGTAATCTTTAAATCCATAAC-3' (SEQ ID NO: 19)
  • Primer 8 5'-ggatccTTGCGCCGGCGAGCTCGGC-3' (SEQ ID NO: 20)
  • sequence aagctt in primer 7 is the restriction site of HindIII
  • sequence ggatcc in primer 8 is the restriction site of BamHI.
  • primer 7 and primer 8 were used for amplification.
  • the reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 72 ° C for 2 minutes and 30 seconds; One cycle; extending at 72 ° C for 10 minutes.
  • the PCR product was detected by 1% agarose gel electrophoresis, and the product was ligated into the pMD20-T vector.
  • the positive clone was screened and verified by sequencing. The sequence is shown as SEQ ID NO: 18, and the plasmid is called T- pTaMS7-D.
  • the T-pTaMS7-D was digested with restriction endonucleases HindIII and BamHI, and the resulting TaMS7-D promoter was ligated into the p2300 35S:GUS vector digested with HindIII and BamHI using T4 DNA ligase to obtain the plant expression vector p2300pTaMS7- D: GUS.
  • the plant expression vector p2300TaMS7-Dp:GUS was transferred into Agrobacterium AGL0 strain by heat shock method.
  • Infected rice embryogenic callus with Agrobacterium cultured in dark for 2-3 days, and then subjected to two steps of resistance screening, pre-differentiation, differentiation and rooting culture, and finally obtained kanamycin-resistant, transferred p2300TaMS7 -Dp: GUS rice T 0 generation plants.
  • the TaMS7-D promoter can only initiate the expression of GUS gene in the anthers of pollen in meiosis and mononuclear stage, indicating that TaMS7-D is activated. Is a promoter of anther-specific expression in early pollen development. According to the functional verification process of the TaMS7-D promoter, the TaMS7-A promoter and the TaMS7-B promoter were also functionally verified. It was found that the TaMS7-A promoter and the TaMS7-B promoter are also an anther-specific promoter. It is consistent with the experimental results in Example 2.
  • the present invention uses the CRISPR-Cas9 technology to perform a site-directed mutagenesis experiment on the wheat genome.
  • the target sequence of the TaMS7 fertility gene is selected as CTGGTGGACCAGCCCATGGT (SEQ ID NO: 21), which is a consensus sequence of TaMS7-A, TaMS7-B, and TaMS7-D, respectively located in TaMS7-A.
  • the sgRNA expression cassette carrying the target sequence and the Cas9 expression cassette were constructed on the same vector pAHC20, and the wheat immature embryos were co-transformed by the gene gun method together with the plasmid expressing the bar gene, and the transgenic wheat plants were obtained.
  • Molecular identification of transgenic wheat plants revealed mutations in the positions of the TaMS7-A, TaMS7-B, and TaMS7-D gene targets. Among them, there were two types of mutations in the TaMS7-A gene, respectively. One base and 55 bases inserted; one of the TaMS7-B genes has a mutation of 13 bases and 54 bases. There are two types of mutations in the TaMS7-D gene, one is deletion of 9 bases, and the other is deletion of 7 bases while inserting 103 bases (see Figure 3).
  • the plants with homozygous mutation or biallelic mutation in the target sequences of TaMS7-A, TaMS7-B and TaMS7-D were phenotypically observed, and the anthers of plants with this genotype were found to be small and not cracked. There is no pollen inside, which is characterized by complete male sterility (see Figure 4), which is pollinated with wild type; while only one or two genes of TaMS7-A, TaMS7-B and TaMS7-D occur. Plants with homozygous mutations or biallelic mutations do not have a male sterile phenotype.
  • the TaMS7 gene of the present invention is a recessive nuclear male sterility gene.
  • the genomic DNA sequences (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) of the TaMS7-A, TaMS7-B and TaMS7-D coding regions were respectively constructed on the pAHC20 vector, and the respective upstream were added
  • the promoter sequence 2.5 kb (SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18) was used to drive expression of the gene, followed by a 1.4 kb downstream sequence (SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24), the vectors were designated as pAHC20pTaMS7-A: gTaMS7-A, pAHC20pTaMS7-B: gTaMS7-B and pAHC20pTaMS7-D: gTaMS7-D, respectively.
  • the above three functional complementary vectors were transformed or co-transformed into the wheat Tams7-abd mutant, and the pollen of the obtained transgenic positive plants was observed by I 2 -KI staining. The results showed that the transgenic positive plants had normal pollen development. Behaved as fertile. These analyses further demonstrate that the TaMS7 gene is involved in the regulation of pollen development, which results in a male sterility phenotype.
  • Example 8 Application of TaMS7 gene in a new generation of cross breeding technology
  • the TaMS7 gene can be used in a new generation of cross breeding technology.
  • the core idea of this technology is to use wheat nuclear recessive male sterility mutants as transforming receptor materials by transforming the three closely linked genes into infertile mutants. Among them, the recessive nuclear male sterility gene can make the transformation be restored by sports.
  • the pollen inactivating gene can inactivate the pollen containing the foreign gene, that is, lose the ability to insefine, and the screening gene can be used for the transgenic seed and the non-GM seed. Sorted, sorted non-transgenic seeds are sterile lines, and transgenic seeds are used as maintainers.
  • the sterile line By maintaining a pollination hybridization to the sterile line, the sterile line can be sturdy, thereby breeding the sterile line.
  • the maintainer system can be continuously reproduced through self-intersection. Because the technology uses biotechnology to produce non-GMO products, it solves the problem of artificial or mechanical emasculation during the hybrid seed production of wheat, eliminating the need for manual emasculation or mechanical emasculation, and can provide higher quality and purity seeds to growers. , saving labor costs.
  • a plant expression vector was constructed using the wheat TaMS7 gene.
  • the inventors Before constructing the plant expression vector of wheat, the inventors first separately transformed the three expression cassettes ZmBT1-ZmAA, TaMS7 and mCherryW in the expression vector, and further verified the function of each expression cassette. The results showed that each expression cassette was able to work well when transformed into wheat alone, achieving the desired design effect.
  • the inventors constructed a transformation vector by assembling the following DNA elements:
  • TaMS7 gene fertility restoration expression cassette containing the fertility related gene TaMS7 and its promoter and terminator are all from wheat variety CB037, and three TaMS7 genes from the A, B and D genomes in wheat, namely TaMS7-A, Both TaMS7-B and TaMS7-D can be used to restore the fertility of the wheat ms7 sterile line, or to restore the fertility of the wheat ms7 sterile line alone.
  • genomic DNA sequence, promoter sequence and terminator sequence of the TaMS7-A gene are shown in SEQ ID NO: 1, SEQ ID NO: 16 and SEQ ID NO: 22, respectively; genomic DNA sequence, promoter of TaMS7-B gene The sequence and terminator sequences are set forth in SEQ ID NO: 2, SEQ ID NO: 17 and SEQ ID NO: 23, respectively; the genomic DNA sequence, promoter sequence and terminator sequence of the TaMS7-D gene are respectively SEQ ID NO: 3 , SEQ ID NO: 18 and SEQ ID NO: 24.
  • Pollen inactivation gene expression cassette PG47 ZmBT1-ZmAA-IN2-1, the pollen inactivating gene is ZmAA, the transit peptide is ZmBT1, and ZmBT1-ZmAA (the nucleotide sequence thereof is shown as SEQ ID NO: 25) is open.
  • the reading frame is ligated downstream of the promoter PG47 (the nucleotide sequence of which is represented by SEQ ID NO: 26) and the terminator IN2-1 (the nucleotide sequence of which is shown by SEQ ID NO: 27).
  • CaMV35S enhancer-LTP2 mCherryW-PINII, open reading frame of mCherryW gene (SEQ ID NO: 28) linked to CaMV35S enhancer-LTP2 promoter (SEQ ID NO: 29) and PINII termination Between the sub-SEQ ID NO: 30, the gene expression cassette (CaMV35S enhancer-LTP2: mCherryW-PINII) of mCherryW was reconstituted.
  • the present invention simultaneously constructs a vector containing only a single TaMS7 gene, and two other pollen inactivating gene expression cassettes and a screening marker gene expression cassette in the TaMS7 gene fertility restoration expression cassette.
  • a TaMS7 gene fertility restorer expression cassette containing two TaMS7 genes, and two other pollen inactivating gene expression cassettes and a vector for screening marker gene expression cassettes were also constructed.
  • the above two vectors were transformed into wheat Tams7-abd mutants, respectively, and 8 transgenic plants of 12 strains, 12 strains and 9 transgenics were obtained.
  • the pollen activity of the transgenic plants and the control was tested.
  • the results showed that the non-transgenic wild-type wheat plants had less than 2% pollen sterility, and the non-transgenic wheat Tams7-abd mutant plants had no pollen in the anthers, while the transgenic plants were in the anthers.
  • the proportion of pollen, but sterile pollen is about 50%, indicating that the TaMS7 gene in the vector provided by the present invention restores the phenotype of the male sterility of the Tams7-abd mutant, and the ZmBT1-ZmAA gene can reach the expected pollen inactivation.
  • T 1 generation seed of the above transgenic plants that bear fluorescent segregation ratio survey results showed that these seeds showed 1: 1 segregation ratio, i.e. carrying a fluorescent seed foreign gene and do not carry non-fluorescent seed exogenous gene expression was 1: 1
  • Isolation indicates that the elements of the vector provided by the present invention are well expressed as a whole, and the purpose of creating and breeding a sterile line can be achieved; wherein the TaMS7 gene can restore the fertility of the male sterile mutant receptor, the ZmBT1-ZmAA gene and The expression of the mCherryW gene can achieve the expected pollen inactivation function and the seed fluorescent labeling function, respectively.
  • a new generation of hybrid breeding technology system is established, that is, based on the wheat ms7 male genic male sterile line, the above vector is transferred to form a maintainer, and the maintainer containing the exogenous vector can be used for a continuous production and maintenance system.
  • the ms7 sterile line to solve the problem that the wheat ms7 male sterility line cannot be propagated, and the obtained ms7 male sterile line can be used for production seed production and production of hybrids.
  • the above technical system can also be obtained by transferring the above-mentioned vector on the basis of common wild type wheat, and then crossing the plant transferred into the above vector with the ms7 male sterile line, and the plants obtained by the hybridization are self-crossed. Screening is performed to obtain a maintainer line containing the exogenous transfer vector in the background of a homozygous ms7 mutation, and the maintainer line containing the exogenous vector can be used for a continuous production maintainer line and a ms7 sterile line.

Abstract

一种小麦育性相关基因TaMS7及其应用方法，属于生物技术领域。通过对不同发育时期小麦花药的全基因组表达谱分析，获得了小麦育性相关基因TaMS7，并且通过调节该基因的表达以调控植物育性，用以生产并保持小麦雄性不育系以及制备杂交种子，对于建立高效的小麦杂交种制种技术、研究小麦雄性不育机理和杂种优势具有重要的理论和实践意义。

Description

小麦育性相关基因TaMS7及其应用方法 技术领域
本发明属于生物技术领域，具体涉及小麦的杂交育种方法，包括小麦核不育系繁殖和杂交种子制备，更具体地涉及一个小麦雄性核不育基因及其突变体及其在杂交育种中的应用。
技术背景
小麦是自花授粉作物，其杂种优势利用的核心是建立高效生产小麦杂交种的技术体系。目前小麦生产上杂种优势利用的途径主要有三种：一是三系法，即利用核质互作雄性不育系，通过不育系、保持系和恢复系三系互作配套生产杂交种；二是化杀法，即应用化学杀雄剂诱导小麦雄性不育以进行杂交制种；三是二系法，即利用光温敏不育系随光温的变化进行杂交制种。三系法自上世纪50年代开始研究，迄今已经育成的小麦核质互作不育系有70多种，研究最多的有T型、K型、Q型等不育系，其胞质主要来自各种山羊草、提莫菲维小麦、野生燕麦、簇毛麦等。虽然这些不育系大都实现了三系配套，但是由于其自身在遗传上存在一些难以克服的缺陷，比如恢复源较少、异源细胞质的不良影响、强优势组合选出率低、不育系种子纯度低等，都未能在生产上大面积推广应用。上世纪80年代，化杀法的研究进入高潮，由于克服了三系法的种种问题，化杀法曾被认为是一种很有希望的杂交制种新技术。但是化学杀雄剂存在稳定性差、易残留、容易产生毒副作用、环境污染重等问题，因此想要大面积应用于生产，关键还需要研制出一种理想的化学杀雄剂。在三系法和化杀法困难重重的情况下，二系法杂交小麦发展迅速并逐渐成为今后小麦杂种优势研究和利用的主要发展趋势。二系法的核心是光温敏不育系，这是一种基因型与环境互作导致的雄性不育类型。由于实现了一系两用(兼具不育系和保持系的功能)，简化了不育系的繁殖程序，制种简单且恢复源广，容易获得优势组合，因此具有很高的推广价值。目前我国独创的二系法杂交小麦应用技术体系，整体领先于国际水平，选育的杂交小麦品种抗逆性强，水肥利用率高，增产幅度可达15％-20％，对粮食生产节本增效作用显著。但是，光温敏不育系的雄性不育高度依赖环境因素，环境的变化严重影响杂种种子的产量、质量和纯度，因此，两系法杂交小麦突破生产尚需时日，关键是要解决环境的影响。
在上述传统的常规育种模式遇到瓶颈的情况下，以现代分子生物学为主导的生物育种技术很可能是突破瓶颈的关键。小麦杂种优势利用的核心是建立高效的生产小麦杂交种的技术体系，而建立高效的小麦杂交种制种技术的关键是要有合适的雄性不育系母本。雄性不育指的是植物在有性繁殖过程中不能产生具有正常功能的花药、花粉或雄配子，对雄性不育机理的揭示，是利用杂种优势提高小麦产量和品质的基础。小麦基因组庞大且复杂，所以对小麦 雄性不育的机理研究迄今为止积累的资料非常有限。因此，将现代分子生物学和细胞生物学手段应用于小麦雄性不育机理研究，对于加强小麦杂种优势研究和利用具有重要的理论和实践意义。
发明内容
本文提到的所有参考文献都通过引用并入本文。
除非有相反指明，本文所用的所有技术和科学术语都具有与本发明所属领域普通技术人员通常所理解的相同的含义。除非有相反指明，本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用，而非加以限制。
本发明提供了一个育性相关基因TaMS7，所述育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
(d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性相关功能的DNA序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
本领域技术人员应该知晓，本发明所述的育性相关基因还包括与TaMS7基因的核苷酸序列或蛋白序列高度同源，并且具有同样的育性调控或恢复功能的同源基因序列。所述高度同源且具有育性调控功能的的同源基因包括在严谨条件下能够与具有SEQ ID NO：1、2、3、4、5或6所示序列的DNA杂交的DNA序列。或是其编码的氨基酸序列与SEQ ID NO：7、8或9所示的蛋白氨基酸序列具有85％以上相似性的核苷酸序列。本文中使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中于杂交，优选杂交温度是53℃-60℃，杂交时间为12-16小时，然后在62℃-68℃下用含0.5×SSC、和0.1％SDS的洗涤液洗涤15-60分钟。
上述同源基因还包括与SEQ ID NO：1、2、3、4、5或6所示序列的全长有至少80％、85％、90％、95％、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、Needleman-Wunsch全局比对法、Smith-Waterman局部比对法、Pearson和Lipman相似性搜索法、Karlin和Altschul的算法。这对于本领域技术人员来说是公知的。
本发明还提供了一种表达盒，所述表达盒含有本发明所公开的育性相关基因的DNA序列， 所述育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
(d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
具体地，上述表达盒中的育性相关基因还可操作性的连有一个可驱动其表达的启动子，所述启动子包括但不限于组成型表达启动子、诱导型启动子、组织特异表达启动子、或时空特异表达启动子。更具体地，所述启动子是一个花药特异表达启动子。优选地，所述花药特异表达启动子的核苷酸序列如SEQ ID NO：16、17或18所示。
本发明上述表达盒，还进一步的包含一个花粉失活基因，所述花粉失活基因可以干扰植株中含有该花粉失活基因的雄性配子的功能或形成。所述花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等。更具体的，所述花粉失活基因是玉米α淀粉酶基因，优选其核苷酸序列如SEQ ID NO：25所示。
本发明上述表达盒，还进一步的包含一个筛选基因，所述筛选基因可以用于将含有该表达盒的植株、植物组织细胞或载体筛选出来。所述筛选基因包括但不限于抗生素抗性基因、或是抗除草剂基因、或是荧光蛋白基因等。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还公开了一种植物育性调控的方法，所述方法通过将育性相关基因转入到ms7雄性不育突变体，使ms7雄性不育突变体的雄性育性恢复，其中所述的育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
(d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
本发明还提供了一种调控植物育性的方法，通过影响育性基因TaMS7的表达来调控植物的育性。包括但不限于通过突变TaMS7基因的方法，获得ms7雄性不育突变体材料；或是通过基因互补的方法，将TaMS7基因用于恢复由相应的TaMS7基因突变所导致的雄性不育，使ms7雄性不育突变体恢复成可育。在本发明中，所述的突变包括在育性调控基因的核苷酸序列上进行取代、缺失或添加一个或多个核苷酸。获得突变的方法包括但不限于物理诱变、化学诱变或RNAi、TALEN、CRISPR-Cas9等基因编辑方法。
本发明还包括一种获得ms7雄性不育突变体的方法，所述方法通过突变植物内源的育性调控基因TaMS7，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。所述育性调控基因TaMS7的氨基酸序列如SEQ ID NO：7、8或9所示。所述育性调控基因TaMS7的核苷酸序列如SEQ ID NO：1、2、3、4、5或6所示。所述“突变”包括但不限于以下方法，如用物理或化学的方法所导致的基因突变，化学方法包括用EMS等诱变剂处理所导致的诱变，所述突变还可以是点突变，也可以是DNA缺失或插入突变，也可以是通过RNAi等基因沉默手段或者通过基因定点突变的方法，所述基因定点突变的方法包括但不限于ZFN定点突变方法、TALEN定点突变方法、和/或CRISPR/Cas9等基因编辑方法。
本发明还提供了一种ms7突变体材料的应用方法，其特征在于所述突变材料是由核苷酸序列的突变所造成，含有该突变后的核苷酸序列的植株具有雄性不育的表型，其中所述核苷酸序列为TaMS7基因的核苷酸序列，优选如SEQ ID NO：1、2、3、4、5或6所示。所述突变体材料的应用，包括但不限于在杂交育种中的应用，更具体的是指将ms7突变体植株作为不育系母本，与恢复系杂交，生产杂交种子。
本发明还公开了一种雄性不育系的保持方法，所述方法以ms7雄性不育突变体为转化受体材料，将紧密连锁的3个目标基因转化至该不育突变体受体植株中。所述3个目标基因分别是育性相关基因TaMS7、花粉失活基因和筛选标记基因。其中，育性相关基因TaMS7可使不育的转化受体育性恢复，花粉失活基因可使含有转化的外源基因的花粉失活，即失去授精能力，筛选基因可以用于转基因种子或组织和非转基因种子或组织的分拣，分拣出的非转基因种子用作不育系生产杂交种，转基因种子用作保持系来源源不断地、稳定地生产不育系。
本发明中，在小麦植株中所述ms7雄性不育突变体是指由TaMS7突变所导致的雄性不育突变体。本发明中ms7雄性不育突变体也可以称为ms7不育系或ms7雄性不育系。
上述雄性不育系的保持方法中，所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等。更具体的，所述花粉失活基因是玉米α淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO：25所示。所述花粉失活基因与偏好于雄性配子表达的启动子相 连。更具体地，所述偏好于雄性配子表达的启动子包括但不限于PG47启动子、Zm13启动子等。所述筛选基因可以用于将含有该表达盒的植株或载体筛选出来。所述筛选基因包括但不限于抗生素抗性基因、或是抗除草剂基因、或是荧光蛋白基因等。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
更具体地，本发明还公开了一种雄性不育系的繁殖方法，所述方法包括以下步骤：
(a)向ms7雄性不育系中转入下述载体，以获得含有下述载体的保持系，所述载体包含：育性相关基因TaMS7，所述育性相关基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的具有活性的雄性配子都是不含所述载体的；和筛选基因，所述筛选基因可以用于转基因种子或组织和非转基因种子或组织的分拣。
(b)将转入上述载体后形成的保持系植株自交，同时产生不含载体的ms7雄性不育系和含载体的保持系种子；或是将保持系植株的花粉赶到ms7雄性不育系植株上，使ms7雄性不育系授粉繁殖出ms7雄性不育系的种子。
上述雄性不育系的繁殖方法中，所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等。更具体的，所述花粉失活基因是玉米α淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO：25所示。所述花粉失活基因与偏好于雄性配子表达的启动子相连。更具体地，所述偏好于雄性配子表达的启动子包括但不限于PG47启动子、Zm13启动子等。所述筛选基因可以用于将含有该表达盒的植株或载体筛选出来。所述筛选基因包括但不限于抗生素抗性基因、或是抗除草剂基因、或是荧光蛋白基因等。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还公开了一种保持系的生产方法，所述方法包括以下步骤:
(a)向ms7雄性不育系中转入下述载体，即获得了ms7雄性不育系的保持系，所述载体包含：育性相关基因TaMS7，所述育性相关基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的可育雄性配子都是不含所述载体的；和筛选基因，所述筛选基因可以用于转基因种子和非转基因种子的分拣。
上述保持系的生产方法中，所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等。更具体的，所述花粉失活基因是玉米α淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO：25所示。所述花粉失活基因与偏好于雄性配子表达的启动子相连。更具体地，所述偏好于雄性配子表达的启动子包括但不限于PG47启动子、Zm13启动子等。所述筛选基因可以用于将含有该表达盒的植株或载体筛选出来。所述筛选基因包括但不限于抗生素抗性基因、或是抗除草剂基因、或是荧光蛋白基因等。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还公开了一种保持系的繁殖方法，所述方法包括以下步骤：
(a)向ms7雄性不育系中转入下述载体，即获得了ms7雄性不育系的保持系，所述载体包含：育性相关基因TaMS7，所述育性相关基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的可育雄性配子都是不含所述载体的；和筛选基因，所述筛选基因可以用于转基因种子和非转基因种子的分拣；和
(b)将转入上述载体后形成的保持系植株自交，即按1:1的比例繁殖获得了不含载体的ms7雄性不育系种子和含载体的保持系种子。
本发明还公开了一种种子的生产方法，所述方法包括：
(a)向ms7雄性不育系中引入下述载体，获得ms7雄性不育系的保持系，所述载体包含：育性相关基因TaMS7，所述育性相关基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的可育雄性配子都是不含所述载体的。
(b)将转入上述载体后的保持系植株自交；和
(c)自交后即获得含有所述载体的保持系种子和不含载体的ms7雄性不育系。
本发明上述的雄性不育系的繁殖或保持方法、保持系的生产方法或繁殖方法、种子的生产方法等中，其中步骤(a)也可以是向普通的植株中引入含有育性相关基因TaMS7、花粉失活基因和筛选基因的载体，获得含有所述载体的转基因植株后，再与ms7雄性不育系杂交，经过定向选育，获得背景为ms7雄性不育系、并且含有所述载体的保持系植株。
本发明上述的雄性不育系的繁殖方法或保持方法、保持系的生产方法或繁殖方法、种子的生产方法等中，其中所述的育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
(d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
上述育性相关基因TaMS7还可操作性的连有一个花药特异表达的启动子，可以驱动TaMS7基因在植物花粉中的表达。所述花药特异表达的启动子选自由MS26、NP1、MSP1、PAIR1、PAIR2、ZEP1、MELL、PSS1、TDR、UDT1、GAMYB4、PTC1、API5、WDA1、CYP704B2、MS26、MS22、DPW、MADS3、OSC6、RIP1、CSA、AID1、5126或Ms45等育性调控基因的启动子构成的组之一。更具体的，所述花药特异表达启动子的核苷酸序列如SEQ ID NO：16、17或18所示。上述育性相关基因TaMS7还可操作性的连有一个终止子，所述终止子可以是已经公开的任一个基因的终止子，具体地，所述终止子的核苷酸序列如SEQ ID NO：22、23或24所示。
本发明上述的雄性不育系的繁殖或保持方法、保持系的生产方法或繁殖方法、种子的生产方法等中，所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等。更具体的，所述花粉失活基因是玉米α淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO：25所示。所述花粉失活基因与偏好于雄性配子表达的启动子相连。更具体地，所述偏好于雄性配子表达的启动子包括但不限于PG47启动子、Zm13启动子等。
本发明上述的雄性不育系的繁殖或保持方法、保持系的生产方法或繁殖方法、种子的生产方法等中，其中所述的筛选基因包括但不限于抗生素抗性基因、除草剂抗性基因或荧光基因。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还提供了一种花药特异表达启动子，其核苷酸序列如SEQ ID NO：16、17或18所示。将SEQ ID NO：16、17或18与报告基因GUS相连，构建载体转化水稻和小麦，检测分析转基因植株中的GUS表达活性和表达模式，通过对转基因植株的根、茎、叶和花进行GUS染色分析，结果发现本发明所提供的启动子驱动GUS基因在植物花粉中表达。说明本发明所提供的SEQ ID NO：16、17或18是一个花药特异性表达的启动子。
本发明所提供的植物花药特异表达启动子，含有序列表中如SEQ ID NO：16、17或18所示的核苷酸序列，或包含与SEQ ID NO：16、17或18中所列核苷酸序列具有90％以上相似性的核苷酸序列，或包含来源于SEQ ID NO：16、17或18序列上的500个及500以上连续的核苷酸片段，并且可以驱动与该启动子操作性连接的核苷酸序列在植物花粉中的表达。含有上述序列的表达载体、转基因细胞系以及宿主菌等均属于本发明的保护范围。扩增本发明所公开的SEQ ID NO：16、17或18启动子的任一核苷酸片段的引物对也在本发明的保护范围之内。
本发明所述的“启动子”是指一种DNA调控区域，其通常包含能指导RNA聚合酶II在特定编码序列的合适转录起始位点起始RNA合成的TATA盒。启动子还可包含其它识别序列，这些识别序列通常位于TATA盒的上游或5’端，通常被称为上游启动子元件，起调控转录效率的作用。本领域技术人员应该知晓，虽然已经鉴定了针对本发明公开的启动子区域的核苷酸序列，但是分离和鉴定处于本发明鉴定的特定启动子区域的TATA盒上游区域的其它调控元件也在本发明的范围内。因此，本文公开的启动子区域通常被进一步界定为包含上游调控元件，例如用于调控编码序列的组织表达性和时间表达功能的那些元件、增强子等。以相同的方式，可以鉴定、分离出使得能在目标组织(例如雄性组织)中进行表达的启动子元件，将其与其它核心启动子一起使用，以验证雄性组织优先的表达。核心启动子指起始转录所需的最小限度的序列，例如被称为TATA盒的序列，这是编码蛋白质的基因的启动子通常都具有的。因此，可选地，TaMS7基因的上游启动子可与其自身的或来自其它来源的核心启动子关联使用。
核心启动子可以是任何一种已知的核心启动子，例如花椰菜花叶病毒35S或19S启动子(美国专利No.5,352,605)、泛素启动子(美国专利No.5,510,474)、IN2核心启动子(美国专利No.5,364,780)或玄参花叶病毒启动子。
所述基因启动子的功能可以通过以下方法进行分析：将启动子序列与报告基因可操作性连接，形成可转化的载体，再将该载体转入植株中，在获得转基因后代中，通过观察报告基因在植物各个组织器官中的表达情况来确认其表达特性；或者将上述载体亚克隆进用于瞬时表达实验的表达载体，通过瞬时表达实验来检测启动子或其调控区的功能。
用来测试启动子或调控区域功能的适当表达载体的选择将取决于宿主和将该表达载体引入宿主的方法，这类方法是本领域普通技术人员所熟知的。对于真核生物，在载体中的区域包括控制转录起始和控制加工的区域。这些区域被可操作地连接到报告基因，所述报告基因包括YFP、UidA、GUS基因或荧光素酶。包含位于基因组片段中的推定调控区的表达载体可 以被引入完整的组织，例如阶段性花粉，或引入愈伤组织，以进行功能验证。
此外，本发明的启动子还可与并非TaMS7基因的核苷酸序列相连，以表达其它异源核苷酸序列。本发明的启动子核苷酸序列及其片段和变体可与异源核苷酸序列一起组装在一个表达盒中，用于在目的植株中表达，更具体地，在该植株的雄性器官中表达。所述表达盒有合适的限制性酶切位点，用于插入所述启动子和异源核苷酸序列。这些表达盒可用于对任何植株进行遗传操作，以获得想要的相应表型。
本发明所公开的花药特异表达启动子，可用于驱动下列异源核苷酸序列的表达，以使转化的植株获得雄性不育的表型，所述异源核苷酸序列可编码促使碳水化合物降解的酶或修饰酶、淀粉酶、脱支酶和果胶酶，更具体的如barnase基因、玉米α淀粉酶基因、生长素基因、rot B、细胞毒素基因、白喉毒素、DAM甲基化酶，或是显性的雄性不育基因。
在某些实施方式中，本发明中所提到的可操作性地连接在本发明启动子下游的核苷酸序列，其中所述的“核苷酸序列”可以是操作性连接于本文所公开的启动子之后的结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA。
本发明还提供了一个转录终止子序列，所述转录终止子的核苷酸序列如SEQ ID NO：22、23或24所示，具有终止基因转录表达的功能。
本发明还提供了一种表达盒、载体或工程菌株，所述表达盒、载体或工程菌株中包含了本发明所提供的花药特异表达启动子SEQ ID NO：16、17或18。具体地，可以将本发明所提供的育性相关基因TaMS7的核苷酸序列构建到本发明所提供的启动子SEQ ID NO：16、17或18的下游，从而驱动该育性基因在转化受体植株中的表达。
本发明的所提供的花药特异表达启动子可用于外源基因在花粉中的特异性表达，从而避免该外源基因在植物其他组织中持续表达所带来的不利影响，还可以用于植物花粉生长发育相关基因的功能分析和鉴定；可用于雄性不育系和保持系的创建；并可应用于花粉败育实验中，从而避免由植物转基因漂移或花粉逃逸所带来的生物安全问题，对植物雄性不育系和保持系的创造具有重要意义。
本发明所提供的TaMS7基因的核苷酸序列和启动子序列或表达盒可被插入载体、质粒、酵母人工染色体、细菌人工染色体或其他适合转化进宿主细胞中的任何载体中。优选的宿主细胞是细菌细胞，尤其是用于克隆或储存多核苷酸、或用于转化植物细胞的细菌细胞，例如大肠杆菌、根瘤土壤杆菌和毛根土壤杆菌。当宿主细胞是植物细胞时，表达盒或载体可插入至被转化的植物细胞的基因组中。插入可以是定位的或随机的插入。
本发明所述的将核苷酸序列、载体或表达盒转入植株或引入植株或对植株进行转化，均 指通过常规的转基因方法，将核苷酸序列、载体或表达盒转入到受体细胞或受体植株中。植物生物技术领域技术人员已知的任何转基因方法均可被用于将重组表达载体转化进植物细胞中，以产生本发明的转基因植物。转化方法可包括直接和间接的转化方法。合适的直接方法包括聚乙二醇诱导的DNA摄入、脂质体介导的转化、使用基因枪导入、电穿孔、以及显微注射。所述转化方法也包括农杆菌介导的植物转化方法等。
与现有技术相比，本发明具有如下的有益效果：本发明提供了一种育性相关基因TaMS7及其启动子，及将该基因用于ms7雄性不育系的繁殖和保持的方法。本发明所提供的育性相关基因、核雄性不育系的育性保持和不育系的繁殖方法，对作物的杂交育种生产来说，具有重大的生产推广价值和应用价值。本发明提供的育性基因以及该基因突变产生的不育系为小麦杂交育种提供了资源，也为构建第三代杂交育种体系提供了必要的元件，该基因突变产生的雄性不育系，用来生产杂交种子，对于突破并改良现有的“三系”和“两系”杂交技术有重要意义。
附图说明
图1是comp155942_c0_seq4在花粉处于减数分裂期(WT-0)、单核期(WT-1)、双核期(WT-2)和三核期(WT-3)的花药中的表达水平分析，横坐标是花粉不同发育时期，纵坐标是FPKM，反映基因的表达水平。
图2是TaMS7的3个同源基因在小麦不同组织器官和不同发育时期的花药中的RT-PCR分析；1表示根，2表示茎，3表示叶片，4表示花粉处于减数分裂期的穗子，5表示花粉处于单核期的花药，6表示花粉处于双核期的花药，7表示花粉处于三核期的花药，8表示花粉处于单核期的花中除花药以外其它的花器官，9表示花粉处于双核期的花中除花药以外其它的花器官，10表示花粉处于三核期的花中除花药以外其它的花器官。
图3是采用CRISPR-Cas9技术得到的7个小麦核隐性雄性不育突变体株系的基因型和表型：a1、a2分别表示TaMS7-A突变的两种类型，b1表示TaMS7-B突变的一种类型，d1、d2分别表示TaMS7-D突变的两种类型。
图4是TaMS7-A、TaMS7-B、TaMS7-D三个基因均发生突变后的小麦植株具有完全雄性不育的性状：A图为野生型与突变体植株的穗，B图为野生型与突变体植株的花药和雌蕊，C图为野生型与突变体植株花粉的I2-KI染色照片。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1.不同发育时期小麦花药的全基因组表达谱分析和花粉发育早期花药表达contig的获得
取花粉处于减数分裂期、单核期、双核期和三核期的小麦花药，用Trizol(Invitrogen)提取总RNA，并进行DNaseI(Promega)处理，进而纯化mRNA(Ambion)。将纯化的mRNA进行反转录(Invitrogen)、超声打断(Fisher)、制备文库(illumina)并扩增(illumina)，最后在illumina机器上进行测序反应。
小麦转录组高通量测序的结果首先通过Trinity软件进行序列拼接，得到的拼接序列进一步去除冗余以及相似性聚类。对于拼接得到的转录本contig的表达变化分析，各样品中高通量测序的序列首先通过TopHat(http://tophat.cbcb.umd.edu/)软件与转录本拼接的结果进行比对。而后Cufflink软件能够计算比对上的转录本contigs的均一化表达量，用“外显子每百万比对片段的千碱基数(fragments per kilobase of exon model per million mapped fragments，FPKM)”表示。
通过对不同发育时期小麦花药的全基因组表达谱分析，找到花粉处于减数分裂期和单核期的花药中高表达而在花粉处于双核和三核期的花药中不表达的转录本contig7231个。如图1所示，comp155942_c0_seq4在花粉处于减数分裂期和单核期的花药中高表达而在花粉处于双核和三核期的花药中不表达。将comp155942_c0_seq4所对应的基因命名为TaMS7。
实施例2.RT-PCR验证TaMS7基因的组织表达特异性
由于小麦是由A、B、D三套基因组组成的异源六倍体，基因的平均拷贝数为2.8个，其中接近一半的基因(46％)有3-4个拷贝，12％的基因有1-2个拷贝，42％的基因拷贝数≥5个。从comp155942_c0_seq4的序列出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了3个TaMS7基因，分别命名为TaMS7-A，TaMS7-B和TaMS7-D。3个TaMS7基因的基因组DNA序列分别为SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3所示，三者之间的同源性为95％-97％；CDS序列分别如SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6所示，三者之间的同源性为97％-98％；蛋白序列分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示，三者之间的同源性为98％-99％。分别设计针对TaMS7-A，TaMS7-B和TaMS7-D CDS的特异性引物，利用RT-PCR方法，对这三个基因在在小麦根、茎、叶、不同发育时期的花药及除花药以外的其他花器官等多种组织材料中进行表达特异性分析，结果如图2所示，TaMS7基因只在花粉处于减数分裂期的穗和花粉处于单核期的花药中特异 表达，在花粉处于双核期和三核期的花药中不表达，在花粉处于各发育时期的除花药以外的其他花器官和根、茎、叶中都不表达，说明TaMS7基因是花药特异表达、且只在花粉发育早期的花药中特异表达的基因，驱动其表达的启动子也是一个花药特异表达的启动子。
TaMS7-A基因的RT-PCR引物为：
引物1：5'-ATACTGACACAAGTTTATGGGGCTG-3'(SEQ ID NO:10)
引物2：5'-AATTACATTCAAATATGGCTCCTTG-3'(SEQ ID NO:11)
TaMS7-B基因的RT-PCR引物为：
引物3：5'-GGCCTCGTGAACTCGTCGTATC-3'(SEQ ID NO:12)
引物4：5'-TGAATTACATGCAAATTTGGCTCCG-3'(SEQ ID NO:13)
TaMS7-D基因的RT-PCR引物为：
引物5：5'-ATGTCCAACCAGGAGCACTTCAC-3'(SEQ ID NO:14)
引物6：5'-GCACAGTTTTTTGAAGCAATGTTG-3'(SEQ ID NO:15)
实施例3.TaMS7基因启动子序列的获得
从TaMS7-A、TaMS7-B和TaMS7-D基因的基因组DNA序列出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了TaMS7-A、TaMS7-B和TaMS7-D基因的启动子，分别命名为TaMS7-A启动子、TaMS7-B启动子和TaMS7-D启动子，在本发明中所述启动子也可分别称为pTaMS7-A、pTaMS7-B和pTaMS7-D，其长度分别为2601bp、2635bp和2821bp，其核苷酸序列分别如SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示。
实施例4.TaMS7-D启动子的克隆和植物表达载体的构建
为了进一步核实上述启动子的功能，本发明进行了TaMS7-D启动子的功能验证实验。将植物表达载体pBI121用限制性内切酶HindIII和EcoRI双酶切，得到的35S:GUS片段用T4DNA ligase连入同样用HindIII和EcoRI双酶切的CAMBIA公司的pCAMBIA2300载体，新的载体被命名为p2300 35S:GUS。
从TaMS7-D启动子的5’端和ATG上游设计引物：
引物7：5’-aagcttCTGACATAGTACATGTAATCTTTAAATCCATAAC-3’(SEQ ID NO:19)
引物8：5’-ggatccTTGCGCCGGCGAGCTCGGC-3’(SEQ ID NO:20)
引物7中序列aagctt是HindIII的酶切位点，引物8中序列ggatcc是BamHI的酶切位点。
以小麦的基因组DNA为模板，用引物7和引物8进行扩增，反应条件是：94℃预变性5分钟；94℃变性30秒；60℃退火30秒；72℃延伸2分30秒；35个循环；72℃延伸10分钟。反应结束后，PCR产物经1％琼脂糖凝胶电泳检测回收，产物连入pMD20-T载体中，筛选阳性克隆并进行测序验证，序列如SEQ ID NO:18所示，该质粒称为T-pTaMS7-D。
用限制性内切酶HindIII和BamHI双酶切T-pTaMS7-D，得到的TaMS7-D启动子用T4DNA ligase连入用HindIII和BamHI双酶切的p2300 35S:GUS载体，得到植物表达载体p2300pTaMS7-D:GUS。
实施例5.水稻的遗传转化和转基因水稻植株不同组织器官GUS基因表达的组织化学检测
利用热激法将植物表达载体p2300TaMS7-Dp:GUS转入农杆菌AGL0菌株。
用农杆菌侵染水稻胚性愈伤，暗中共培养2-3天，然后经过两步抗性筛选、预分化、分化和生根培养等步骤，最终获得具有卡那霉素抗性的、转p2300TaMS7-Dp:GUS水稻T0代植株。
选择合适大小的转基因幼苗或特定组织浸入GUS染液中，37℃染色过夜，吸去反应液，乙醇梯度脱色，显微镜观察。结果表明，在转基因水稻的根、茎和叶等营养器官中都检测不到GUS基因的表达，在花粉处于双核期和三核期的花药和其他花器官、花粉处于减数分裂期和单核期的除花药以外的其它花器官中也检测不到GUS基因的表达，TaMS7-D启动子只能启动GUS基因在花粉处于减数分裂期和单核期的花药中表达，说明TaMS7-D启动子是一个花粉发育早期花药特异表达的启动子。按照TaMS7-D启动子的功能验证过程，对TaMS7-A启动子和TaMS7-B启动子也进行了功能验证，发现TaMS7-A启动子和TaMS7-B启动子也是一个花药特异表达的启动子，和实施例2中的实验结果一致。
实施例6.TaMS7育性基因的定点敲除及突变体的表型分析
本发明采用CRISPR-Cas9技术对小麦基因组进行定点突变实验。具体的，在本发明中，TaMS7育性基因的靶序列选为CTGGTGGACCAGCCCATGGT(SEQ ID NO：21)，该序列为TaMS7-A、TaMS7-B、TaMS7-D的共有序列，分别位于TaMS7-A正链中的第1017-1036位核苷酸、TaMS7-B正链中的第1035-1054位核苷酸、TaMS7-D正链中的第1038-1057位核苷酸。将带有靶序列的sgRNA表达框与Cas9表达框构建到同一载体pAHC20上，与表达bar基因的质粒一起，利用基因枪法共转化小麦幼胚，获得了转基因小麦植株。
对转基因小麦植株进行分子鉴定，发现在TaMS7-A、TaMS7-B、TaMS7-D三个基因靶序列的位置都发生了突变：其中TaMS7-A基因发生的突变有两种类型，分别为缺失5个碱基和插入55个碱基；TaMS7-B基因发生的突变有一种，为缺失13个碱基的同时又插入了54个碱 基；TaMS7-D基因的突变有两种类型，一种为缺失9个碱基，另一种为缺失7个碱基的同时插入103个碱基(见图3)。
对TaMS7-A、TaMS7-B、TaMS7-D三个基因的靶序列都发生纯合突变或双等位基因突变的植株进行表型观察，发现具有这种基因型的植株的花药瘦小、不开裂、内部没有花粉，表现为完全的雄性不育(见图4)，用野生型对其授粉保种；而TaMS7-A、TaMS7-B、TaMS7-D三个基因中只有一个或两个基因发生纯合突变或双等位基因突变的植株，没有雄性不育表型。说明本发明的TaMS7基因就是一个隐性的核雄性不育基因。
实施例7.转基因功能互补
将TaMS7-A，TaMS7-B和TaMS7-D编码区的基因组DNA序列(SEQ ID NO:1,SEQ ID NO:2和SEQ ID NO:3)分别构建到pAHC20载体上，前面分别加各自的上游启动子序列2.5kb(SEQ ID NO:16,SEQ ID NO:17和SEQ ID NO:18)用于驱动基因的表达，后面分别加1.4kb的下游序列(SEQ ID NO:22,SEQ ID NO:23和SEQ ID NO:24)，载体分别命名为pAHC20pTaMS7-A:gTaMS7-A,pAHC20pTaMS7-B:gTaMS7-B和pAHC20pTaMS7-D:gTaMS7-D。
利用基因枪法，将上述三个功能互补载体分别转化或是共转化小麦Tams7-abd突变体，对获得的转基因阳性植株的花粉进行I2-KI染色观察，结果显示转基因阳性植株花粉均发育正常、表现为可育。这些分析进一步证明TaMS7基因参与花粉发育调控，该基因突变导致雄性不育表型。
实施例8.TaMS7基因在新一代杂交育种技术中的应用
TaMS7基因可以用于新一代杂交育种技术，该技术的核心思想是：以小麦核隐性雄性不育突变体为转化受体材料，通过将紧密连锁的3个目标基因转化至不育突变体中，其中，隐性核雄性不育基因可使转化受体育性恢复，花粉失活基因可使含有外源基因的花粉失活，即失去授精能力，筛选基因可以用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子即为不育系，而转基因种子用作保持系。通过保持系给不育系授粉杂交，可以在不育系上结实，由此繁殖不育系。而保持系通过自交可以源源不断地得以繁殖。由于该技术利用生物技术生产非转基因产品，解决了小麦杂交制种过程中面临人工或机械去雄问题，省去人工去雄或机械去雄步骤，可以提供更高质量及纯度的种子给种植者，节约劳动成本。
根据以上原理，更具体地，采用小麦TaMS7基因构建植物表达载体。在构建小麦的植物表达载体之前，发明人首先分别对表达载体内的ZmBT1-ZmAA、TaMS7和mCherryW三个表达盒单独进行了小麦转化，并进一步对各个表达盒的功能进行了验证。结果表明各个表达盒单独转化小麦时，都能够工作良好，达到预期的设计效果。
进一步，发明人通过装配下述DNA元件，构建了转化载体：
1)以pAHC20载体为基础，可操作性连入以下各个表达盒；
2)TaMS7基因育性恢复表达盒，含有育性相关基因TaMS7及其启动子和终止子均来自小麦品种CB037，小麦中来自A、B、D基因组上的三个TaMS7基因，即TaMS7-A，TaMS7-B和TaMS7-D均可同时用于恢复小麦ms7不育系的育性，或是单独用于恢复小麦ms7不育系的育性。其中TaMS7-A基因的基因组DNA序列、启动子序列和终止子序列分别如SEQ ID NO：1，SEQ ID NO：16和SEQ ID NO：22所示；TaMS7-B基因的基因组DNA序列、启动子序列和终止子序列分别如SEQ ID NO：2，SEQ ID NO：17和SEQ ID NO：23所示；TaMS7-D基因的基因组DNA序列、启动子序列和终止子序列分别如SEQ ID NO：3，SEQ ID NO：18和SEQ ID NO：24所示。
3)花粉失活基因表达盒PG47：ZmBT1-ZmAA-IN2-1，花粉失活基因为ZmAA，转运肽为ZmBT1，ZmBT1-ZmAA(其核苷酸序列如SEQ ID NO：25所示)的开放读码框连接于启动子PG47(其核苷酸序列如SEQ ID NO：26所示)的下游、终止子IN2-1(其核苷酸序列如SEQ ID NO：27所示)的上游。
4)筛选标记基因表达盒CaMV35S增强子-LTP2：mCherryW-PINII，mCherryW基因(SEQ ID NO：28)的开放读码框连接于CaMV35S增强子-LTP2启动子(SEQ ID NO：29)和PINII终止子(SEQ ID NO：30)之间，重组成mCherryW的基因表达盒(CaMV35S增强子-LTP2：mCherryW-PINII)。
本发明同时构建了TaMS7基因育性恢复表达盒中只含单个TaMS7基因，及另外两个花粉失活基因表达盒和筛选标记基因表达盒的载体。此外，也构建了TaMS7基因育性恢复表达盒中含有两个TaMS7基因，及另外两个花粉失活基因表达盒和筛选标记基因表达盒的载体。
利用基因枪法，将上述两种载体分别转化小麦Tams7-abd突变体，分别得到8株、12株和9株转基因为单拷贝的转基因阳性植株。对转基因植株及对照进行花粉活性检测，结果显示，非转基因的野生型小麦植株花粉不育比例小于2％，非转基因的小麦Tams7-abd突变体植株的花药中没有花粉，而转基因植株的花药中有花粉，但不育花粉的比例为50％左右，表明本发明所提供的载体中TaMS7基因恢复了Tams7-abd突变体雄性不育的表型，同时ZmBT1-ZmAA基因能够到达预期的花粉失活功能。
对上述转基因植株所结T1代种子进行荧光分离比例调查，结果表明这些种子均显示1:1分离比，即携带外源基因的荧光种子和不携带外源基因的非荧光种子表现为1:1分离，表明本发明所提供的载体各元件作为整体表达良好，可以实现创制和繁殖不育系的目的；其中， TaMS7基因可以恢复雄性不育突变体受体的育性，ZmBT1-ZmAA基因和mCherryW基因的表达可以分别实现预期的花粉失活功能和种子荧光标记功能。由此，建立新一代杂交育种技术体系，即在小麦ms7雄性核不育系的基础上，转入上述载体以形成保持系，含有该外源载体的保持系可以用于源源不断的生产保持系和ms7不育系，以解决小麦ms7雄性核不育系无法繁殖的问题，获得的ms7雄性不育系可以用于生产制种，及生产杂交种。
上述技术体系也可以通过以下方式获得，即在普通野生型小麦的基础上，转入上述载体，再将转入了上述载体的植株与ms7雄性不育系杂交，杂交获得的植株再自交后进行筛选，即获得背景为纯合ms7突变的含有外源转入载体的保持系，含有该外源载体的保持系可以用于源源不断的生产保持系和ms7不育系。

Claims (20)

  1. 一种育性相关基因，其特征在于所述育性相关基因TaMS7的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  2. 一种表达盒、表达载体或工程菌，其特征在于所述表达盒、表达载体或工程菌包含权利要求1所述的育性相关基因。
  3. 一种育性恢复基因、表达盒、表达载体或工程菌在调控植物育性中的应用，其特征在于所述育性恢复基因、表达盒、表达载体、工程菌含有如下所示的核苷酸序列之一：
    (a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  4. 一种调控植物育性的方法，所述方法通过过表达、抑制或突变植株中的育性恢复基因，影响其表达水平，进而调控植物育性，其特征在于：所述育性恢复基因TaMS7的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  5. 根据权利要求4所述的方法，其中所述的突变包括在育性恢复基因的核苷酸序列上进行取代、缺失或添加一个或多个核苷酸。
  6. 根据权利要求4-5之任一所述的方法，其中所述的“突变”包括但不限于以下方法，如用物 理或化学的方法所导致的基因突变，化学方法包括用EMS等诱变剂处理所导致的诱变，或是通过RNAi等基因沉默手段或者通过基因编辑等方法，所述基因定点突变的方法包括但不限于ZFN、TALEN、和/或CRISPR/Cas9等基因编辑方法。
  7. 根据权利要求4所述的方法，其特征在于所述方法包括用TaMS7基因的核苷酸序列互补由TaMS7基因突变所导致的雄性不育表型，使ms7雄性不育系恢复成可育。
  8. 权利要求4-7之任一所述的方法在调控植物育性中的应用。
  9. 一种雄性不育系的生产或繁殖方法，所述方法包括以下步骤：
    (a)向ms7雄性不育系中转入下述载体，以获得含有下述载体的保持系，所述载体包含：育性恢复基因TaMS7，所述育性恢复基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的可育雄性配子都是不含所述载体的；和筛选基因，所述筛选基因可以用于转基因种子和非转基因种子的分拣；和
    (b)将转入上述载体后形成的保持系植株自交，同时产生不含载体的ms7雄性不育系种子和含载体的保持系种子；或是用保持系植株的花粉给ms7不育系植株授粉上，使ms7不育系授粉繁殖出ms7不育系种子。
  10. 根据权利要求9所述的生产或繁殖方法，其中所述的育性恢复基因TAMS7的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  11. 根据权利要求10所述的生产或繁殖方法，其中所述的育性恢复基因TAMS7由一个花粉特异性表达的启动子驱动表达，优选所述花粉特异性表达启动子的核苷酸序列如SEQ ID NO:16、17或18所示。
  12. 根据权利要求9-11之任一所述的生产或繁殖方法，其中所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等，优选的所述花粉失活基因是玉米α淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO：25所示。
  13. 根据权利要求12所述的生产或繁殖方法，其中所述的花粉失活基因与偏好于雄性配子表 达的启动子相连，优选地所述启动子是PG47启动子或Zm13启动子。
  14. 根据权利要求9-13之任一所述的繁殖方法，其中所述的筛选基因包括但不限于抗生素抗性基因、或抗除草剂基因、或荧光蛋白基因等，优选的所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
  15. 一种保持系的生产或繁殖方法，所述方法包括以下步骤：
    (a)向ms7雄性不育系中转入下述载体，即获得了ms7雄性不育系的保持系，所述载体包含：育性恢复基因TaMS7，所述育性恢复基因TaMS7可以恢复ms7雄性不育系的雄性生育力；和花粉失活基因，所述花粉失活基因表达时，会干扰植株中含有该花粉失活基因的雄性配子的功能或形成，从而使得所述植株中产生的可育雄性配子都是不含所述载体的；和筛选基因，所述筛选基因可以用于转基因种子和非转基因种子的分拣；和
    (b)将转入上述载体后形成的保持系植株自交，同时产生不含载体的ms7雄性不育系种子和含载体的保持系种子。
  16. 根据权利要求15所述的生产或繁殖方法，其中所述的育性恢复基因TaMS7的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、3、4、5或6所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：7、8或9所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有育性恢复功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  17. 根据权利要求16所述的生产或繁殖方法，其中所述的育性恢复基因TaMS7由一个花粉特异性表达的启动子驱动表达，优选所述花粉特异性表达启动子的核苷酸序列如SEQ ID NO:16、17或18所示。
  18. 根据权利要求15-17之任一所述的生产或繁殖方法，其中所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等，优选的所述花粉失活基因是玉米α淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO：25所示。
  19. 根据权利要求18所述的生产或繁殖方法，其中所述的花粉失活基因与偏好于雄性配子表达的启动子相连，优选地所述启动子是PG47启动子或Zm13启动子。
  20. 根据权利要求15-19之任一所述的繁殖方法，其中所述的筛选基因包括但不限于抗生素抗性基因、或抗除草剂基因、或荧光蛋白基因等，优选的所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
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