WO2018019195A1 - 雄性育性的保持方法及其应用 - Google Patents

雄性育性的保持方法及其应用 Download PDF

Info

Publication number
WO2018019195A1
WO2018019195A1 PCT/CN2017/094020 CN2017094020W WO2018019195A1 WO 2018019195 A1 WO2018019195 A1 WO 2018019195A1 CN 2017094020 W CN2017094020 W CN 2017094020W WO 2018019195 A1 WO2018019195 A1 WO 2018019195A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
sequence
frg1
fertility
seq
Prior art date
Application number
PCT/CN2017/094020
Other languages
English (en)
French (fr)
Inventor
马力耕
王峥
李健
何航
陈少霞
邓兴旺
Original Assignee
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
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 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 filed Critical 未名兴旺系统作物设计前沿实验室（北京）有限公司
Publication of WO2018019195A1 publication Critical patent/WO2018019195A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/007—Vector systems having a special element relevant for transcription cell cycle specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/65—Vector systems having a special element relevant for transcription from plants

Definitions

  • the invention belongs to the field of plant biotechnology, and particularly relates to the cloning of a plant recessive nuclear male sterility gene, the breeding method of the male sterility line and the application thereof in the cross breeding, and more particularly to a recessive nuclear male sterility in wheat. Cloning of genes and their promoters, and their use in cross breeding.
  • Heterosis is a common phenomenon in the biological world. Hybrid breeding is the main way to breed new varieties, and it is the most important method in modern breeding. Compared with rice, corn, sorghum, etc., the research on the utilization of wheat heterosis is relatively lagging. The average production growth in the past decade has also stagnated and even declined for many years. Wheat is a self-pollinated crop. The core problem of wheat heterosis utilization is the technical system for efficient production of wheat hybrids. A large number of scientists at home and abroad have made great efforts and achieved a series of important results.
  • the research on the utilization of wheat heterosis mainly focuses on the use of nuclear infertility male sterility ("three-line method”), the use of chemical killing technology (“slaughter method”) and light temperature sensitivity Use of nuclear male sterility (“two-line method”).
  • the three-line method has not been widely applied in production due to the difficulty in breeding of the sterile line, narrow recovery sources, and cytoplasmic side effects.
  • the method of killing avoids the relationship between recovery and maintenance. It has been considered as a promising new technology for wheat hybrid seed production, but due to its poor stability during seed production, high seed production costs and environmental pollution. For many reasons, it is difficult to promote and utilize it in actual production.
  • the two-line method based on light temperature sensitivity has the advantages of low seed production cost, wide recovery source, and easy to obtain superior combination, it also faces two key problems - the instability of environmental factors on the fertility of sterile lines.
  • the wheat sterile lines selected and influenced by the light-temperature sensitivity characteristics are very limited.
  • Recessive nuclear male-sterile mutants are used for crop heterosis utilization, and their sterility has characteristics that are easily recovered and are not easily maintained.
  • the recessive nuclear male sterile mutant has the following advantages for hybrid wheat development: 1) there is no negative impact of exogenous cytoplasm, hybrid F1 has strong advantage; 2) paternal pair Hybrid F1 has high fertility restoration degree; 3) The selection of breeding parent material of sterile line, maintainer line and paternal line is not restricted by specific recovery/protection relationship, and the selection of materials is very wide, and the utilization rate of germplasm resources is high, which is beneficial to Breeding hybrids with high combining ability.
  • the dominant male sterility line of wheat can not find a complete recovery system or a complete maintainer system, such as the Taigu nuclear sterile line (MS2) found in China in 1972. Therefore, it is only applicable to conventional breeding recurrent selection and backcross breeding methods, and cannot be used as a parent of hybrid wheat breeding.
  • the recessive nuclear male sterile material is crossed with any normal material, and the F1 generation is fertile. Any material with normal fertility is its recovery system. As long as the identification of nuclear sterility and the problem of effective maintenance can be solved, it can be applied.
  • a new generation of wheat cross breeding technology is a new generation of wheat cross breeding technology.
  • the wheat genome is huge (17Gb), about five times that of humans, 40 times that of rice, and 100 times that of Arabidopsis.
  • the composition is extremely complex, consisting of three groups of A, B, and D with partial homology.
  • the genome consists of 7 pairs of chromosomes with 21 pairs of chromosomes, which are typical heterologous hexaploids (Zhang ZB, et al., 2002), and about 75% are simple repeats (Rachel B, et al., 2012; IWGSC, 2014).
  • the invention successfully clones the fertility restoration gene FRG1 by flow cytometry and high-throughput sequencing method, and the gene can completely restore the male fertility of the Lanzhou nuclear male sterile mutant or its allelic mutant, and construct a novel The wheat hybrid breeding technology system laid the foundation.
  • the technical bottleneck problems such as the unstable fertility of the sterile lines in the “three-line” and two-line hybrid technologies of wheat, the limitation of hybrid resources, the complicated seed production technology and the high seed production cost, There are many possibilities.
  • the breeding and maintaining methods of the gene and the sterile line provided by the invention have important significance and application value for the wheat cross breeding work.
  • the present invention provides a fertility restoring gene FRG1 (Fertility Restoration gene1), the nucleotide sequence of the fertility restoring gene being selected from one of the following group of sequences:
  • the fertility restoring gene of the present invention also includes homologous gene sequences which are highly homologous to the nucleotide or protein sequence of the FRG1 gene and which have the same fertility regulating or restoring function.
  • the homologous gene which is highly homologous and has a fertility regulating function includes, under stringent conditions, having SEQ ID NOS: 2, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, DNA sequence of DNA hybridization of the sequence shown in 20, 21, 23, 24, 26, 27 or 35. 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: 4, 7, 10, 13, 16, 19, 22, 25 or 28.
  • the above homologous gene further includes the sequence shown in SEQ ID NO: 2, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 or 35
  • a DNA sequence having a sequence similarity of at least 80%, 85%, 90%, 95%, 98%, or 99% of the full length and having a fertility regulating function can be isolated from any plant.
  • the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller 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 restoring gene disclosed in the present invention, the nucleotide sequence of the fertility restoring gene being selected from one of the following group of sequences:
  • the fertility restoring 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 a pollen-specific expression promoter.
  • the nucleotide sequence of the pollen-specific promoter is shown in SEQ ID NO: 1.
  • the above expression cassette of the present invention further comprises a pollen inactivating gene, and the pollen inactivating gene can interfere with the planting The function or formation of a male gamete containing the pollen inactivating gene in the strain.
  • 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 a amylase gene, preferably having a nucleotide sequence as shown in SEQ ID NO:29.
  • 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, which recovers the male fertility of the frg1 male sterile mutant by transferring the fertility restoring gene into the frg1 male sterile mutant, wherein the The nucleotide sequence of the fertility restorer gene is selected from one of the following set of sequences:
  • the present invention also provides a method for regulating plant fertility, which regulates the fertility of plants by affecting the expression of the fertility gene FRG1.
  • a method for regulating plant fertility which regulates the fertility of plants by affecting the expression of the fertility gene FRG1.
  • 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 frg1 male sterile mutant, which is obtained by mutating a plant endogenous fertility control gene FRG1, or mutating a nucleotide sequence of a gene highly homologous thereto.
  • the process of male fertility is set forth in SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25 or 28.
  • the nucleotide sequence of the fertility regulatory gene FRG1 is SEQ ID NO: 2, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 or 35.
  • 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 frg1 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 FRG1 gene, preferably as SEQ ID NOs: 2, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21 23, 24, 26, 27 or 35.
  • the use of the mutant material includes, but is not limited to, the use in cross breeding, and more specifically, the frg1 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 frg1 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 a fertility restorer gene FRG1, a pollen inactivating gene, and a selection marker gene, respectively.
  • the fertility restoration gene FRG1 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 insemination, 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 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 the maize a amylase gene Zm-AA, preferably the nucleotide sequence thereof is shown in SEQ ID NO:29.
  • 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 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 the maize a amylase gene Zm-AA, preferably the nucleotide sequence thereof is shown in SEQ ID NO:29.
  • 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 the maize a amylase gene Zm-AA, preferably the nucleotide sequence thereof is shown in SEQ ID NO:29.
  • 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.
  • nucleotide sequence of the fertility restoring gene is selected from the following group of sequences one:
  • the above fertility restorer gene FRG1 is also operably linked to a pollen-specific promoter, which can drive the expression of the FRG1 gene in plant pollen.
  • the pollen-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 pollen-specific promoter is shown in SEQ ID NO: 1.
  • the above fertility restorer gene FRG1 is also operably linked to a terminator which may be a terminator of any of the disclosed genes. Specifically, the nucleotide sequence of one of the terminators is SEQ ID NO: 3 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 the maize a amylase gene Zm-AA, preferably the nucleotide sequence thereof is shown in SEQ ID NO:29.
  • 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 a pollen-specific expression promoter, the nucleotide sequence of which is shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 was ligated to the reporter gene GUS, and the vector was transformed into rice and wheat, and the GUS expression activity and expression pattern in the transgenic plants were detected and analyzed. GUS staining analysis was performed on the roots, stems, leaves and flowers of the transgenic plants.
  • the promoter-driven GUS gene provided by the present invention was found to be expressed in plant pollen.
  • the SEQ ID NO: 1 provided by the present invention is a promoter for pollen-specific expression.
  • the plant pollen-specific expression promoter provided by the present invention comprises the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, or comprises 90% or more similar to the nucleotide sequence listed in SEQ ID NO: 1. a nucleotide sequence comprising or comprising 500 and more than contiguous nucleotide fragments derived from the sequence of SEQ ID NO: 1 and which can drive a nucleotide sequence operably linked to the promoter in plant pollen expression.
  • Expression vector containing the above sequence, transgenic fine Cell lines, host bacteria and the like are all within the scope of the present invention.
  • Primer pairs that amplify any of the nucleotide fragments of the SEQ ID NO: 1 promoter disclosed herein 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 FRG1 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 can be introduced into a complete tissue, such as a staged pollen, or introduced into a callus for functional verification.
  • the promoter of the present invention may also be ligated to a nucleotide sequence other than the FRG1 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 pollen-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 pectinase, more specifically such as barnase gene, maize a-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 pectinase, more specifically such as barnase gene, maize a-amylase gene, auxin gene, rot B, cytotoxin gene, diphtheria toxin, DAM methylase Or a dominant male sterility
  • 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: 3, and has a function of terminating transcriptional expression of a gene.
  • the present invention also provides an expression cassette, vector or engineered strain comprising the pollen-specific expression promoter SEQ ID NO: 1 provided by the present invention.
  • the nucleotide sequence of the fertility restoring gene FRG1 provided by the present invention can be constructed downstream of the promoter SEQ ID NO: 1 provided by the present invention, thereby driving the fertility gene in the transformed recipient plant. expression.
  • the pollen-specific expression promoter provided by the invention can be used for specific expression of a foreign gene in pollen, thereby avoiding the adverse effects of the persistent 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 FRG1 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 means transferring a nucleotide sequence, a vector or an expression cassette to a receptor by a conventional transgenic method.
  • 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 restoring gene FRG1 and a promoter thereof, and a method for using the gene for propagation and maintenance of a frg1 male sterile line.
  • the fertility restoration gene provided by the invention, the fertility preservation of the nuclear male sterile line and the breeding method of the sterile line have significant production promotion value and application value for the cross breeding breeding of the crop, and the invention provides The fertility restoration gene also solves the problem of breeding and maintaining the male sterile line of the Lanzhou nuclear male sterile line or its allele sterile line, and is of great significance for breaking through and improving the existing "three-line” and "two-line” hybrid technology.
  • Figure 1 is a genomic DNA sequence alignment of the EeFRG1 gene with a homologous gene in wheat.
  • Figure 2 is a CDS sequence alignment of the EeFRG1 gene with a homologous gene in wheat.
  • Figure 3 is a protein sequence alignment of the EeFRG1 gene with a homologous gene in wheat.
  • Figure 4 is a genomic DNA sequence alignment of the EeFRG1 gene with a homologous gene in a related species.
  • Figure 5 is a CDS sequence alignment of the EeFRG1 gene with a homologous gene in a related species.
  • the length of the 4AgS chromosome in the 4AgS-ms double-endsome addition line was significantly shorter than that of all wheat chromosomes. After measurement, the size was estimated to be about 1/4 of the longest wheat chromosome, about 250Mb.
  • the 4AgS chromosome of Elymus sibiricum was isolated by flow cytometry, and the 4AgS reference genome was spliced by a combination of second-generation and third-generation sequencing. Combined with the transcriptome sequencing of the 4AgS-ms double-end heterologous line mononuclear anthers, 8 candidate genes were obtained.
  • the genomic DNA sequences of the 8 candidate genes were constructed on the pAHC20 vector, and the 4Ag-ms monomer-added line plant immature embryos were transformed by the gene gun method.
  • plants without 4Ag chromosome ie, Lanzhou genic male sterile homozygous mutant background
  • only the vector with the genomic DNA sequence of gene ID CUFF.199 could restore Lanzhou.
  • the gene corresponding to CUFF.199 is the Fertility Restoration Gene1 (FRG1)
  • the genomic DNA sequence is shown in SEQ ID NO: 2
  • the CDS sequence is SEQ ID NO:
  • the protein sequence is shown in SEQ ID NO: 4
  • the promoter sequence is shown in SEQ ID NO: 1
  • the terminator sequence is shown in SEQ ID NO: 3.
  • the FRG1 gene promoter was amplified by 2265 bp, the sequence was as shown in SEQ ID NO: 1, and the amplified product was ligated into pAHC20-GUS vector by In-fusion method to obtain pAHC20- pFRG1-GUS expression vector.
  • the pAHC20-pFRG1-GUS plasmid was transformed into wheat immature embryos by gene gun method, and 16 transgenic positive plants were obtained. GUS staining analysis of roots, stems, leaves and flowers of different developmental stages of transgenic plants showed that the FRG1 gene promoter could drive GUS specifically expressed in wheat pollen, indicating that the FRG1 gene promoter is a pollen-specific promoter.
  • the genomic DNA sequence, CDS sequence and protein sequence of TaFRG1-2 gene were compared with EeFRG1 gene by Clustal Omega. The results showed that the sequence of TaFRG1-2 gene and EeFRG1 genomic DNA sequence was 88.9. %, the consistency of the CDS sequence was 95.2%, and the protein sequence identity was 95.0%.
  • TaFRG1-1 gene and the TaFRG1-1 gene and the EeFRG1 gene protein sequence homology are both above 95%, it is presumed that it has a similar function to the EeFRG1 gene.
  • Example 3 Sequence alignment of EeFRG1 gene with homologous genes of Uraltu wheat, crude goat grass, sorghum, corn and millet
  • the protein sequence of the EeFRG1 gene was searched in a database such as NCBI, and the homologous proteins predicted by the Uraltu wheat, crude goat grass, sorghum, corn, and millet genomes were obtained.
  • the maize contained two homologous genes of EeFRG1, named respectively. For ZmFRG1-1 and ZmFRG1-2.
  • the genomic DNA sequence of the fertility gene TuFRG1 in Uraltu wheat is as shown in SEQ ID NO: 11, the CDS sequence is shown in SEQ ID NO: 12, and the protein sequence is shown in SEQ ID NO: 13;
  • the genomic DNA sequence of the fertility gene AetFRG1 in the grass is shown in SEQ ID NO: 14, the CDS sequence is shown in SEQ ID NO: 15, and the protein sequence is shown in SEQ ID NO: 16: the fertility gene is in sorghum.
  • the genomic DNA sequence of SbFRG1 is shown in SEQ ID NO: 17, the CDS sequence is shown in SEQ ID NO: 18, and the protein sequence is shown in SEQ ID NO: 19; two homologous genes of TaFRG1 gene are present in maize.
  • ZmFRG1-1 and ZmFRG1-2 the genomic DNA sequence of ZmFRG1-1 is shown in SEQ ID NO: 20, the CDS sequence is shown in SEQ ID NO: 21, and the protein sequence is shown in SEQ ID NO: 22.
  • the genomic DNA sequence of ZmFRG1-2 is shown in SEQ ID NO: 23, the CDS sequence is shown in SEQ ID NO: 24, and the protein sequence thereof is set forth in SEQ ID NO:
  • the genomic DNA sequence of the fertility gene SiFRG1 is shown in SEQ ID NO: 26
  • the CDS sequence is shown in SEQ ID NO: 27, and the protein sequence is shown in SEQ ID NO: 28.
  • the protein sequences of the EeFRG1 gene were aligned with the protein sequences of these homologous genes, respectively, and the results showed that homologous proteins from different plants have very similar conserved sequences (Fig. 4-6).
  • the sequence of the protein sequence of EeFRG1 gene and the predicted homologous protein sequence of Uraltu wheat, Aegilops tauschii, sorghum, and millet genome were 96.4%, 95.5%, 59.0%, 60.9%, respectively.
  • the sequence identity of two homologous proteins in maize was 58.1% and 50.8%, respectively.
  • the above results indicate that the protein plays a very important role in the biological function of the plant during the development of male organs.
  • FRG1 and its homologous gene can be used in a new generation of cross breeding technology.
  • the core idea of this technology is to use frg1 recessive nuclear male sterility mutant as a transforming receptor material, by transforming the three closely related target genes into no Among the mutants, the fertility restorer gene FRG1 can restore the transformation to physique, and the pollen-inactivated gene can inactivate the pollen containing the foreign gene, that is, lose the ability to fertilize, and the seed marker gene can be used for the transgenic seed and non-
  • the sorting of the transgenic seeds, the sorted non-transgenic seeds are the sterile lines, and the transgenic seeds are used as the maintainer lines.
  • 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-GM products, it solves the bottleneck problem faced by wheat and other crops in the process of hybrid seed production, that is, the low utilization rate of the three-line method and the instability of the sterile line in the two-line method.
  • the inventors first separately performed wheat transformation on three expression cassettes of ZmBT1-ZmAA, FRG1 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 FRG1 expression cassette contains a functional FRG1 gene, for example, EeFRG1, TaFRG1-1, TaFRG1-2, TuFRG1, AetFRG1, SbFRG1, SiFRG1, ZmFRG1-1 or ZmFRG1-2 can restore frg1 male no Breeding of the mutant.
  • the inventors constructed the pAHC20-FRG1-AA-mCherryW vector by assembling the following DNA elements:
  • the functional FRG1 gene is ligated downstream of the EeFRG1 gene promoter (such as SEQ ID NO: 1), upstream of the EeFRG1 gene terminator (such as SEQ ID NO: 3), and the FRG1 gene may be EeFRG1.
  • CaMV35S enhancer-LTP2 mCherryW-PINII, open reading frame of mCherryW gene (SEQ ID NO: 32) ligated to CaMV35S enhancer-LTP2 promoter (SEQ ID NO: 33) and PINII terminator ( Between SEQ ID NO: 34), a gene expression cassette (CaMV35S enhancer-LTP2: mCherryW-PINII) reconstituted into mCherryW;
  • the plasmid pAHC20-FRG1-AA-mCherryW was transformed into 4Ag-ms monomer-added lineage immature embryos by gene gun method. After screening, differentiation, strong seedling rooting and PCR identification, the genotype was obtained from Lanzhou genic male sterile site. Mutant, non-Long-eared buckwheat 4Ag chromosome, and the transgene is a single copy of the transgenic positive plant.
  • the pollen activity of the above plants was tested.
  • the proportion of sterile pollen was less than 2%, and the proportion of sterile pollen in the transgenic plants was about 50%, indicating that the carrier provided by the present invention can achieve the expected pollen loss. Live function.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Reproductive Health (AREA)
  • Pregnancy & Childbirth (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种小麦育性恢复基因及其应用，属于植物生物技术领域，具体涉及一个隐性核雄性不育基因及其启动子的克隆，及其在杂交育种中的应用。通过流式细胞术和高通量测序的方法，成功克隆了一个育性恢复基因FRG1，该基因可以完全恢复兰州核雄性不育突变体或其等位突变体的雄性育性，为构建新型小麦杂交育种技术体系奠定了基础。同时，为解决小麦现有的"三系"和"两系"杂交技术所存在不育系育性不稳定、杂交品种资源受局限、制种技术复杂、制种成本高等技术瓶颈问题，提供了更多的可能性。提供的基因及不育系的繁殖和保持方法，对小麦杂交育种工作具有重要的意义和应用价值。

Description

雄性育性的保持方法及其应用 技术领域
本发明属于植物生物技术领域，具体涉及植物隐性核雄性不育基因的克隆，及其雄性不育系的繁殖方法和在杂交育种中的应用，更具体地涉及一个小麦隐性核雄性不育基因及其启动子的克隆，及其在杂交育种中的应用。
技术背景
杂种优势是生物界的普遍现象，杂交种育种是选育新品种的主要途径，是近代育种工作最重要方法。与水稻、玉米、高粱等相比，小麦杂种优势利用的研究相对滞后，近十年来其平均产量的增长也趋于停滞、甚至出现连续多年下滑的局面。小麦是自花授粉作物，小麦杂种优势利用的核心问题是高效生产小麦杂交种的技术体系，国内外大量科学家为此已做出巨大的努力，并取得一系列重要成果。综合近50年来的研究进展，小麦杂种优势利用研究主要集中于：核质互作雄性不育的利用(“三系法”)、化学杀雄技术的利用(“化杀法”)和光温敏核雄性不育的利用(“两系法”)。三系法由于不育系难以繁殖、恢复源较窄、细胞质副效应等原因，未能在生产上大面积应用。化杀法避开了恢复与保持间的相互关系，曾被认为是一种很有希望的小麦杂交制种新技术，但由于其在制种过程中稳定性差、制种成本高及环境污染等多方面原因，在实际生产上也难以推广利用。基于光温敏的两系法虽然具有制种成本低、恢复源广泛，较易获得优势组合等优点，但也面临着两大关键问题——环境因素的不稳定性对不育系育性的影响和利用光温敏特性所选育的小麦不育系十分有限。
隐性核雄性不育突变体用于作物杂种优势利用，其不育性具有易被恢复而不易被保持的特性。与细胞质雄性不育杂交小麦体系相比，隐性核雄性不育突变体用于杂交小麦开发具有以下优点：1)不存在外源细胞质的负面影响，杂种F1优势强；2)父本系对杂种F1的育性恢复度高；3)不育系、保持系、父本系的育种亲本材料选择不受特定恢/保关系的限制，选材范围极广、种质资源利用率高，有利于选育高配合力的杂交种。但是，按照常规方法无法实现纯合核不育系种子的大量有效生产。因此，针对小麦杂种优势利用的现状，建立高效小麦雄性不育繁育新体系，是杂交小麦获得成功应用的最关键因素之一。
小麦显性核雄性不育系由于其自身的遗传特点，既找不到完全的恢复系，也找不到完全的保持系，比如1972年在我国发现的太谷核不育系(即MS2)，因此只适用于常规育种轮回选择和回交育种手段，不能用作杂交小麦育种的亲本。而隐性核雄性不育材料与任何正常材料杂交，F1代均可育，任何育性正常的材料都是其恢复系，只要解决核不育性的标识和有效保持的问题，就能应用于新一代小麦杂交育种技术。
小麦基因组巨大(17Gb)，约是人类的5倍，水稻的40倍，拟南芥的100倍；组成极其复杂，由A、B、D三个具有部分同源关系的染色体组组成，每个染色体组由7对染色体构成，共有21对染色体，是典型的异源六倍体(Zhang ZB,et al.,2002)，并且约75％是简单重复序列(Rachel B,et al.,2012；IWGSC,2014)。近年来，虽然小麦及其近缘种的基因组测序工作取得了很大进展，但截至目前，仍然没有完整的参考基因组序列公布(Vogel JP,et al.,2010；The International Barley Genome Sequencing Consortium,2012；Rachel B,et al.,2012；Ling HQ,et al.,2013；Jia J,et al.,2013；IWGSC,2014)。如此复杂的基因组使得小麦功能基因的研究工作异常困难，目前为止国际范围内小麦突变体成功克隆基因的例子只有寥寥几个。
本发明通过流式细胞术和高通量测序的方法，成功克隆了育性恢复基因FRG1，该基因可以完全恢复兰州核雄性不育突变体或其等位突变体的雄性育性，为构建新型小麦杂交育种技术体系奠定了基础。同时，为解决小麦现有的“三系”和两系”杂交技术所存在不育系育性不稳定、杂交品种资源受局限、制种技术复杂、制种成本高等技术瓶颈问题，提供了更多的可能性。本发明所提供的基因及不育系的繁殖和保持方法，对小麦杂交育种工作具有重要的意义和应用价值。
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恢复基因FRG1(Fertility Restoration gene1)，所述育性恢复基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、
26、27或35所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：4、7、10、13、16、19、22、25或28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。
本领域技术人员应该知晓，本发明所述的育性恢复基因还包括与FRG1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：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35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:4、7、10、13、16、19、22、25或28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53℃-60℃杂交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：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35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恢复基因的DNA序列，所述育性恢复基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO:4、7、10、13、16、19、22、25或28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恢复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粉特异表达启动子。优选地，所述花粉特异表达启动子的核苷酸序列如SEQ ID NO:1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a淀粉酶基因，优选其核苷酸序列如SEQ ID NO:29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恢复基因转入到frg1雄性不育突变体，使frg1雄性不育突变体的雄性育性恢复，其中所述的育性恢复基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO:4、7、10、13、16、19、22、25或28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FRG1的表达来调控植物的育性。包括但不限于通过突变FRG1基因的方法，获得frg1雄性不育突变体材料；或是通过基因互补的方法，将FRG1基因用于恢复由相应的FRG1基因突变所导致的雄性不育，使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frg1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FRG1，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FRG1的氨基酸序列如SEQ ID NO：4、7、10、13、16、19、22、25或28所示。所述育性调控基因FRG1的核苷酸序列如SEQ ID NO：2、5、6、8、9、11、12、 14、15、17、18、20、21、23、24、26、27或35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frg1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FRG1基因的核苷酸序列，优选如SEQ ID NO:2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35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frg1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frg1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恢复基因FRG1、花粉失活基因和筛选标记基因。其中，育性恢复基因FRG1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frg1雄性不育突变体是指兰州核雄性不育突变体或其等位突变体。本发明中frg1雄性不育突变体也可以称为frg1不育系或frg1雄性不育系，在小麦植株中，也可称为兰州核雄性不育系、兰州核不育、或兰州核雄性不育系的等位不育系等。
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a淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO:29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frg1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FRG1，所述育性恢复基因FRG1可以恢复frg1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frg1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frg1雄性不育系植株上，使frg1雄性不育系授粉繁殖出frg1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a淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO:29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frg1雄性不育系中转入下述载体，即获得了frg1雄性不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复frg1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a淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO:29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frg1雄性不育系中转入下述载体，即获得了frg1雄性不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复frg1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frg1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frg1雄性不育系中引入下述载体，获得frg1雄性不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复frg1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frg1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恢复基因FRG1、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frg1雄性不育系杂交，经过定向选育，获得背景为frg1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，其中所述的育性恢复基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：2、5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27或35所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO:4、7、10、13、16、19、22、25或28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。
上述育性恢复基因FRG1还可操作性的连有一个花粉特异表达的启动子，可以驱动FRG1基因在植物花粉中的表达。所述花粉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，所述花粉特异表达启动子的核苷酸序列如SEQ ID NO:1所示。上述育性恢复基因FRG1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，其中一个终止子的核苷酸序列如SEQ ID NO:3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a淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO:29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粉特异表达启动子，其核苷酸序列如SEQ ID NO:1所示。将SEQ ID NO:1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:1是一个花粉特异性表达的启动子。
本发明所提供的植物花粉特异表达启动子，含有序列表中如SEQ ID NO:1所示的核苷酸序列，或包含与SEQ ID NO:1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:1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:1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，FRG1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FRG1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。
本发明所公开的花粉特异表达启动子，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a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:3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粉特异表达启动子SEQ ID NO:1。具体地，可以将本发明所提供的育性恢复基因FRG1的核苷酸序列构建到本发明所提供的启动子SEQ ID NO:1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粉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FRG1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恢复基因FRG1及其启动子，及将该基因用于frg1雄性不育系的繁殖和保持的方法。本发明所提供的育性恢复基因、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，本发明提供的育性恢复基因还解决了小麦兰州核雄性不育系或其等位不育系的繁殖和保持问题，对于突破并改良现有的“三系”和“两系”杂交技术具有重要意义。
参考文献：
周宽基,周文麟,王淑英(1996)4E-ms小麦雄性核不育、保持体系的建立.中国农业科学29:93The International Barley Genome Sequencing Consortium(2012)A physical,genetic and functional sequence assembly of the barley genome.Nature 491:711
The International Wheat Genome Sequencing Consortium(IWGSC)(2014)A chromosome-based draft sequence of the hexaploid bread wheat(Triticum aestivum)genome.Science 345:1251788
Jia J,Zhao S,Kong X,et al.(2013)Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation.Nature 496:91
Ling HQ,Zhao S,Liu D,et al.(2013)Draft genome of the wheat A-genome progenitor Triticum urartu.Nature 496:87
Rachel B,Manuel S,Matthias P,et al.(2012)Analysis of the bread wheat genome using whole-genome shotgun sequencing.Nature 491:705
Vogel JP,Garvin DF,Mockler TC,et al.(2010)Genome sequencing and analysis of the model grass Brachypodium distachyon.Nature 463:763
Zhang ZB,Xu P(2002)Reviewed on wheat genome.Hereditas 24:389
附图说明
图1是EeFRG1基因与小麦中同源基因的基因组DNA序列比对。
图2是EeFRG1基因与小麦中同源基因的CDS序列比对。
图3是EeFRG1基因与小麦中同源基因的蛋白序列比对。
图4是EeFRG1基因与近缘种中同源基因的基因组DNA序列比对。
图5是EeFRG1基因与近缘种中同源基因的CDS序列比对。
图6是EeFRG1基因与近缘种中同源基因的蛋白序列比对。
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、长穗偃麦草FRG1基因的克隆
小麦兰州核雄性不育突变体是典型的单基因控制的隐性突变。4AgS-ms双端体异附加系是在兰州核雄性不育突变体中附加了半条长穗偃麦草的4Ag染色体(4AgS)(周宽基等，1996)，附加的4AgS染色体能够恢复突变体雄性不育的表型，表明长穗偃麦草4AgS染色体中含有育性恢复基因Fertility Restoration gene1(FRG1)。
4AgS-ms双端体异附加系中4AgS染色体的长度比小麦所有染色体都显著短，测量后估计其大小约为最长小麦染色体的1/4，大概250Mb左右。利用流式细胞仪分离长穗偃麦草4AgS染色体，采用二代和三代测序相结合的策略拼接4AgS参考基因组。结合4AgS-ms双端体异附加系单核期花药的转录组测序，获得了8个候选基因。
将8个候选基因的基因组DNA序列构建到pAHC20载体上，利用基因枪法，转化4Ag-ms单体异附加系植株幼胚。对T0代转基因植株中，不带有4Ag染色体的植株(即兰州核不育纯合突变背景)进行花粉育性观察，结果只有连有基因ID为CUFF.199的基因组DNA序列的载体能够恢复兰州核不育突变体雄性不育的表型。因此，基因ID为CUFF.199所对应的基因为长穗偃麦草中的育性恢复基因(Fertility Restoration Gene1，FRG1)，其基因组DNA序列如SEQ ID NO:2所示，CDS序列如SEQ ID NO:35所示，蛋白序列如SEQ ID NO:4所示，启动子序列如SEQ ID NO:1所示，终止子序列如SEQ ID NO:3所示。
以4AgS-ms双端体异附加系DNA为模板，扩增FRG1基因启动子2265bp，序列如SEQ ID NO:1所示，扩增产物通过In-fusion方法连入pAHC20-GUS载体，获得pAHC20-pFRG1-GUS表达载体。
利用基因枪法将pAHC20-pFRG1-GUS质粒转化小麦幼胚，获得16株转基因阳性植株。对转基因阳性植株的根、茎、叶和不同发育时期的花进行GUS染色分析，发现FRG1基因启动子能驱动GUS在小麦花粉中特异表达，说明FRG1基因启动子为花粉特异表达型启动子。
实施例2、EeFRG1在小麦中的同源基因
利用EeFRG1基因(SEQ ID NO:2)在IWGSC(International Wheat Genome Sequencing Consortium)数据库中进行比对，找到两个同源基因，命名为TaFRG1-1和TaFRG1-2，其基因组DNA序列分别如SEQ ID NO:5和SEQ ID NO:8所示，根据EeFRG1基因推定的CDS 序列分别如SEQ ID NO:6和SEQ ID NO:9所示，蛋白序列分别如SEQ ID NO:7和SEQ ID NO:10所示。
利用Clustal Omega，将TaFRG1-1基因的基因组DNA序列、CDS序列和蛋白序列分别与EeFRG1基因进行比对，结果如图1-3所示，TaFRG1-1基因与EeFRG1基因基因组DNA序列一致性为88.5％，CDS序列的一致性为95.0％，蛋白序列一致性为95.9％。
利用Clustal Omega，将TaFRG1-2基因的基因组DNA序列、CDS序列和蛋白序列分别与EeFRG1基因进行比对，结果如图1-3所示，TaFRG1-2基因与EeFRG1基因基因组DNA序列一致性为88.9％，CDS序列的一致性为95.2％，蛋白序列一致性为95.0％。
由于TaFRG1-1基因和TaFRG1-1基因与EeFRG1基因蛋白序列同源性均达到95％以上，推测很可能具有与EeFRG1基因类似的功能。为了证实这种推测，我们分别构建了pAHC20-pEeFRG1-gTaFRG1-1-tEeFRG1和pAHC20-pEeFRG1-gTaFRG1-2-tEeFRG1载体，用EeFRG1基因的启动子分别驱动TaFRG1-1基因和TaFRG1-2基因表达。将pAHC20-pEeFRG1-gTaFRG1-1-tEeFRG1和pAHC20-pEeFRG1-gTaFRG1-2-tEeFRG1载体分别转化4Ag-ms单体异附加系植株幼胚，分别获得22株和26株转基因阳性植株，通过PCR鉴定分别发现11株和12株转基因阳性植株不带有4Ag染色体，但花粉均发育正常、表现为可育，表明TaFRG1-1基因和TaFRG1-2基因的核酸和蛋白序列的确具有与EeFRG1基因类似的功能。
实施例3、EeFRG1基因与乌拉尔图小麦、粗山羊草、高粱、玉米、粟中的同源基因的序列比对
将EeFRG1基因的蛋白序列在NCBI等数据库中查找，获得了乌拉尔图小麦、粗山羊草、高粱、玉米、粟基因组中预测的同源蛋白，其中玉米中包含两个EeFRG1的同源基因，分别命名为ZmFRG1-1和ZmFRG1-2。其中，在乌拉尔图小麦中该育性基因TuFRG1的基因组DNA序列如SEQ ID NO:11所示，CDS序列如SEQ ID NO:12所示，蛋白序列如SEQ ID NO:13所示；在粗山羊草中该育性基因AetFRG1的基因组DNA序列如SEQ ID NO:14所示，CDS序列如SEQ ID NO:15所示，其蛋白序列如SEQ ID NO:16所示；在高粱中该育性基因SbFRG1的基因组DNA序列如SEQ ID NO:17所示，CDS序列如SEQ ID NO:18所示，其蛋白序列如SEQ ID NO:19所示；在玉米中存在两个TaFRG1基因的同源基因，分别命名为ZmFRG1-1和ZmFRG1-2，ZmFRG1-1的基因组DNA序列如SEQ ID NO:20所示，CDS序列如SEQ ID NO:21所示，其蛋白序列如SEQ ID NO:22所示,ZmFRG1-2的基因组DNA序列如SEQ ID NO:23所示，CDS序列如SEQ ID NO:24所示，其蛋白序列如SEQ ID NO:25所 示；在粟中该育性基因SiFRG1的基因组DNA序列如SEQ ID NO:26所示，CDS序列如SEQ ID NO:27所示，其蛋白序列如SEQ ID NO:28所示。
将EeFRG1基因的蛋白序列分别与这些同源基因的蛋白序列进行比对，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(图4-图6)。利用Clustal Omega进行序列比对，EeFRG1基因的蛋白序列与乌拉尔图小麦、粗山羊草、高粱、粟基因组中预测的同源蛋白序列一致性分别为96.4％、95.5％、59.0％、60.9％、与玉米中两个同源蛋白的序列一致性分别为58.1％和50.8％。上述结果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。
实施例4、EeFRG1及其同源基因在新一代小麦杂交育种技术中的应用
FRG1及其同源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frg1隐性核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，育性恢复基因FRG1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，种子标记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，解决了小麦等作物在杂交制种过程中面临的瓶颈问题，即三系法资源利用率低而两系法中不育系育性不稳定的问题。
根据以上原理，发明人首先分别对表达载体内的ZmBT1-ZmAA、FRG1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。其中FRG1表达盒中只要包含的是有功能的FRG1基因即可，例如EeFRG1,TaFRG1-1,TaFRG1-2,TuFRG1,AetFRG1,SbFRG1,SiFRG1,ZmFRG1-1或ZmFRG1-2等均可恢复frg1雄性不育突变体的育性。
进一步,发明人通过装配下述DNA元件，构建了pAHC20-FRG1-AA-mCherryW载体：
1)以pAHC20载体为基础；
2)FRG1基因表达盒，有功能的FRG1基因连接于EeFRG1基因启动子(如SEQ ID NO:1)的下游、EeFRG1基因终止子(如SEQ ID NO:3)的上游，FRG1基因可以是EeFRG1,TaFRG1-1,TaFRG1-2,TuFRG1,AetFRG1,SbFRG1,SiFRG1,ZmFRG1-1或ZmFRG1-2，序列如SEQ ID NO：2，SEQ ID NO：5，SEQ ID NO：8，SEQ ID NO：11，SEQ ID NO：14，SEQ ID NO：17，SEQ ID NO：20，SEQ ID NO：23，SEQ ID NO：26所示；
3)基因表达盒PG47：ZmBT1-ZmAA-IN2-1，目标基因为ZmAA，转运肽为ZmBT1， ZmBT1-ZmAA(其核苷酸序列如SEQ ID NO：29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：30所示)的下游、终止子IN2-1(其核苷酸序列如SEQ ID NO：31所示)的上游。
4)基因表达盒CaMV35S增强子-LTP2：mCherryW-PINII，mCherryW基因(SEQ ID NO：32)的开放读码框连接于CaMV35S增强子-LTP2启动子(SEQ ID NO：33)和PINII终止子(SEQ ID NO：34)之间，重组成mCherryW的基因表达盒(CaMV35S增强子-LTP2：mCherryW-PINII)；
利用基因枪法将质粒pAHC20-FRG1-AA-mCherryW转化4Ag-ms单体异附加系植株幼胚，经过筛选、分化、壮苗生根、PCR鉴定等过程，得到基因型为兰州核不育位点纯合突变、无长穗偃麦草4Ag染色体、且转基因为单拷贝的转基因阳性植株。
对上述植株进行花粉活性检测，在非转基因植株中，不育花粉的比例小于2％，而转基因植株中不育花粉的比例为50％左右，表明本发明所提供的载体能够达到预期的花粉失活功能。
对植株所结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。

Claims (20)

  1. 一种育性恢复基因，其特征在于所述育性恢复基因FRG1的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：5、6、8、9、11、12、14、15、17、18、20、21、23、24、26或27所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO:7、10、13、16、19、22、25或28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所述的育性恢复基因。
  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：5、6、8、9、11、12、14、15、17、18、20、21、23、24、26或27所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO:7、10、13、16、19、22、25或28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FRG1的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：5、6、8、9、11、12、14、15、17、18、20、21、23、24、26或27所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO:7、10、13、16、19、22、25或28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FRG1基因的核苷酸序列互补由FRG1基因突变所导致的雄性不育表型，使frg1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frg1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FRG1，所述育性恢复基因FRG1可以恢复frg1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frg1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frg1不育系植株授粉上，使frg1不育系授粉繁殖出frg1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FRG1的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：5、6、8、9、11、12、14、15、17、18、20、21、23、24、26或27所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO:7、10、13、16、19、22、25或28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FRG1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:1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a淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO:6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frg1雄性不育系中转入下述载体，即获得了frg1雄性不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复frg1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frg1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FRG1的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：5、6、8、9、11、12、14、15、17、18、20、21、23、24、26或27所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO:7、10、13、16、19、22、25或28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FRG1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:1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a淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO:6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。
PCT/CN2017/094020 2016-07-25 2017-07-24 雄性育性的保持方法及其应用 WO2018019195A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201610588768.4 2016-07-25
CN201610588768 2016-07-25
CN201710599348.0A CN107267527B (zh) 2016-07-25 2017-07-21 雄性育性的保持方法及其应用
CN201710599348.0 2017-07-21

Publications (1)

Publication Number Publication Date
WO2018019195A1 true WO2018019195A1 (zh) 2018-02-01

Family

ID=60078999

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2017/094020 WO2018019195A1 (zh) 2016-07-25 2017-07-24 雄性育性的保持方法及其应用
PCT/CN2017/094012 WO2018019193A1 (zh) 2016-07-25 2017-07-24 小麦育性恢复基因及其应用

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/094012 WO2018019193A1 (zh) 2016-07-25 2017-07-24 小麦育性恢复基因及其应用

Country Status (4)

Country Link
US (1) US11130967B2 (zh)
CN (2) CN107267527B (zh)
CA (1) CA3031844C (zh)
WO (2) WO2018019195A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110386967A (zh) * 2018-03-26 2019-10-29 中国农业科学院作物科学研究所 与植物雄性育性相关的蛋白SiMS1及其编码基因与应用

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109593778A (zh) * 2018-11-08 2019-04-09 浙江大学 一种植物人工智能雄性不育系及应用
CN110178721B (zh) * 2019-06-10 2023-09-05 中国农业大学 形态标记法扩繁植物核雄性不育系
CN110714022B (zh) * 2019-11-25 2023-01-24 中国农业大学 对花粉竞争力基因stk1；2的表达调控及在提高扩繁植物核雄性不育系效率中的应用
CN113621642A (zh) * 2020-05-07 2021-11-09 海南波莲水稻基因科技有限公司 一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用
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
CN111903507A (zh) * 2020-08-11 2020-11-10 淄博爱民种业有限公司 含有对蓝败Ms2性状表达具有抑制效应的隐性上位非等位基因种质的选育方法
CN112501178B (zh) * 2020-10-16 2022-10-14 上海师范大学 一种水稻温敏不育突变体tms18及其应用
CN114540366B (zh) * 2020-11-24 2024-02-13 海南波莲水稻基因科技有限公司 一种水稻育性调控基因gms3及其突变体与应用
WO2022109764A1 (zh) * 2020-11-24 2022-06-02 北京大学现代农业研究院 一种育性相关基因及其在杂交育种中的应用
CN114959095A (zh) * 2022-05-19 2022-08-30 海南波莲水稻基因科技有限公司 检测植物内、外源基因的分子标记、引物组和方法
CN116875580B (zh) * 2023-09-08 2023-12-01 北京首佳利华科技有限公司 利用人工突变创制玉米msp1雄性不育系

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1570103A (zh) * 2004-05-08 2005-01-26 南京农业大学 小麦育性恢复基因Rf6的分子标记及其获得方法
WO2016100309A1 (en) * 2014-12-16 2016-06-23 Pioneer Hi-Bred International, Inc. Restoration of male fertility in wheat

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL120835A0 (en) * 1997-05-15 1997-09-30 Yeda Res & Dev Method for production of hybrid wheat
AU2002246937A1 (en) * 2001-01-04 2002-08-06 Yeda Research And Development Co. Ltd. Maintenance of genic male-sterile female parental lines using a maintainer line with a stable engineered chromosome
CN101243770A (zh) * 2007-02-14 2008-08-20 甘肃省农业科学院 一种细胞核雄性不育杂交小麦的培育方法
BR112013033176A2 (pt) * 2011-06-21 2018-06-12 E I Du Pont De Nemouras And Company método para produzir uma modificação direcionada em um gene de fertilidade masculina, planta, molécula de ácido nucleio isolada, construto de expressão
WO2013064085A1 (zh) * 2011-11-02 2013-05-10 未名兴旺系统作物设计前沿实验室（北京）有限公司 小麦细胞核雄性不育系的保持方法
CN113416738A (zh) * 2012-09-06 2021-09-21 先锋国际良种公司 包含雄性育性序列的组合物和方法
CN103820445B (zh) * 2014-01-15 2016-07-06 深圳市作物分子设计育种研究院 一个植物花药特异表达启动子的鉴定和应用
US9747297B2 (en) * 2014-09-23 2017-08-29 Amazon Technologies, Inc. Synchronization of shared folders and files
AU2015321591B2 (en) * 2014-09-26 2022-03-10 Pioneer Hi-Bred International, Inc. Wheat MS1 polynucleotides, polypeptides, and mehtods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1570103A (zh) * 2004-05-08 2005-01-26 南京农业大学 小麦育性恢复基因Rf6的分子标记及其获得方法
WO2016100309A1 (en) * 2014-12-16 2016-06-23 Pioneer Hi-Bred International, Inc. Restoration of male fertility in wheat

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
AHMED, T.A.: "QTL analysis of fertility-restoration against cytoplasmic male sterility in wheat", GENES GENET. SYST., vol. 76, 31 December 2001 (2001-12-31), XP055358955, DOI: doi:10.1266/ggs.76.33 *
BORNER, A.: "Genetics and molecular mapping of a male fertility restoration locus (Rfgl) in rye (Secale cereale L.", THEOR APPL GENET, vol. 97, 31 December 1998 (1998-12-31) *
DATABASE Nucleotide 13 July 2009 (2009-07-13), "PREDICTED: Sorghum bicolor vegetative cell wall protein gpl (LOC8075844), mRNA", XP055602891, retrieved from NCBI Database accession no. XM_002447848. 1 *
DATABASE Nucleotide 20 April 2017 (2017-04-20), "Zea mays hypothetical protein (LOC100272822), mRNA", XP055602876, retrieved from NCBI Database accession no. NM _ 001147275.1 *
DATABASE Nucleotide 20 May 2011 (2011-05-20), "Hordeum vulgare subsp. vulgare mRNA for predicted protein, complete cds, clone: NIASHv3009E08", XP055602920, retrieved from NCBI Database accession no. AK372683. 1 *
DATABASE Nucleotide 24 February 2017 (2017-02-24), "PREDICTED: Aegilops tauschii subsp. tauschii vegetative cell wall protein gpl-like (LOC109749084), mRNA", XP055602827, retrieved from NCBI Database accession no. XM_020308073. 1 *
DATABASE Nucleotide 25 February 2009 (2009-02-25), "Zea mays full-length cDNA clone ZM _BFc0090K09 mRNA, complete cds", XP055602914, retrieved from NCBI Database accession no. BT069327. 1 *
DATABASE Nucleotide 30 November 2015 (2015-11-30), "PREDICTED: Setaria italica leucine-rich repeat extensin-like protein 5 (LOC101781029), mRNA", XP055602895, retrieved from NCBI Database accession no. XM_004977964.1 *
DATABASE Protein 2 August 2014 (2014-08-02), "PREDICTED: Zea mays uncharacterized LOC103641673 (LOC103641673), mRNA", XP055602887, retrieved from NCBI Database accession no. XM_008665004.1 *
KOJIMA, T .: "High-resolution RFLP mapping of the fertility restoration (Rf3) gene against Triticum timopheevi cytoplasm located on chromosome 1BS of common wheat", G ENES GENET. SYST., vol. 72, no. 6, 31 December 1997 (1997-12-31), pages 353 - 359, XP055358980, DOI: 10.1266/ggs.72.353 *
L. BING-HUA ET AL.: "A Dominant Gene for Male Sterility in Wheat", PLANT BREEDING., vol. 97, no. 3, 31 December 1986 (1986-12-31), pages 204 - 209, XP055602831 *
LIU, DONGTAO ET AL.: "xiong2xing4bu4yu4deyu4xing4ji1in1ding4wei4yan2jiu1jin4zhan3", TRITICAL CROPS, vol. 19, no. 4, 31 July 1997 (1997-07-31), pages 6 - 11 *
ZHENG, HONGYUAN ET AL.: "cDNA-AFLP Analysis of restorer genes of K-Cytoplasmic Male Sterile (CMS) in wheat", JOURNAL OF HENAN AGRICULTURAL UNIVERSITY, vol. 48, no. 2, 30 April 2014 (2014-04-30), pages 117 - 122 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110386967A (zh) * 2018-03-26 2019-10-29 中国农业科学院作物科学研究所 与植物雄性育性相关的蛋白SiMS1及其编码基因与应用
CN110386967B (zh) * 2018-03-26 2021-04-06 中国农业科学院作物科学研究所 与植物雄性育性相关的蛋白SiMS1及其编码基因与应用

Also Published As

Publication number Publication date
CN107267527B (zh) 2021-03-19
CN107304428A (zh) 2017-10-31
US20190338305A1 (en) 2019-11-07
CA3031844A1 (en) 2018-02-01
CN107267527A (zh) 2017-10-20
WO2018019193A1 (zh) 2018-02-01
CN107304428B (zh) 2021-04-02
CA3031844C (en) 2023-06-20
US11130967B2 (en) 2021-09-28

Similar Documents

Publication Publication Date Title
WO2018019195A1 (zh) 雄性育性的保持方法及其应用
Chen et al. Generation of transgene-free maize male sterile lines using the CRISPR/Cas9 system
CN105746362B (zh) 一种育性基因及其应用
US10856481B2 (en) Use of genic male sterility gene and mutation thereof in hybridization
US20220298526A1 (en) Stsci protein for changing self-incompatibility of diploid potato materials
CA3047163A1 (en) Genome editing-based crop engineering and production of brachytic plants
WO2020042412A1 (zh) 一种水稻育性调控基因及其突变体与应用
CN104837334A (zh) 植物新型保持系及不育系建立及其用途
US20210324398A1 (en) Edited nac genes in plants
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
CN105695477B (zh) 雄性不育突变体oss125及其应用
WO2019129145A1 (en) Flowering time-regulating gene cmp1 and related constructs and applications thereof
US20220275383A1 (en) Sterile genes and related constructs and applications thereof
CN109295071B (zh) 一种水稻花器官发育调控基因peh1及其编码的蛋白质和应用
WO2022109764A1 (zh) 一种育性相关基因及其在杂交育种中的应用
CN108660139B (zh) 植物育性调控基因np2及其编码蛋白和应用
US11926823B2 (en) Compositions and methods of modifying a plant genome to produce a MS9, MS22, MS26, or MS45 male-sterile plant
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
US11753650B2 (en) Wheat fertility-related gene TaMS7 and application method thereof
CN111560372A (zh) 一种恢复水稻OsCYP704B2突变体雄性育性的表达盒、载体及其检测方法和应用
CN112204144A (zh) 非生物胁迫耐受植物和使用方法
CN117402887A (zh) 一种玉米雄性育性调控基因ZmMS2085及其突变体与应用
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
CN111575285B (zh) 一种含长雄野生稻启动子的可恢复水稻OsCYP704B2突变体雄性育性的载体及应用
CN118165997A (zh) 一种表达盒及其应用

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: 17833498

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 24/05/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17833498

Country of ref document: EP

Kind code of ref document: A1