WO2018019193A1 - 小麦育性恢复基因及其应用 - Google Patents

小麦育性恢复基因及其应用 Download PDF

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WO2018019193A1
WO2018019193A1 PCT/CN2017/094012 CN2017094012W WO2018019193A1 WO 2018019193 A1 WO2018019193 A1 WO 2018019193A1 CN 2017094012 W CN2017094012 W CN 2017094012W WO 2018019193 A1 WO2018019193 A1 WO 2018019193A1
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gene
sequence
promoter
fertility
pollen
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PCT/CN2017/094012
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French (fr)
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马力耕
王峥
李健
何航
陈少霞
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 CA3031844A priority Critical patent/CA3031844C/en
Priority to US16/320,105 priority patent/US11130967B2/en
Publication of WO2018019193A1 publication Critical patent/WO2018019193A1/zh

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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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, more specifically to a recessive nuclear male sterility gene. Cloning of its promoter and its application 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, capable of having the SEQ ID NO: 1 or 2 Sequence DNA hybridization of DNA sequences. Or a nucleotide sequence whose encoded amino acid sequence has an affinity of 85% or more with the amino acid sequence of the protein represented by SEQ ID NO: 3.
  • stringent conditions include, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA at 53 ° C to 60 ° C for 12-16 hours, then at 62 ° C - Wash with a wash solution containing 0.5 x SSC, and 0.1% SDS at 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 shown in SEQ ID NO: 1 or 2, and has fertility regulating function.
  • the DNA sequence can be isolated from any plant. Among them, 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 as shown in SEQ ID NO: 4.
  • 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 a amylase gene, preferably the nucleotide sequence thereof is shown in SEQ ID NO: 6.
  • 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 comprises a Lanzhou nuclear by transferring a fertility restoration gene into a Lanzhou nuclear male sterile mutant (Zhou Kuanji et al., 1996) or an allelic mutant thereof.
  • the Lanzhou nuclear male sterile mutant described in the present invention is also known as Lanzhou nuclear infertility or Lanzhou nuclear sterile line or Lanzhou nuclear infertility mutant.
  • the invention also discloses a method for maintaining a male sterile line, which uses a Lanzhou nuclear male sterile mutant or an allelic mutant thereof as a transforming receptor material, and converts the closely linked three target genes into the no Breeding mutant recipient plants.
  • 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 Lanzhou nuclear male sterile mutant or allelic mutant thereof may also be referred to as a sterile line or a male sterile line or a Lanzhou nuclear male sterile line, or an allelic 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 a amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 6.
  • 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 present invention also discloses a method for breeding a male sterile line, the method comprising the steps of:
  • the gene FRG1 can restore the male fertility of the Lanzhou male male sterile line or its allelic sterile line; and the pollen inactivating gene, which interferes with the male gamete containing the pollen inactivating gene in the plant when the pollen inactivating gene is expressed Function or formation such that the active male gametes produced in the plants are free of the vector; and screening for genes that can be used for transgenic seeds or tissues and non-transgenic seeds or tissues Pick.
  • 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 Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 6.
  • 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 vector comprising:
  • the sexual recovery gene FRG1, the fertility restorer gene FRG1 can restore the male fertility of the Lanzhou male male sterile line or its allelic sterile line; and the pollen inactivating gene, which interferes with the plant when the pollen inactivating gene is expressed
  • the function or formation of a male gamete containing the pollen inactivating gene such that the fertile male gametes produced in the plant are free of the vector; and the screening gene can be used for the transgenic seed and Sorting of non-GM seeds.
  • the pollen inactivation gene includes, but is not limited to, a barnase gene, an amylase group. Cause, DAM methylase and the like. More specifically, the pollen inactivating gene is a maize a amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 6.
  • 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 vector comprising:
  • the sexual recovery gene FRG1, the fertility restorer gene FRG1 can restore the male fertility of the Lanzhou male male sterile line or its allelic sterile line; and the pollen inactivating gene, which interferes with the plant when the pollen inactivating gene is expressed
  • the function or formation of a male gamete containing the pollen inactivating gene such that the fertile male gametes produced in the plant are free of the vector; and the screening gene can be used for the transgenic seed and Sorting of non-GM seeds; and
  • the invention also discloses a method for producing a seed, the method comprising:
  • the vector comprising: a fertility restorer gene FRG1, the fertility restorer gene FRG1 can restore the male fertility of the Lanzhou male male sterile line or its allelic sterile line; and the pollen inactivating gene, which, when expressed by the pollen inactivating gene, interferes with the plant
  • a fertility restorer gene FRG1 the fertility restorer gene FRG1 can restore the male fertility of the Lanzhou male male sterile line or its allelic sterile line
  • the pollen inactivating gene which, when expressed by the pollen inactivating gene, interferes with the plant The function or formation of a male gamete of the pollen-inactivated gene such that the fertile male gametes produced in the plant are free of the vector.
  • the step (a) may also be a method of introducing a fertility restorer gene FRG1 into a common plant. a pollen inactivating gene and a vector for screening the gene, obtaining a transgenic plant containing the vector, and then crossing with a Lanzhou nuclear male sterile line or an allelic sterile line thereof, and directional breeding, obtaining a background for the Lanzhou nuclear male Breeding line or its allele sterile line, and contains
  • the carrier of the vector is a plant.
  • 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: 4.
  • 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: 5 is shown.
  • the pollen-inactivated gene includes, but is not limited to, a barnase gene, an amylase gene, and DAM A. Base enzymes, etc.
  • the pollen inactivating gene is a maize a amylase gene Zm-AA, preferably having a nucleotide sequence as shown in SEQ ID NO: 6.
  • 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: 4.
  • SEQ ID NO: 4 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. GUS staining analysis was performed on the roots, stems, leaves and flowers of the transgenic plants. Find The promoter provided by the present invention drives the GUS gene to be expressed in plant pollen.
  • the SEQ ID NO: 4 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: 4 in the sequence listing, or comprises 90% or more similar to the nucleotide sequence listed in SEQ ID NO: a nucleotide sequence, or comprising 500 and more than 500 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 4, and which can drive a nucleotide sequence operably linked to the promoter in plant pollen expression.
  • 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: 4 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 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, the reporter gene Includes 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: 5, 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: 4 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: 4 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 Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a fertility restoration gene FRG1 and a promoter thereof, and the gene is applied to the Lanzhou nuclear male sterile line or its allelic sterile line.
  • the method of reproduction and maintenance The fertility restoration gene provided by the invention, the fertility preservation of the wheat recessive male male sterile line and the breeding of the sterile line have great production promotion value and application value for the hybrid breeding production of wheat, and the invention
  • the fertility restoration gene provided solves the problem of reproduction and conservation of the Lanzhou male 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 shows the GISH analysis of the 4AgS-ms double-end heterologous addition line. Hybridization was carried out using genomic DNA of E. striata as a probe, and the red fluorescent signal was 4AgS chromosome; the chromosome was dark blue by DAPI staining.
  • Figure 2 is a flow karyotype of the Chinese spring and 4AgS-ms double-end body addition system.
  • the picture above shows the Chinese spring material.
  • the figure below shows the 4AgS-ms double-end body addition system.
  • the abscissa is the relative fluorescence intensity and the ordinate is the number of chromosomes. I, II, III and 3B mark different size wheat genomes.
  • the 4AgS marker is derived from the 4Ag chromosome of Elytrigia.
  • Figure 3 shows the distribution of the percentage of genes whose parent material expression is higher than that of Lanzhou nuclear sterile material in chr4B.
  • the Lanzhou nuclear male sterile mutant was found in the hybrid F4 generation of hybrid combinations between spring wheat varieties. It was crossed with 9 wheat lines including Chinese spring, and the F1 generation was selfed. The separation ratio of F2 sterile plants to fertile plants was observed, which was consistent with the ratio of 1:3, indicating that the wheat Lanzhou nuclear male sterility mutation
  • the body is a typical single gene-controlled recessive mutation (Zhou Kuanji et al., 1996).
  • the blue-granular dimeric addition line is a wheat line in which two E. longissima 4Ag chromosomes are added to the wheat genome, and the grain is blue because the additional 4Ag chromosome contains the blue grain gene Ba.
  • the pollen of the blue-granular dimeric addition wheat was granted to the Lanzhou nuclear male sterile mutant to obtain the light blue hybrid seed, and the F1 plant was able to be normal.
  • a 4Ag chromosome line with a genetic background of a homozygous mutant of Lanzhou genic male sterility and a long-eared buckwheat grass was obtained, which was named as 4Ag-ms monomer-added line.
  • the strains of the strain are blue and self-fertile, indicating that the 4Ag chromosome of the long-eared buckwheat hybrids entering the Lanzhou nuclear male-sterile mutant contains a fertility restorer gene.
  • 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. Since the 4AgS-ms double-end heterologous line material is white and fertile, it indicates that the lost 4Ag chromosome contains the blue grain gene Ba, and the retained 4Ag contains the fertility restoring gene Fertility Restoration gene1 (FRG1).
  • the cell cycle was synchronized by the double-blocking method, and then subjected to the steps of formaldehyde fixation, mechanical homogenization and DAPI staining, followed by an upflow cytometer.
  • the karyotypes of wheat chromosomes usually form four independent peaks according to their size (Vrana et al., 2000): I peak contains 4 chromosomes of 1D, 4D, 6D and 7D; II peak contains 1A, 3A, 6A, 2D, 3D and 5D have 6 chromosomes; III peak contains 2 chromosomes of 2A, 4A, 5A, 7A, 1B, 2B, 4B, 5B, 6B and 7B; 3B is the largest and independent peak.
  • Chromosomes isolated by flow cytometry are highly concentrated chromatin and cannot be directly applied to high-throughput sequencing. DNA must be separated from the chromosome by proteinase K digestion. A total of 2.2 million 4ES chromosomes were co-purified to obtain approximately 500 ng of DNA with good integrity.
  • Gene amplification was carried out using Qiagen REPL1-g Single Cell Kit using a 100 ng 4ES genome as a template, and a total of 30 ⁇ g of amplification product was obtained.
  • the amplified products were digested, cloned and sequenced. None of the 31 clones were contaminated by E. coli and humans. One clone was able to match the wheat genome sequence completely. The remaining clones were probably from the 4Ag of E. longissima. The ratio was 97%.
  • the second generation of sequencing constructs different insert libraries, which are 300bp, 500bp, 2Kb and 4Kb fragments, and sequenced in two-way 2 ⁇ 125bp.
  • the sequencing amounts are 20Gb, 14Gb, 6Gb and 6Gb respectively.
  • the total amount is 46Gb, and the data covers the chromosome. 184 times.
  • Three generations of sequencing constructed a 10Kb insert library to obtain a read length of 5 to 10 Kb, which effectively assisted in the splicing of the entire chromosome sequence.
  • the second generation sequencing data was first assembled by platanus, and a reference genomic sequence of 212 Mb and N50 of 30 Kb was obtained.
  • sspaceLR was used to add three generations of data for gap filling, and finally a reference genome with a total size of 234M, an N50 of 48k, and a number of scaffolds of 17,302 was obtained (Table 1).
  • the single-nuclear anther of the 4AgS-ms double-end heterologous line was used for transcriptome sequencing, and the Lanzhou nuclear infertility mutant and the corresponding parent were used as controls.
  • the quality control of the data is: removal of the linker sequence and removal of the first 14 bp sequence at the 5' end, excision of the base of the mass value at both ends of the sequence, removal of the sequencing sequence with a sequence length of less than 50 bp, and removal of human mitochondria from E. coli, wheat And chloroplast genome, gramineous ribosomal rRNA contamination, and finally get clean sequencing data.
  • Example 5 Using transcriptome and collinearity to obtain candidate genes
  • 2150 genes were obtained. That is to say, 2150 genes on the 4AgS chromosome in the 4AgS-ms duplex heterologous line are expressed in the mononuclear anther.
  • the Fertility restore gene1 (FRG1) gene is located on the 4AgS chromosome, only in the 4AgS-ms pair.
  • the expression in the end-body heterologous line is not expressed in the control material Lanzhou nuclear infertility mutant and the corresponding parent, and the gene expressed in the Lanzhou nuclear sterile material and/or the corresponding parent is also removed.
  • the sequence comparison tool tophat2 was used to compare the transcriptome data of Lanzhou nuclear sterile material and the corresponding parent to the reference genome, respectively, allowing a maximum of 2 mismatches.
  • the number of reads from two samples in locus was converted to RPKM, and the difference in expression abundance of the genes represented by each locus in two samples was calculated. The FDR value less than 0.001 was considered to be significantly different.
  • the results showed that the difference between the parent and the Lanzhou nuclear sterile mutant was significant: each half of the chromosome was divided into 100 parts, and the gene expression in the parent was significantly higher than that of the Lanzhou nuclear sterile material. The proportion.
  • the 4AgS chromosome of E. longissima has a certain collinearity with the 4BS chromosome of wheat, the full length (exon+intron) of 374 genes is aligned to the TGAC-4BS reference genome, and only 189 genes are identical in 4BS. Source gene.
  • the comparison tool is blastn and the alignment parameter is blastn default parameter.
  • 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 phenotype of male sterile male sterile male sterility, the detailed results are shown in Table 3 below.
  • the gene corresponding to CUFF.199 is the Fertility Restoration Gene1 (FRG1)
  • the genomic DNA sequence is shown in SEQ ID NO: 1
  • the CDS sequence is SEQ ID NO: 1
  • the protein sequence is shown in SEQ ID NO: 3
  • the promoter sequence is shown in SEQ ID NO: 4
  • the terminator sequence is shown in SEQ ID NO: 5.
  • the FRG1 gene promoter was amplified by 2265 bp, the sequence is shown in SEQ ID NO: 4, 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.
  • Example 7 Application of FRG1 gene in a new generation of wheat cross breeding technology
  • the FRG1 gene can be used in a new generation of cross breeding technology.
  • the core idea of this technology is to use the wheat recessive nuclear male sterility mutant as a transforming receptor material by transforming the three closely linked genes into infertile mutants. Among them, the fertility restoring gene can make the transformation be restored by sports.
  • the pollen inactivating gene can inactivate the pollen containing the foreign gene, that is, the insemination ability is lost, and the seed marker gene can be used for the sorting of the transgenic seed and the non-GM seed.
  • the sorted non-transgenic seeds are sterile lines, and the transgenic seeds are used as maintainer lines.
  • 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 in the process of wheat 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 above hybrid breeding technique of the present invention is applicable to the propagation and maintenance of a male sterile male mutant and its allelic mutant in Lanzhou.
  • the inventors first separately transformed wheat into three expression cassettes of ZmBT1-ZmAA, FRG1 and mCherr y W 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 the pAHC20-FRG1-AA-mCherr y W vector by assembling the following DNA elements:
  • FRG1 gene expression cassette the target gene FRG1 and its promoter and terminator are all derived from Elymus sibiricum, wherein the promoter sequence of the FRG1 gene is shown in SEQ ID NO: 4, and the terminator sequence thereof is SEQ ID NO: 5.
  • the genomic DNA sequence of the FRG1 gene is shown in SEQ ID NO: 1
  • the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO:
  • CaMV35S enhancer-LTP2 mCherryW-PINII, open reading frame of mCherryW gene (SEQ ID NO: 9 ) linked to CaMV35S enhancer-LTP2 promoter (SEQ ID NO: 10 ) and PINII terminator ( Between SEQ ID NO: 11 ), a gene expression cassette (CaMV35S enhancer-LTP2: mCherryW-PINII) reconstituted into mCherryW;
  • Transformation of wheat The plasmid pAHC20-FRG1-AA-mCherryW was transformed into 4Ag-ms monomer-added plant immature embryos by gene gun method. After screening, differentiation, rooting, PCR identification, etc., the genotype was obtained from Lanzhou nuclear infertility. Site homozygous mutation, 4Ag chromosome without E. longissima, and the transgene is a single copy of the transgenic positive plant.
  • pollen fertility testing of transgenic wheat plants pollen activity was tested on the above plants. The specific method is: take one flower for each of the transgenic plants and the non-transgenic plants, take one anther per flower, place it in the center of the slide, add a drop of 1% I 2 -KI solution, release with tweezers and anatomic needle. After the pollen, cover the coverslip, observe under the microscope, count the number of sterile pollen and the total number of pollen, can be colored dark blue for fertile pollen, and can not be colored for sterile pollen.

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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

小麦育性恢复基因及其应用 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：1或2所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：3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：1或2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:3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：1或2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：1或2所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：3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:4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:6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兰州核雄性不育突变体(周宽基等，1996)或其等位突变体中，使兰州核雄性不育突变体或其等位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：1或2所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：3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。
本发明中所述的兰州核雄性不育突变体，又称为兰州核不育或兰州核不育系或兰州核不育突变体。
本发明还公开了一种雄性不育系的保持方法，所述方法以兰州核雄性不育突变体或其等位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。
在本发明中，所述兰州核雄性不育突变体或其等位突变体也可以称为不育系或雄性不育系或兰州核雄性不育系、或等位不育系。
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a淀粉酶基因Zm-AA，优选其核苷酸序列如SEQ ID NO:6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)向兰州核雄性不育系或其等位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可以恢复兰州核雄性不育系或其等位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兰州核雄性不育系或其等位不育系种子和含载体的保持系种子；或是将保持系植株的花粉赶到兰州核雄性不育系或其等位不育系植株上，使兰州核雄性不育系或其等位不育系授粉繁殖出兰州核雄性不育系或其等位不育系种子。
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:6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)向兰州核雄性不育系或其等位不育系中转入下述载体，即获得了兰州核雄性不育系或其等位不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复兰州核雄性不育系或其等位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:6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)向兰州核雄性不育系或其等位不育系中转入下述载体，即获得了兰州核雄性不育系或其等位不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复兰州核雄性不育系或其等位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兰州核雄性不育系或其等位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)向兰州核雄性不育系或其等位不育系中引入下述载体，获得兰州核雄性不育系或其等位不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复兰州核雄性不育系或其等位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兰州核雄性不育系或其等位不育系种子。
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，再与兰州核雄性不育系或其等位不育系杂交，经过定向选育，获得背景为兰州核雄性不育系或其等位不育系、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：1或2所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：3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:4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:5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:6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:4所示。将SEQ ID NO:4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:4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:4所示的核苷酸序列，或包含与SEQ ID NO:4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:4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:4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:5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:4。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:4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，及将该基因用于兰州核雄性不育系或其等位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，小麦隐性核雄性不育系的育性保持和不育系的繁殖，对小麦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解决了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：
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附图说明
图1是4AgS-ms双端体异附加系的GISH分析。以长穗偃麦草基因组DNA为探针进行杂交，红色荧光信号为4AgS染色体；染色体用DAPI染色呈深蓝色。
图2是中国春与4AgS-ms双端体异附加系的流式核型图。上图为中国春材料，下图为4AgS-ms双端体异附加系。横坐标为相对荧光强度，纵坐标为染色体数目。Ⅰ、Ⅱ、Ⅲ和3B标记不同大小小麦染色体组群。4AgS标记来自成穗偃麦草的4Ag染色体。
图3是亲本材料表达值高于兰州核不育材料的基因的百分比在chr4B分布情况。
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、4AgS-ms双端体异附加系的选育
兰州核雄性不育突变体是在春小麦品种间杂交组合的杂种F4代群体中发现的。将其与包括中国春在内的9个小麦品系杂交，F1代自交，观察F2代不育株与可育株的分离比，均符合1：3的比例，表明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(周宽基等，1996)。
蓝粒二体异附加系是在小麦基因组中附加了两条长穗偃麦草4Ag染色体的小麦品系，籽粒为蓝色是由于附加的4Ag染色体含有蓝粒基因Ba。将蓝粒二体异附加系小麦的花粉授予兰州核雄性不育突变体，获得浅蓝粒杂种种子，F1植株能正常结实。经过有目标的选育，获得了遗传背景为兰州核不育纯合突变体并附加了一条长穗偃麦草的4Ag染色体的株系，命名为4Ag-ms单体异附加系。该株系籽粒为蓝色的、自交可育,表明杂交进入兰州核雄性不育突变体的长穗偃麦草4Ag染色体中含有育性恢复基因。
由于4Ag-ms单体异附加系中只存在一条4Ag染色体，因此其自交获得的种子存在蓝粒(4Ag+)和白粒(4Ag-)的分离，其中蓝粒的植株可育，白粒的植株不育。经过对4Ag-ms单体异附加系的多代自交选育，获得了一个籽粒为白粒、植株为可育的株系。GISH分析显示(图1)，该材料中的4Ag染色体发生断裂，导致大部分4Ag染色体丢失，只留下一小部分保留在小麦染色体组中，因此将该株系命名为4AgS-ms双端体异附加系。
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。由于4AgS-ms双端体异附加系材料表现为白粒、可育，表明丢失的4Ag染色体含有蓝粒基因Ba，保留的4Ag含有育性恢复基因Fertility Restoration gene1(FRG1)。
实施例2、利用流式细胞术分离4Ag染色体
采用双阻断法对根尖细胞进行细胞周期同步化处理，再经过甲醛固定、机械匀浆和DAPI染色等步骤后，上流式细胞仪。
首先进行流式核型分析以确认需要分离的染色体大小，以4AgS-ms双端体异附加系为实验组，中国春为对照。小麦染色体根据其大小差异，核型图通常形成4个独立的峰(Vrana et al.,2000)：Ⅰ峰包含1D、4D、6D和7D共4条染色体；Ⅱ峰包含1A、3A、6A、2D、3D和5D共6条染色体；Ⅲ峰包含2A、4A、5A、7A、1B、2B、4B、5B、6B和7B共10条染色体；3B最大，独立成峰。对比中国春的核型图，可见4AgS-ms双端体异附加系材料中除了标准核型图外，多出一个明显小的峰，根据相对荧光强度测算其大小在250Mb左右，这个峰就是要分离的目标染色体(图2)。
经过多次试验，我们合计分离了200万条4Ag染色体。以长穗偃麦草基因组DNA为探针通过GISH分析鉴定分离染色体的纯度为88％。
实施例3、4Ag染色体的高通量测序和序列拼接
通过流式细胞仪分离出来的染色体是高度浓缩状态的染色质，不能直接应用于高通量测序，必须通过蛋白酶K消化处理将DNA从染色体中分离出来。220万条4ES染色体共纯化获得约500ng DNA，完整性好。
以100ng 4ES基因组为模板，用Qiagen REPL1-g Single Cell Kit进行基因扩增，合计获得30μg扩增产物。将扩增产物进行酶切、克隆、测序，31个克隆中无来自大肠杆菌及人类污染，有1个克隆能够与小麦基因组序列完全匹配，余下克隆推测很可能来自长穗偃麦草4Ag，比例为97％。
考虑到小麦族物种基因组都有重复序列比例高、拼接困难的特点(小麦中重复序列比例约为80％)，因此我们采取了二代和三代测序相结合的策略。二代测序构建不同的插入片段库，分别为300bp、500bp、2Kb和4Kb种片段，双向2×125bp测序，测序量分别为20Gb、14Gb、6Gb和6Gb，合计测序46Gb，数据量覆盖该条染色体184倍。三代测序构建10Kb插入片段库，获取5～10Kb读长，有效帮助整条染色体序列的拼接。
利用上述数据，首先通过platanus组装二代测序数据，获得了大小为212Mb、N50为30Kb的参考基因组序列。在此基础上利用sspaceLR添加三代数据进行gap filling，最终获得总大小为234M，N50为48k，scaffold数目为17302条的参考基因组(表1)。
表1、4Ag参考基因组的拼接
Figure PCTCN2017094012-appb-000001
实施例4、4AgS-ms双端体异附加系的转录组测序
取4AgS-ms双端体异附加系的单核期花药进行转录组测序，同时以兰州核不育突变体和相应亲本作为对照。Illumina Hi-Seq 2000双向2×100bp测序，每个样品各测约12G有效数据。对数据进行质量控制，分别是：去除接头序列和切除5’端前14bp序列、切除序列两端质量值的碱基、去除序列长度小于50bp的测序序列以及去除来自于人类、大肠杆菌、小麦线粒体和叶绿体基因组、禾本科核糖体rRNA的污染，最后得到干净的测序数据。
实施例5、利用转录组和共线性获得候选基因
利用4AgS-ms双端体异附加系转录组数据比对到17302条4AgS scaffold上，并控制比对参数为100％一致性，得到2150个基因。也就是说4AgS-ms双端体异附加系中4AgS染色体上有2150个基因在单核期花药中表达。
由于流式细胞术分离4AgS染色体纯度为88％，因此组装的4AgS scaffold中会有少量来自小麦基因组的污染，根据要寻找的Fertility restore gene1(FRG1)基因位于4AgS染色体上，只在4AgS-ms双端体异附加系中表达，在对照材料兰州核不育突变体和相应亲本中均不表达的原则，去除在兰州核不育材料和/或相应亲本中也有表达的基因。由于表达偏高的有可能是重复序列、表达偏低的可信度差，因此去除4AgS-ms双端体异附加系表达列表中10％与90％分位数的表达值以外的基因。经过上述两步筛选，候选基因从2150减少到374个基因。比对参数为100％一致性。
以IWGSC发布的小麦序列作为参考基因组，使用序列比对工具tophat2将兰州核不育材料和相应亲本的转录组数据分别比对到参考基因组上，最大允许2个错配，比对后统计每个locus中分别来自2个样品的reads数目，转换成RPKM，计算每个locus代表的基因在2个样品中的表达丰度差异，FDR值小于0.001的即被认定为具有显著性差异。结果显示亲本与兰州核不育突变体之间的差异显著：将每半条染色体均分成100份，计算每一份内所有基因中，在亲本中表达值显著高于兰州核不育材料的基因所占比例。结果发现，4B短臂远端2-7％区间内，几乎所有基因在亲本材料中均显著高表达，这一值显著高于整条染色体上24％的水平(图3)。由此我们推断，兰州核不育突变体是由于4BS染色体远端约2-7％缺失所导致。
由于长穗偃麦草的4AgS染色体与小麦4BS染色体存在一定共线性，将374个基因全长(exon+intron)比对到TGAC-4BS参考基因组上，有且仅有189个基因在4BS中有同源基因。比对工具为blastn，比对参数为blastn默认参数。其中2-7％区段内共有8个基因，这8个基因就是长穗偃麦草Fertility Restore Gene(FRG1)的候选基因。
这8个候选基因进行功能注释及在4AgS-ms双端体异附加系及对照材料中的表达丰度信息见下表2。
表2、候选基因表达信息和功能注释
Figure PCTCN2017094012-appb-000002
实施例6、转基因互补
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，详细结果见下表3。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:1所示，CDS序列如SEQ ID NO:2所示，蛋白序列如SEQ ID NO:3所示，启动子序列如SEQ ID NO:4所示，终止子序列如SEQ ID NO:5所示。
表3、候选基因转基因T0代植株花粉育性观察
Figure PCTCN2017094012-appb-000003
实施例6.FRG1基因启动子表达载体的构建和功能分析
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:4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7.FRG1基因在新一代小麦杂交育种技术中的应用
FRG1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。也可以通过用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。
本发明上述的杂交育种技术适用于兰州核雄性不育突变体及其等位突变体的繁殖和保持。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。
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，目标基因FRG1及其启动子和终止子均来自长穗偃麦草，其中FRG1基因的启动子序列如SEQ ID NO：4所示，其终止子序列如SEQ ID NO：5所示，FRG1基因的基因组DNA序列如SEQ ID NO：1所示，其核苷酸序列编码的蛋白氨基酸序列如SEQ ID NO：3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：6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：7所示)的下游、终止子IN2-1(其核苷酸序列如SEQ ID NO：8所示)的上游。
4)基因表达盒CaMV35S增强子-LTP2：mCherryW-PINII，mCherryW基因(SEQ ID NO：9)的开放读码框连接于CaMV35S增强子-LTP2启动子(SEQ ID NO：10)和PINII终止子(SEQ ID NO：11)之间，重组成mCherryW的基因表达盒(CaMV35S增强子-LTP2：mCherryW-PINII)；
小麦转化：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。
转基因小麦植株的花粉育性检测：对上述植株进行花粉活性检测。具体做法为：对转基 因植株和非转基因植株各取一朵花，每朵花取1个花药，置于载玻片中央，滴加一滴1％的I2-KI溶液，用镊子和解剖针释放花粉后，盖上盖玻片，在显微镜下观察、计数不育花粉数和花粉总数，可以着色为深蓝色的为可育花粉，而不能够着色的为不育花粉。结果显示，在非转基因植株中，不育花粉的比例小于2％，而多个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。
转基因小麦植株的荧光种子与非荧光种子分离分析：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；其中，FRG1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。

Claims (22)

  1. 一种育性恢复基因，其特征在于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：1或2所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3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)或(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，所述表达盒含有一个育性恢复基因，其特征在于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：1或2所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3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。
  3. 根据权利要求2所述的表达盒，其中所述的育性恢复基因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。
  4. 根据权利要求3所述的表达盒，其中所述的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:4所示。
  5. 根据权利要求2-4之任一所述的表达盒，其中所述的表达盒还包含一个花粉失活基因，所述花粉失活基因可以干扰含有该基因的雄配子的产生或功能。
  6. 根据权利要求5所述的表达盒，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。
  7. 根据权利要求2-6之任一所述的表达盒，其中所述的表达盒还包含一个筛选 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。
  8. 根据权利要求7所述的表达盒，其中所述的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，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。
  9. 一种调控植物育性的方法，所述方法通过将育性恢复基因转入到兰州核雄性不育突变体或其等位突变体中，使兰州核雄性不育突变体或其等位突变体的雄性育性恢复，其特征在于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：1或2所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3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。
  10. 根据权利要求9所述的方法，其中所述的育性恢复基因可操作性的连有一个花粉特异表达的启动子，可以驱动育性恢复基因在植物花粉中的表达。
  11. 根据权利要求10所述的方法，其中所述的启动子选自由MS26、NP1、MSP1、PAIR1、PAIR2、ZEP1、MELL、PSS1、TDR、UDT1、GAMYB4、PTC1、API5、WDA1、CYP704B2、MS26、MS22、DPW、MADS3、OSC6、RIP1、CSA、AID1、5126、Ms45、FRG1等育性调控基因的启动子构成的组之一。
  12. 根据权利要求11所述的方法，其中所述的启动子的核苷酸序列如SEQ ID NO:4所示。
  13. 一种雄性不育系的繁殖方法，其特征在于所述方法包括以下步骤：
    (a)向兰州核雄性不育系或其等位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 以恢复兰州核雄性不育系或其等位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；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兰州核雄性不育系或其等位不育系种子和含载体的保持系种子；或是将保持系植株的花粉赶到兰州核雄性不育系或其等位不育系植株上，使兰州核雄性不育系或其等位不育系授粉繁殖出兰州核雄性不育系或其等位不育系种子。
  14. 根据权利要求13所述的繁殖方法，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：1或2所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3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。
  15. 根据权利要求13或14所述的繁殖方法，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。
  16. 根据权利要求15所述的繁殖方法，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。
  17. 根据权利要求13-16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。
  18. 一种保持系的繁殖方法，其特征在于所述方法包括以下步骤：
    (a)向兰州核雄性不育系或其等位不育系中转入下述载体，即获得了兰州核雄性不育系或其等位不育系的保持系，所述载体包含：育性恢复基因FRG1，所述育性恢复基因FRG1可以恢复兰州核雄性不育系或其等位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；
    (b)将转入上述载体后形成的保持系植株自交，即获得了不含上述载体的兰州核雄性不育系或其等位不育系种子和含上述载体的保持系种子。
  19. 根据权利要求18所述的繁殖方法，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：1或2所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3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。
  20. 根据权利要求18或19所述的繁殖方法，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。
  21. 根据权利要求18-20之任一所述的繁殖方法，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。
  22. 根据权利要求18-21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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CN112501178A (zh) * 2020-10-16 2021-03-16 上海师范大学 一种水稻温敏不育突变体tms18及其应用
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
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

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