WO2022109764A1 - 一种育性相关基因及其在杂交育种中的应用 - Google Patents

一种育性相关基因及其在杂交育种中的应用 Download PDF

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WO2022109764A1
WO2022109764A1 PCT/CN2020/130982 CN2020130982W WO2022109764A1 WO 2022109764 A1 WO2022109764 A1 WO 2022109764A1 CN 2020130982 W CN2020130982 W CN 2020130982W WO 2022109764 A1 WO2022109764 A1 WO 2022109764A1
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gene
fertility
seq
nucleotide sequence
vector
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PCT/CN2020/130982
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English (en)
French (fr)
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王峥
李健
何航
邓兴旺
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北京大学现代农业研究院
北京智育小麦生物科技有限公司
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Priority to PCT/CN2020/130982 priority Critical patent/WO2022109764A1/zh
Priority to CN202080106639.1A priority patent/CN116529376A/zh
Publication of WO2022109764A1 publication Critical patent/WO2022109764A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/02—Flowers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention belongs to the field of plant biotechnology, in particular to the cloning of a plant recessive nuclear sterility gene, a breeding method for a male sterile line thereof and its application in cross-breeding, and more particularly to a wheat recessive male sterility gene The cloning of its promoter and its application in cross-breeding.
  • Heterosis is a natural phenomenon ubiquitous in the biological world. Making full use of heterosis to greatly increase crop yield and improve crop quality has achieved huge economic benefits in various crops such as rice, corn, sorghum, cotton, and rape.
  • Wheat is the most widely cultivated and most adaptable food crop in the world, and about 40% of the world's population eats wheat as the staple food. Like rice, wheat also has significant heterosis.
  • the performance of hybrid wheat is far less than that of hybrid rice, and the theoretical and practical research on wheat heterosis is far behind crops such as rice, corn, and rape.
  • Wheat is a self-pollinating crop, and the core of its heterosis utilization is to establish a technical system for efficient production of wheat hybrids.
  • the key to the seed production technology of wheat heterosis utilization approach is to have a suitable female parent of recessive GM male sterile line.
  • Male sterility refers to the fact that plants cannot produce anther, pollen or male gametes with normal functions during sexual reproduction.
  • the disclosure of the mechanism of male sterility is the basis for utilizing heterosis to improve wheat yield and quality.
  • the wheat genome is huge and complex, so the data accumulated so far on the mechanism of wheat male sterility is very limited. Therefore, the application of modern molecular biology and cell biology methods to the research on the mechanism of wheat male sterility has important theoretical and practical significance for strengthening the research and utilization of wheat heterosis.
  • MS1 is located on the short arm of wheat chromosome 4B and encodes a 220 amino acid non-specific lipid transfer protein (nsLTP) (Wang et al., 2017; Tuker et al., 2017).
  • nsLTP is widespread in plants and is characterized by an 8CM domain consisting of eight cysteine residues (Boutrot et al., 2008).
  • MS1 is a newly evolved protein of Poaceae, and in situ hybridization showed that this gene was specifically expressed in the microspore mother cells of anthers. Lipid binding experiments showed that MS1 can specifically bind phosphatidic acid (PA) and several types of inositol phospholipids (phosphoinositide [PIPs]). Sequence analysis showed that there is one MS1 homologous gene in the ABD genome of the wheat genome. However, due to the methylation modification of the MS1 homologous gene promoter in the AD genome, these two genes are completely different in hexaploid wheat. Express.
  • PA phosphatidic acid
  • PIPs phosphoinositide
  • MS5 is located on the wheat chromosome 3A and also encodes a non-specific lipid transporter, but its amino acid sequence is not highly homologous to MS1 except for the 8CM domain, which also has 8 cysteine residues ( Pallotta et al., 2019). MS5 is conserved only in Triticale.
  • the present invention screened out more than 20 genetically stable recessive single gene control male sterility materials through phenotypic screening and genetic analysis of the EMS mutagenesis library. After allelic testing of these materials with the existing ms1 and ms5 mutants, a new single-gene control wheat recessive male sterile line was found. All sites are not allelic, and the present invention names it as MS8.
  • the invention clones the wheat TaMS8 gene by using the Mutmap method, and the gene and the male sterile line produced by the mutation of the gene provide necessary elements for constructing a new hybrid breeding system.
  • the present invention provides a fertility-related gene TaMS8, and the nucleotide sequence of the fertility-related gene is selected from one of the following sequences:
  • the fertility-related genes of the present invention also include homologous gene sequences that are highly homologous to the nucleotide sequence or protein sequence of the TaMS8 gene and have the same fertility regulation or restoration function.
  • the homologous gene with high homology and fertility regulation function includes a DNA sequence capable of hybridizing with DNA having the sequence shown in SEQ ID NO: 1 or 2 under stringent conditions. Or a nucleotide sequence whose encoded amino acid sequence has more than 85% similarity with the protein amino acid sequence shown in SEQ ID NO: 3.
  • “Stringent conditions” as used herein are well known and include, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA at 53°C-60°C for 12-16 hours, followed by 62°C- Wash with washing solution containing 0.5xSSC, and 0.1% SDS at 68°C for 15-60 minutes.
  • the above-mentioned homologous gene also includes at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity with the full length of the sequence shown in SEQ ID NO: 1 or 2, and has a fertility regulation 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, the expression cassette contains the DNA sequence of the fertility-related gene disclosed in the present invention, and the nucleotide sequence of the fertility-related gene is selected from one of the following sequences:
  • the fertility-related gene in the above-mentioned expression cassette can also be operably connected with a promoter that can drive its expression, and the promoters include but are not limited to constitutive expression promoters, inducible promoters, tissue-specific expression promoters promoter, or spatiotemporally specific expression promoter. More specifically, the promoter is a pollen-specific expression promoter.
  • the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO: 10.
  • the above-mentioned expression cassette of the present invention further comprises a pollen inactivation gene, and the pollen inactivation gene can interfere with the function or formation of male gametes containing the pollen inactivation gene in the plant.
  • the pollen inactivating genes include but are not limited to barnase genes, amylase genes, DAM methylase and the like. More specifically, the pollen inactivation gene is a corn alpha amylase gene, and its nucleotide sequence is shown in SEQ ID NO: 14.
  • the above-mentioned expression cassette of the present invention further comprises a screening gene, and the screening gene can be used to screen out plants, plant tissue cells or vectors containing the expression cassette.
  • the screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like.
  • the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance 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, etc.
  • the invention also discloses a method for regulating plant fertility.
  • the method restores the male fertility of the wheat ms8 mutant by transferring a fertility-related gene into the wheat ms8 mutant, wherein the fertility is related to
  • the nucleotide sequence of the gene is selected from one of the following groups:
  • the invention also discloses a method for maintaining a male sterile line.
  • the method uses a wheat ms8 mutant plant as a transformation receptor material, and transforms three closely linked target genes into the sterile mutant receptor plant.
  • the three target genes are fertility related gene TaMS8, pollen inactivation gene and screening gene respectively.
  • the fertility-related gene TaMS8 can restore sterile transformation to vegetative performance, and the pollen inactivation gene can inactivate the pollen containing the transformed exogenous gene, that is, the ability to fertilize.
  • the screening gene can be used for transgenic seeds or tissues and Sorting of non-transgenic seeds or tissues, the sorted non-transgenic seeds are used as sterile lines to produce hybrids, and the transgenic seeds are used as maintainer lines to continuously and stably produce sterile lines.
  • the pollen inactivating genes include but are not limited to barnase gene, amylase gene, DAM methylase and the like. More specifically, the pollen inactivation gene is the maize alpha amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO: 14.
  • the pollen inactivating gene is linked to a promoter that prefers male gamete expression. More specifically, the promoters that prefer male gamete expression include, but are not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene can be used to screen out plants or vectors containing the expression cassette.
  • the screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance 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, etc.
  • the present invention also discloses a method for breeding male sterile line, the method comprises the following steps:
  • the following vector was transferred into the wheat ms8 male sterile line to obtain a maintainer line containing the following vector, the vector comprising: the fertility-related gene TaMS8, which can restore the wheat ms8 mutation male fertility; and a pollen-inactivating gene that, when expressed, interferes with the function or formation of male gametes containing the pollen-inactivating gene in a plant, thereby allowing the production of active Male gametes are all free of the vector; and a selection gene that can be used for sorting of transgenic seeds or tissues and non-transgenic seeds or tissues.
  • the pollen inactivating genes include but are not limited to barnase gene, amylase gene, DAM methylase and the like. More specifically, the pollen inactivation gene is the maize alpha amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO: 14.
  • the pollen inactivating gene is linked to a promoter that prefers male gamete expression. More specifically, the promoters that prefer male gamete expression include, but are not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene can be used to screen out plants or vectors containing the expression cassette.
  • the screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance 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, etc.
  • the present invention also discloses a kind of production method of maintainer, described method comprises the following steps:
  • the following vector is transferred into the wheat ms8 male sterile line, that is, the maintenance line of the wheat ms8 nuclear male sterile line or its allelic sterile line is obtained, and the vector comprises: the fertility-related gene TaMS8, and the The fertility-related gene TaMS8 can restore the male fertility of the wheat ms8 nuclear male sterile line or its allelic sterile line; and the pollen inactivation gene, which, when expressed, will interfere with plants containing the pollen inactivation gene.
  • the function or formation of male gametes of live genes such that all fertile male gametes produced in the plants are free of the vector; and screening genes that can be used for the differentiation of transgenic and non-transgenic seeds pick.
  • the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM methylase and the like. More specifically, the pollen inactivation gene is the maize alpha amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO: 14.
  • the pollen inactivating gene is linked to a promoter that prefers male gamete expression. More specifically, the promoters that prefer male gamete expression include, but are not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening gene can be used to screen out plants or vectors containing the expression cassette.
  • the screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance 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, etc.
  • the present invention also discloses a breeding method of the maintainer, the method comprising the following steps:
  • the following vector is transferred into the wheat ms8 GM male sterile line or its allelic sterile line, that is, a maintainer line of the wheat ms8 GM male sterile line or its allelic sterile line is obtained, and the vector comprises : the fertility-related gene TaMS8, which can restore the male fertility of the wheat ms8 nuclear male sterile line or its allelic sterile line; and the pollen inactivation gene, which when expressed, interferes with the function or formation of male gametes in plants that contain the pollen inactivating gene, so that all fertile male gametes produced in said plants are free of said vector; and a screening gene that can be used for Sorting of GMO and non-GMO seeds; and
  • the present invention also discloses a method for producing seeds, the method comprising:
  • step (a) can also be introduced into ordinary plants containing fertility-related gene TaMS8 , pollen inactivation gene and the vector of the screening gene, after obtaining the transgenic plant containing the vector, it is then crossed with the wheat ms8 nuclear male sterile line or its allelic sterile line, and through directional breeding, the background is the wheat ms8 nuclear male sterile line.
  • nucleotide sequence of the fertility-related gene is selected from the sequence of the following group one:
  • the above fertility-related gene TaMS8 can also be operably linked with a pollen-specific expression promoter, which can drive the expression of the TaMS8 gene in plant pollen.
  • the pollen-specific expression promoter is selected from MS26, NP1, MSP1, PAIR1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, MS22, DPW, MADS3, OSC6,
  • One of the group consisting of promoters of fertility-regulating genes such as RIP1, CSA, AID1, 5126 or Ms45. More specifically, the nucleotide sequence of the pollen-specific expression promoter is shown in SEQ ID NO: 10.
  • the above-mentioned fertility-related gene TaMS8 can also be operably connected with a terminator, and the terminator can be the terminator of any gene that has been disclosed, specifically, the nucleotide sequence of one of the terminators is such as SEQ ID NO: 11 shown.
  • the pollen inactivation gene includes but is not limited to barnase gene, amylase gene, DAM enzyme, etc. More specifically, the pollen inactivation gene is the maize alpha amylase gene Zm-AA, and its nucleotide sequence is shown in SEQ ID NO: 14.
  • the pollen inactivating gene is linked to a promoter that prefers male gamete expression. More specifically, the promoters that prefer male gamete expression include, but are not limited to, the PG47 promoter, the Zm13 promoter, and the like.
  • the screening genes include but are not limited to blue grain genes, antibiotic resistance genes, herbicidal genes, etc. drug resistance gene or fluorescent gene.
  • the screening genes include but are not limited to: blue grain gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance 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, etc.
  • the present invention also provides a pollen-specific expression promoter, the nucleotide sequence of which is shown in SEQ ID NO: 10.
  • SEQ ID NO: 10 Connect SEQ ID NO: 10 to the reporter gene GUS, construct a vector to transform rice and wheat, detect and analyze the GUS expression activity and expression pattern in the transgenic plants, and analyze the roots, stems, leaves and flowers of the transgenic plants by GUS staining.
  • the results It is found that the promoter provided by the present invention drives the expression of GUS gene in plant pollen.
  • SEQ ID NO: 10 provided by the present invention is a pollen-specific expression promoter.
  • the plant pollen-specific expression promoter provided by the present invention contains the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing, or contains more than 90% similarity with the nucleotide sequence listed in SEQ ID NO: 10 nucleotide sequence, or a nucleotide sequence comprising 500 or more contiguous nucleotides derived from the SEQ ID NO: 10 column, and can drive the nucleotide sequence operably linked to the promoter in plant pollen expression.
  • Expression vectors, transgenic cell lines and host bacteria containing the above sequences all belong to the protection scope of the present invention.
  • a primer pair for amplifying any nucleotide fragment of the SEQ ID NO: 10 promoter disclosed in the present invention is also within the protection scope of the present invention.
  • a “promoter” as used herein refers to a DNA regulatory region that typically contains a TATA box that directs RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence.
  • the promoter may also contain other recognition sequences, usually located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which function to regulate the efficiency of transcription. It will be appreciated by those skilled in the art that, although the nucleotide sequences for the promoter regions disclosed herein have been identified, the isolation and identification of other regulatory elements in the regions upstream of the TATA box of the specific promoter regions identified by the present invention are also within the scope of the present invention. within the scope of the invention.
  • the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those for regulating the tissue expressivity and temporal expression function of the coding sequence, enhancers, and the like.
  • upstream regulatory elements such as those for regulating the tissue expressivity and temporal expression function of the coding sequence, enhancers, and the like.
  • promoter elements that enable expression in target tissues can be identified, isolated, and used with other core promoters to verify male tissue-preferred expression.
  • a core promoter refers to the minimal sequence required to initiate transcription, such as a sequence known as the TATA box, which is commonly found in promoters of genes encoding proteins.
  • the upstream promoter of the TaMS8 gene can be used in conjunction with its own core promoter or from other sources.
  • the core promoter can be any of the known core promoters, such as the cauliflower mosaic virus 35S or 19S promoter (US Pat. No. 5,352,605), the ubiquitin promoter (US Pat. No. 5,510,474), the IN2 core promoter ( US Patent No. 5,364,780) or the Scrophulariaceae mosaic virus promoter.
  • the known core promoters such as the cauliflower mosaic virus 35S or 19S promoter (US Pat. No. 5,352,605), the ubiquitin promoter (US Pat. No. 5,510,474), the IN2 core promoter ( US Patent No. 5,364,780) or the Scrophulariaceae mosaic virus promoter.
  • the function of the gene promoter can be analyzed by the following method: the promoter sequence is operably linked with the reporter gene to form a transformable vector, and then the vector is transferred into the plant. The expression characteristics of the gene in various tissues and organs of the plant can be confirmed; or the above-mentioned vector is subcloned into an expression vector for transient expression experiments, and the function of the promoter or its regulatory region is detected by transient expression experiments.
  • an appropriate expression vector for testing the function of a promoter or regulatory region will depend on the host and the method by which the expression vector is introduced into the host, such methods are well known to those of ordinary skill in the art.
  • the regions in the vector include regions that control transcription initiation and regions that control processing. These regions are operably linked to reporter genes including YFP, UidA, GUS gene or luciferase.
  • Expression vectors containing putative regulatory regions located in genomic fragments can be introduced into intact tissues, such as stage pollen, or into callus for functional verification.
  • the promoter of the present invention can also be linked to a nucleotide sequence other than the TaMS8 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 heterologous nucleotide sequences in an expression cassette for expression in a plant of interest, more particularly in the male organ of the plant Express.
  • the expression cassette has suitable restriction sites for insertion of the promoter and heterologous nucleotide sequence.
  • the pollen-specific expression promoter disclosed in the present invention can be used to drive the expression of the following heterologous nucleotide sequences, which can encode carbohydrates that promote Degradative or modifying enzymes, amylases, debranching enzymes and pectinases, more specifically barnase genes, maize a-amylase genes, auxin genes, rotB, cytotoxic genes, diphtheria toxin, DAM methylase, Or a dominant male sterility gene.
  • heterologous nucleotide sequences which can encode carbohydrates that promote Degradative or modifying enzymes, amylases, debranching enzymes and pectinases, more specifically barnase genes, maize a-amylase genes, auxin genes, rotB, cytotoxic genes, diphtheria toxin, DAM methylase, Or a dominant male sterility gene.
  • nucleotide sequences referred to in the present invention are operably linked downstream of the promoters of the present invention, wherein said "nucleotide sequences" may be operably linked to those disclosed herein.
  • the present invention also provides a transcription terminator sequence, the nucleotide sequence of the transcription terminator is shown in SEQ ID NO: 11, and has the function of terminating gene transcription and expression.
  • the present invention also provides an expression cassette, vector or engineering strain, and the expression cassette, vector or engineering strain comprises the pollen-specific expression promoter SEQ ID NO: 10 provided by the present invention.
  • the nucleotide sequence of the fertility-related gene TaMS8 provided by the present invention can be constructed into the downstream of the promoter SEQ ID NO: 10 provided by the present invention, thereby driving the fertility gene in the transformed recipient plants.
  • the pollen-specific expression promoter provided by the present invention can be used for the specific expression of an exogenous gene in pollen, so as to avoid the adverse effects caused by the continuous expression of the exogenous gene in other plant tissues, and can also be used in plant pollen Functional analysis and identification of growth and development-related genes; can be used for the creation of male sterile lines and maintainer lines; and can be used in pollen abortion experiments to avoid biosafety problems caused by plant transgene 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 TaMS8 gene provided by the present invention can be inserted into a vector, plasmid, yeast artificial chromosome, bacterial artificial chromosome or any other vector suitable for transformation into host cells.
  • Preferred host cells are bacterial cells, especially for cloning or storage of 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 positioned or random.
  • the transfer of a nucleotide sequence, a vector or an expression cassette into a plant or the introduction of a plant or the transformation of a plant as described in the present invention refers to the transfer of a nucleotide sequence, vector or expression cassette into a recipient by conventional transgenic methods cells or recipient plants. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform the recombinant expression vector into plant cells to produce the transgenic plants of the present invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes Agrobacterium-mediated plant transformation methods and the like.
  • the present invention provides a fertility-related gene TaMS8 and its promoter, and a method for using the gene for the reproduction and maintenance of the ms8 male sterile line.
  • the fertility-related genes, the fertility maintenance of the nuclear male sterile line and the breeding method of the sterile line provided by the invention have great production promotion value and application value for the cross-breeding production of crops.
  • the fertility gene provided by the present invention and the sterile line produced by the gene mutation provide resources for wheat hybrid breeding, and also provide necessary elements for the construction of the third-generation hybrid breeding system.
  • the male sterile line produced by the gene mutation using It is of great significance to break through and improve the existing "three-line” and "two-line” hybrid technology to produce hybrid seeds.
  • Figure 1 shows the phenotypes of the wheat ms8 mutants.
  • Picture A is the panicle of the parental Ningchun 4 (left) and ms8 mutant (right) at full bloom, and pictures B and C are the parental Ningchun 4 and ms8, respectively.
  • the anthers of the mutants at full bloom, D and E are the iodine-potassium iodide staining of the mature anthers of the parent Ningchun 4 and ms8 mutants, respectively.
  • Figure 2 shows the distribution of wild-type and mutant SNP indexes on chromosome 1A of wheat.
  • Figure 3 shows the results of TaMS8 gene mutmap and map-based cloning.
  • the first row shows the mapping results of 404 sterile ms8 strains
  • the second row shows the fine mapping results after expanding the population
  • the third row shows the SNP analysis results in the fine mapping interval
  • the fourth row Candidate gene structures and mutation sites.
  • Figure 4 shows the genotype and phenotype analysis of the exchanged individual plants screened by the TaMS8 gene fine mapping.
  • Figure 5 shows the expression pattern of TaMS8 gene in different tissues of Chinese spring wheat by semi-quantitative RT-PCR and real-time PCR
  • ACTIN is a housekeeping gene.
  • the upper picture is the agarose gel electrophoresis image of the RT-PCR products of TaMS8 gene in different organs or tissues; the lower picture is the quantitative RT-PCR results of TaMS8 gene in different organs or tissues, 0 is the meiotic anther , 1 is the anther in the mononucleate stage, 2 is the anther in the binucleate stage, 3 is the anther in the trinucleate stage, the palea is the mixed material of palea and lemma, and gDNA is the genomic DNA control.
  • Figure 6 is a schematic diagram of the construction of a plant transformation vector for functional complementation of ms8 mutants, where LB and RB are the left and right borders of T-DNA, respectively, and the expression of the Bar resistance gene is driven by Ubip (the promoter of the Ubi gene) and terminated by Nos subterminus; the TaMS8 sequence is shown in SEQ ID NO:12.
  • the present invention obtains more than 100 wheat male sterility materials by conducting large-scale phenotype screening on 1 million M2 generation plants of spring wheat variety Ningchun No. 4 EMS mutagenesis library. By subsequent genetic analysis of each material, more than 20 genetically stable recessive single-gene control male sterility materials were screened out. After allelic testing of these materials with the existing ms1 and ms5 mutants, a new single-gene control wheat recessive male sterile line was found. All sites are not allelic, and the present invention names it as MS8.
  • the anthers of the parental plants were able to dehiscence normally, and the pollen grains were obviously scattered, while the anthers of the ms8 mutant were shriveled and did not dehiscence at all.
  • the pollen activity was identified by iodine-potassium iodide (I 2 -KI) staining.
  • I 2 -KI iodine-potassium iodide staining.
  • the mature pollen starch grains of the parental plants were darkly colored and full spherical, while the ms8 mutant was obviously shrunken and had no coloration at all, indicating that its pollen was inactive, so the later stage No seeding at all (see Figure 1).
  • the crossed F2 population of the ms8 mutant and the parent Ningchun 4 was constructed, and the DNA of the sterile plants in the F2 population was mixed into a mutant DNA pool.
  • the single-plant DNA with the wild-type genotype of MS8 locus in the F2 generation population was mixed into a wild-type DNA pool.
  • the two mixed pools were sent to a sequencing company for DNA sequencing, and each mixed pool measured 500G of data.
  • the sequencing data of each pool was aligned to the wheat Chinese spring reference sequence for mutmap analysis to obtain the single nucleotide polymorphism (SNP) of the two samples.
  • SNP single nucleotide polymorphism
  • the linkage analysis of 5 SNP markers and MS8 loci was carried out with 404 ms8 sterile strains isolated from the F2 generation population, and MS8 was located in the range of 3.65Mb between 481,704,309bp(1A-1)-485,352,404bp(1A-3). Inside, the distance from 1A-1 is 0.12cM, and the distance from 1A-3 DYZ8 is 0.25cM (see Figure 3).
  • the CDS sequence is shown in SEQ ID NO: 24, resulting in the mutation of the 208th amino acid of the encoded protein sequence from glycine (Gly) to aspartic acid (Asp), and the amino acid sequence is shown in SEQ ID NO: 25 (see image 3).
  • the genomic DNA sequence of the TaMS8 gene coding region is shown in SEQ ID NO:1
  • the CDS sequence is shown in SEQ ID NO:2
  • the protein sequence is shown in SEQ ID NO:3.
  • the primer sequences used are as follows:
  • TaMS8-RTF 5'-ACTGCACAGACCACCATTGAGATT-3' (SEQ ID NO:4)
  • TaMS8-RTR 5'-ATCAAGTAGCGCGCAGACATTG-3' (SEQ ID NO:5)
  • TaMS8-QF 5'-CGACGACAAGAAGAAGGTTTGAGGAG-3' (SEQ ID NO:6)
  • ACTIN-RTF 5'-TCAGCCATACTGTGCCAATC-3' (SEQ ID NO:7)
  • ACTIN-RTR 5'-CTTCATGCTGCTTGGTGC-3' (SEQ ID NO: 8)
  • ACTIN-QF 5'-TTCCAGCCATCTTTCATTG-3' (SEQ ID NO: 9)
  • the 6783bp TaMS8 genome sequence (SEQ ID NO: 12, which contains 2907bp promoter sequence (SEQ ID NO: 10), 2988bp genome sequence (SEQ ID NO: 1) and 888bp terminator sequence (SEQ ID NO: 1) ID NO: 11) was inserted into the multiple cloning site by in-fusion method, thereby forming the plant expression vector p1300-TaMS8 (see Figure 6).
  • the functional complementation vector p1300-TaMS8 vector was transformed into young embryos of heterozygous plants of wheat ms8 mutants, and a total of 45 transgenic positive plants were obtained. Since the genotype of immature embryos used as transformed explants may be MS8 locus wild type, heterozygous or homozygous mutant state, 9 transgenic positive plants were found to be ms8 homozygous mutant genotype by genotype identification of adjacent SNP markers , observed the pollen fertility of these 9 transgenic positive plants, and found that all of them were fertile. The above results indicate that the mutation of the TaMS8 gene leads to the male sterile phenotype of the ms8 mutant.
  • the invention applies the wheat ms8 male sterility mutant and TaMS8 gene to the new generation hybrid breeding technology of wheat.
  • the core of the new generation of cross-breeding technology is to use wheat recessive nuclear male sterility mutants as transformation receptor materials, and to transform fertility restorer genes, pollen inactivation genes and seed marker genes into male sterility mutants together to form restorations.
  • the TaMS8 gene is used as the fertility restoration gene
  • the maize gene ZmBT1-ZmAA
  • the pollen inactivation gene the maize gene (ZmBT1-ZmAA)
  • the blue grain gene is used as the seed marker gene
  • three expression cassettes are jointly constructed to transform the resistance gene into the Ubi promoter.
  • TaMS8 gene expression cassette the target gene TaMS8 and its promoter and terminator are all from wheat variety Ningchun No. 4, the full-length nucleotide sequence is shown in SEQ ID NO: 12, and the promoter sequence of TaMS8 gene is shown in SEQ ID NO: 12 ID NO: 10, the terminator sequence is shown in SEQ ID NO: 11, the genomic DNA sequence is shown in SEQ ID NO: 1, and the protein amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 3 ;
  • Gene expression cassette PG47 ZmBT1-ZmAA-IN2-1, the target gene is ZmAA, the transit peptide is ZmBT1, and the open reading frame of ZmBT1-ZmAA (its nucleotide sequence is shown in SEQ ID NO: 14) is connected to Downstream of promoter PG47 (whose nucleotide sequence is shown in SEQ ID NO: 15), upstream of terminator IN2-1 (whose nucleotide sequence is shown in SEQ ID NO: 16).
  • the blue grain marker gene expression cassette is shown in patent WO2019090496A1.
  • the target gene is the 3215bp ThMYB1 genomic sequence (containing the 1952bp promoter sequence (shown in SEQ ID NO: 17), the 822bp genomic sequence (shown in SEQ ID NO: 18) and the 441bp terminator sequence (shown in SEQ ID NO: 19)) And 4422bp ThR1 genomic sequence (comprising 2084bp promoter sequence (SEQ ID NO:20), 1720bp CDS sequence (SEQ ID NO:21) and 618bp terminator sequence (SEQ ID NO:22)).
  • the above vector was transformed into young embryos of heterozygous plants of wheat ms8 mutants, and 15 transgenic positive plants with the genotype of ms8 homozygous mutation were obtained.
  • the transgenic positive plants were stained with iodine-potassium iodide to detect the pollen activity, and the results showed that the percentage of sterile pollen in many transgenic plants was about 50%, which indicated that the fertility restorer gene worked first to restore the male sterility phenotype of the ms8 mutant.
  • the pollen-killing gene inactivated half of the pollen containing the transgene.
  • the ratio of normal color seeds to blue seeds is about 1:1, that is, the seeds carrying the exogenous transgene show a blue phenotype due to the seed marker gene.
  • the above results show that the three expression cassettes of the fertility restoration gene, the pollen inactivation gene and the seed marker gene in the present invention can function correctly, and can be successfully applied to the new generation hybrid breeding technology of wheat.

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Abstract

一种小麦育性相关基因TaMS8及其应用方法，属于生物技术领域。通过对EMS诱变库的表型筛选和遗传分析得到隐性单基因控制雄性不育材料，然后通过Mutmap方法获得了小麦育性相关基因TaMS8，并且通过调节该基因的表达以调控植物育性，用以生产并保持小麦雄性不育系以及制备杂交种子，对于建立高效的小麦杂交种制种技术、研究小麦雄性不育机理和杂种优势具有重要的理论和实践意义。

Description

一种育性相关基因及其在杂交育种中的应用 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。
背景技术
杂种优势是生物界普遍存在的一种自然现象。充分利用杂种优势大幅度提高作物产量，改善作物品质，已经在水稻、玉米、高粱、棉花、油菜等多种作物中获得了巨大的经济效益。小麦是世界上栽培面积最广，适应性最强的粮食作物，全球约有40％的人口以小麦为主食。像水稻一样，小麦也具有明显的杂种优势。然而，由于其自身复杂的遗传特性，杂交小麦的表现远不如杂交水稻出色，小麦杂种优势的理论和实践研究都远远落后于水稻、玉米、油菜等作物。
小麦是自花授粉作物，其杂种优势利用的核心是建立高效生产小麦杂交种的技术体系。小麦杂种优势利用途径制种技术的关键是要有合适的隐性核雄性不育系母本。雄性不育指的是植物在有性繁殖过程中不能产生具有正常功能的花药、花粉或雄配子，对雄性不育机理的揭示，是利用杂种优势提高小麦产量和品质的基础。小麦基因组庞大且复杂，所以对小麦雄性不育的机理研究迄今为止积累的资料非常有限。因此，将现代分子生物学和细胞生物学手段应用于小麦雄性不育机理研究，对于加强小麦杂种优势研究和利用具有重要的理论和实践意义。
小麦是异源六倍体，有三套相似而又不近完全相同的基因组存在，因此小麦中的单基因控制隐性突变体相对其他物种明显少。目前已经公开报道的单基因控制小麦隐性核雄性不育材料只有2个位点，MS1和MS5。MS1定位在小麦4B染色体的短臂上，编码一个220个氨基酸的非特异性脂转运蛋白(non-specific lipid transfer protein[nsLTP])(Wang et al.,2017；Tuker et al.,2017)。nsLTP在植物中广泛存在，其特点是具有由8个半胱氨酸残基构成的8CM结构域(Boutrot et al.,2008)。MS1是一个禾本科新进化出的蛋白，原位杂交显示该基因特异地在花药的小孢子母细胞中高量表达。脂结合实验表明MS1能够特异地结合磷脂酸(phosphatidic acid[PA])和几类肌醇磷脂(phosphoinositide[PIPs])。序列分析显示小麦基因组的ABD基因组中各存在一个MS1同源基因，但是由于AD基因组中的MS1同源基因启动子区发生了甲基化修饰，导致这两个基因在六倍体小麦中完全不表达。MS5定位在小麦3A 染色体上，也编码一个非特异性脂转运蛋白，但是除了也具有8个半胱氨酸残基构成的8CM结构域外，其氨基酸序列与MS1之间的同源性并不高(Pallotta et al.,2019)。MS5仅在小麦族中保守存在。
为了获得更多的小麦雄性不育资源，本发明通过对EMS诱变库的表型筛选和遗传分析，筛选出二十余份遗传稳定的隐性单基因控制雄性不育材料。将这些材料与已有的ms1和ms5突变体进行等位检测后，发现其中1个新的单基因控制小麦隐性核雄性不育株系，等位杂交检测实验确认其与MS1位点和MS5位点均不等位，本发明将其命名为MS8。本发明利用Mutmap方法克隆了小麦TaMS8基因，该基因以及该基因突变产生的雄性不育系为构建新型杂交育种体系提供了必要的元件。
发明内容
本文提到的所有参考文献都通过引用并入本文。
除非有相反指明，本文所用的所有技术和科学术语都具有与本发明所属领域普通技术人员通常所理解的相同的含义。除非有相反指明，本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用，而非加以限制。
本发明提供了一个育性相关基因TaMS8，所述育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1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，本发明所述的育性相关基因还包括与TaMS8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相关基因的DNA序列，所述育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1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相关基因还可操作性的连有一个可驱动其表达的启动子，所述启动子包括但不限于组成型表达启动子、诱导型启动子、组织特异表达启动子、或时空特异表达启动子。更具体地，所述启动子是一个花粉特异表达启动子。优选地，所述花粉特异表达启动子的核苷酸序列如SEQ ID NO:10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，其核苷酸序列如SEQ ID NO:14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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等。具体地，所述筛选基因包括但不限于：蓝粒基因、氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还公开了一种植物育性调控的方法，所述方法通过将育性相关基因转入到小麦ms8突变体中，使小麦ms8突变体的雄性育性恢复，其中所述的育性相关基因的核苷酸序列选自 下列组的序列之一：
(a)如SEQ ID NO：1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。
本发明还公开了一种雄性不育系的保持方法，所述方法以小麦ms8突变体植株为转化受体材料，将紧密连锁的3个目标基因转化至该不育突变体受体植株中。所述3个目标基因分别是育性相关基因TaMS8、花粉失活基因和筛选基因。其中，育性相关基因TaMS8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。
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，其核苷酸序列如SEQ ID NO:14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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等。具体地，所述筛选基因包括但不限于：蓝粒基因、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)向小麦ms8雄性不育系中转入下述载体，以获得含有下述载体的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复小麦ms8突变体的雄性生育力；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小麦ms8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小麦ms8雄性不育系植株上，使小麦ms8雄性不育系植株授粉繁殖出ms8雄性不育系或其等位不育系种子。
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，其核苷酸序列如SEQ ID NO:14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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等。具体地，所述筛选基因包括但不限于：蓝粒基因、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)向小麦ms8雄性不育系中转入下述载体，即获得了小麦ms8核雄性不育系或其等位不育系的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复小麦ms8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，其核苷酸序列如SEQ ID NO:14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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等。具体地，所述筛选基因包括但不限于：蓝粒基因、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)向小麦ms8核雄性不育系或其等位不育系中转入下述载体，即获得了小麦ms8核雄性 不育系或其等位不育系的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复小麦ms8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小麦ms8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)向小麦ms8核雄性不育系或其等位不育系中引入下述载体，获得小麦ms8核雄性不育系或其等位不育系的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复小麦ms8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小麦ms8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相关基因TaMS8、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，再与小麦ms8核雄性不育系或其等位不育系杂交，经过定向选育，获得背景为小麦ms8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，其中所述的育性相关基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1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。
上述育性相关基因TaMS8还可操作性的连有一个花粉特异表达的启动子，可以驱动TaMS8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:10所示。上述育性相关基因TaMS8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:11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，其核苷酸序列如SEQ ID NO:14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蓝粒基因、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：蓝粒基因、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:10所示。将SEQ ID NO:10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:10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:10所示的核苷酸序列，或包含与SEQ ID NO:10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:10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:10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，TaMS8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TaMS8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***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、rotB、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:11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:10。具体地，可以将本发明所提供的育性相关基因TaMS8的核苷酸序列构建到本发明所提供的启动子SEQ ID NO:10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TaMS8基因的核苷酸序列和启动子序列或表达盒可被***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***至被转化的植物细胞的基因组中。***可以是定位的或随机的***。
本发明所述的将核苷酸序列、载体或表达盒转入植株或引入植株或对植株进行转化，均指通过常规的转基因方法，将核苷酸序列、载体或表达盒转入到受体细胞或受体植株中。植物生物技术领域技术人员已知的任何转基因方法均可被用于将重组表达载体转化进植物细胞中，以产生本发明的转基因植物。转化方法可包括直接和间接的转化方法。合适的直接方法包括聚乙二醇诱导的DNA摄入、脂质体介导的转化、使用基因枪导入、电穿孔、以及显微注射。所述转化方法也包括农杆菌介导的植物转化方法等。
与现有技术相比，本发明具有如下的有益效果：本发明提供了一种育性相关基因TaMS8及其启动子，及将该基因用于ms8雄性不育系的繁殖和保持的方法。本发明所提供的育性相关基因、核雄性不育系的育性保持和不育系的繁殖方法，对作物的杂交育种生产来说，具有重大的生产推广价值和应用价值。本发明提供的育性基因以及该基因突变产生的不育系为小麦杂交育种提供了资源，也为构建第三代杂交育种体系提供了必要的元件，该基因突变产生的雄性不育系，用来生产杂交种子，对于突破并改良现有的“三系”和“两系”杂交技术有重要意义。
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附图说明
图1是小麦ms8突变体的表型，其中A图为亲本宁春四号(左)和ms8突变体(右)盛花期的穗子，B图和C图分别为亲本宁春四号和和ms8突变体盛花期的花药，D图和E图分别为亲本宁春四号和和ms8突变体成熟花药碘-碘化钾染色情况。
图2为野生型与突变体SNP index在小麦1A染色体上分布图表。
图3为TaMS8基因mutmap及图位克隆结果，其中第一行为404株ms8不育株定位结果，第二行为扩大群体后显示精细定位结果，第三行为精细定位区间内SNP分析结果，第四行为 候选基因结构及突变位点。
图4为TaMS8基因精细定位筛选出的交换单株基因型与表型分析。
图5为利用半定量RT-PCR和荧光定量PCR分析TaMS8基因在中国春小麦不同组织中的表达模式，ACTIN为管家基因。其中上图为TaMS8基因在不同器官或组织中的RT-PCR产物的琼脂糖凝胶电泳图；下图为TaMS8基因在不同器官或组织中的定量RT-PCR结果，0为减数***期花药，1为花单核期花药，2为双核期花药，3为三核期花药，稃为内稃和外稃混合材料，gDNA为基因组DNA对照。
图6为用于ms8突变体功能互补的植物转化载体构建示意图，其中LB和RB分别为T-DNA的左右边界，Bar抗性基因的表达由Ubip(Ubi基因的启动子)驱动，由Nos终止子终止；TaMS8序列如SEQ ID NO：12所示。
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.小麦ms8突变体的获得及表型分析
为了获得更多的小麦雄性不育资源，本发明通过对春小麦品种宁春四号EMS诱变库100万M2代植株进行大规模的表型筛选，获得了一百多份小麦雄性不育材料。后续通过对每一份材料进行遗传分析，从中筛选出二十余份遗传稳定的隐性单基因控制雄性不育材料。将这些材料与已有的ms1和ms5突变体进行等位检测后，发现其中1个新的单基因控制小麦隐性核雄性不育株系，等位杂交检测实验确认其与MS1位点和MS5位点均不等位，本发明将其命名为MS8。
遗传分析结果显示，该突变体与其亲本宁春四号杂交的F2代群体中，可育株与不育株的分离比为242：69，表明该位点为单基因控制的隐性突变。对ms8突变体与其亲本进行表性观察，其营养生长期与亲本没有任何可见差别，直到盛花期，ms8突变体穗型与亲本宁春四号相比差异显著：ms8突变体的护颖和外颖以及外颖上的麦芒张开的角度与正常植株相比明显大，亲本植株花药能够正常开裂，明显可见花粉粒散出，而ms8突变体花药较瘪、完全不开裂。用碘-碘化钾(I 2-KI)染色鉴定花粉活性，亲本植株成熟花粉淀粉粒着色深，呈饱满的圆球状，ms8突变体则明显皱缩、完全没有着色，表明其花粉无活力，因此后期完全不打种子(见图1)。
实施例2.小麦MS8基因的克隆
由于ms8突变体遗传分析显示其为单基因控制的隐性突变，因此我们选择了mutmap即突变位点图谱的技术对其进行基因克隆。
首先构建ms8突变体与亲本宁春四号的杂交F2代群体，将F2代群体中的不育株DNA混合为突变体DNA混池，追踪了F2代群体中的可育株后代表型后，将F2代群体中MS8位点基因型为野生型的单株DNA混合为野生型DNA混池。将两个混池分别送测序公司进行DNA测序，每个混池各测500G数据。将每个混池的测序数据比对到小麦中国春参考序列上进行mutmap分析，获得两个样品的单核苷酸多态性位点(single nucleotide polymorphism，SNP)。根据SNP Index在每条染色体上的分布，可见在小麦的21对染色体中，1A染色体400～600Mb处有明显的峰，表明MS8很可能定位在该区域内(见图2)。
对该区段内的SNP设计引物并进行分子验证。SNP index峰值所在位置大概在小麦1A染色体长臂480～490Mb处，我们在这段区间内挑选了5个高可信度SNP，分别命名为1A-1～1A-5，除了1A-4外，其他4个SNP均开发为CAPs(Cleaved Amplified Polymorphic Sequences)分子标记。在亲本和突变体中的验证结果表明这5个SNP均真实存在。用F2代群体中分离的404株ms8不育株对5个SNP标记与MS8位点进行连锁分析，将MS8定位在481,704,309bp(1A-1)-485,352,404bp(1A-3)之间3.65Mb范围内，距离1A-1 0.12cM，距离1A-3 DYZ8 0.25cM(见图3)。
为了进一步精细定位MS8，我们首先用1A-1(481,704,309bp)和1A-5(488,109,008bp)两个CAPs标记筛选了2160株F2代群体，获得共计37株交换单株，再用1A-3(485,352,404bp)CAPs标记对这37株进行基因型鉴定，获得1A-1和1A-3之间交换单株21株。对21株交换单株进行表型观察结合基因型鉴定结果，将MS8位点的定位区间进一步缩小到481,704,309bp(1A-1)-483,032,932bp(1A-2)之间1.33Mb范围内(见图3)，这此范围内剩余交换单株13株。在1A-1和1A-2之间开发了新的SNP标记1A-6(482,337,315bp)、1A-13(482,081,042bp)、1A-16(482,661,640bp)、1A-17(482,830,475bp)和1A-20(482,184,426bp)，对此13株交换单株进行基因型鉴定后，结合其MS8位点基因型(根据其后代表型鉴定结果推断)，将MS8位点缩小到1A-20(482,184,426bp)和1A-17(482,830,475bp)之间646Kb区段内(见图3和图4)。这646Kb基因组序列内注释了13个编码基因，对此区段内的SNP进行分析，只有1个SNP 1A-6落在基因编码区段内，导致错义突变，暗示该SNP所在基因很可能就是MS8基因，我们将其命名为TaMS8。1A-6所在基因ATG后基因组DNA序列的第1664位核苷酸由G突变为A，其中突变后的基因组序列如SEQ ID NO：23所示， 突变后的CDS序列如SEQ ID NO：24所示，导致其编码的蛋白序列的第208位氨基酸由甘氨酸(Gly)突变为天冬氨酸(Asp)，氨基酸序列如SEQ ID NO：25所示(见图3)。
在小麦中国春品种中，所述TaMS8基因编码区的基因组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。
实施例3.TaMS8基因的表达特异性
为了分析TaMS8基因的表达模式，以该基因的cDNA序列为模板设计半定量RT-PCR引物TaMS8-RTF和TaMS8-RTR(SEQ ID NO:4和5)及荧光定量PCR引物TaMS8-QF和TaMS8-RTR(SEQ ID NO:5和6)，以小麦ACTIN基因作为内参对照设计半定量RT-PCR引物ACTIN-RTF和ACTIN-RTR(SEQ ID NO:7和8)及荧光定量PCR引物ACTIN-QF和ACTIN-RTR(SEQ ID NO:8和9)。取中国春小麦各组织材料提取总RNA并合成cDNA模板，用半定量RT-PCR方法和荧光实时定量RT-PCR方法检测TaMS8基因的表达。结果如图5所示，该基因表达高峰在减数***期花药和单核期花药，至双核期花药表达明显减弱，三核期表达则更低，定量PCR的结果与半定量RT-PCR一致，在其他组织器官，比如根、茎、叶、内/外稃、雌蕊及减数***前各种大小的穗子中表达相对比较低。
所用引物序列如下：
TaMS8-RTF:5’-ACTGCACAGACCACCATTGAGATT-3’(SEQ ID NO:4)
TaMS8-RTR:5’-ATCAAGTAGCGCGCAGACATTG-3’(SEQ ID NO:5)
TaMS8-QF:5’-CGACGACAAGAAGAAGGTTGAGGAG-3’(SEQ ID NO:6)
ACTIN-RTF:5’-TCAGCCATACTGTGCCAATC-3’(SEQ ID NO:7)
ACTIN-RTR:5’-CTTCATGCTGCTTGGTGC-3’(SEQ ID NO:8)
ACTIN-QF:5’-TTCCAGCCATCTTTCATTG-3’(SEQ ID NO:9)
实施例4.小麦ms8突变体的转基因功能互补
为了验证TaMS8基因的功能，我们构建了稳定转化载体用于小麦ms8突变体的功能互补。以双元表达载体pCAMBIA1300为骨架，首先将pCAMBIA1300上的植物抗性筛选基因35S启动子驱动的潮霉素抗性表达框用pAHC20载体上的Ubi启动子驱动的Bar抗性基因表达框取代。在此基础上，将6783bp TaMS8基因组序列(SEQ ID NO：12所示，其中包含2907bp启动子序列(SEQ ID NO:10)、2988bp基因组序列(SEQ ID NO:1)和888bp终止子序列(SEQ ID NO:11))用in-fusion的方法***多克隆位点，从而形成植物表达载体p1300-TaMS8(见图6)。
利用农杆菌介导的遗传转化技术，将功能互补载体p1300-TaMS8载体转化小麦ms8突变体的杂合体植株幼胚，共获得45株转基因阳性植株。由于作为转化外植体的幼胚的基因型可能为MS8位点野生型、杂合或者纯合突变状态，通过相邻SNP标记基因型鉴定发现其中9株转基因阳性植株为ms8纯合突变基因型，观察这9株转基因阳性植株的花粉育性，发现花粉其均可育。上述结果表明TaMS8基因的突变导致ms8突变体雄性不育表型。
实施例5.TaMS8基因启动子表达载体的构建和功能分析
以小麦宁春四号品种基因组DNA为模板，扩增2907bp TaMS8基因启动子片段(SEQ ID NO:10)并通过in-fusion技术连入抗性基因改造为Ubi启动子驱动的Bar基因的pCAMBIA1300载体上，然后用in-fusion技术在2907bp TaMS8基因启动子(SEQ ID NO:10)后面***GUS报告基因(SEQ ID NO:11)(见图8)。利用农杆菌介导的遗传转化技术将上述质粒转化小麦幼胚，获得15株转基因阳性植株。对转基因阳性植株不同组织器官的GUS染色分析结果与TaMS8基因的表达模式分析结果一致，表明TaMS8基因启动子为花药特异表达型启动子。
实施例6.TaMS8基因在新一代小麦杂交育种技术中的应用
本发明将小麦ms8雄性不育突变体和TaMS8基因应用于小麦新一代杂交育种技术。新一代杂交育种技术的核心是以小麦隐性核雄性不育突变体为转化受体材料，将育性恢复基因、花粉失活基因和种子标记基因共同转化至雄性不育突变体中，形成恢复系和保持系一体的转基因株系。利用保持系给不育系授粉，由此繁殖不育系。
本发明以TaMS8基因作为育性恢复基因，以玉米基因(ZmBT1-ZmAA)为花粉失活基因，以蓝粒基因作为种子标记基因，将三个表达盒共同构建到抗性基因改造为Ubi启动子驱动的Bar基因的pCAMBIA1300载体上。
1)以抗性基因改造为Ubi启动子驱动的Bar基因的pCAMBIA1300载体为基础；
2)TaMS8基因表达盒，目标基因TaMS8及其启动子和终止子均来自小麦品种宁春四号，全长核苷酸序列如SEQ ID NO：12所示，其中TaMS8基因的启动子序列如SEQ ID NO：10所示，其终止子序列如SEQ ID NO：11所示，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：14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：15所示)的下游、终止子IN2-1(其核苷酸序 列如SEQ ID NO：16所示)的上游。
4)蓝粒标记基因表达盒见专利WO2019090496A1。目标基因为3215bp ThMYB1基因组序列(包含1952bp启动子序列(SEQ ID NO：17所示)、822bp基因组序列(SEQ ID NO：18所示)和441bp终止子序列(SEQ ID NO：19所示))和4422bp ThR1基因组序列(包含2084bp启动子序列(SEQ ID NO：20所示)、1720bp CDS序列(SEQ ID NO：21所示)和618bp终止子序列(SEQ ID NO：22所示))。
利用农杆菌介导的遗传转化技术，将上述载体转化小麦ms8突变体的杂合体植株幼胚，得到15株基因型为ms8纯合突变的转基因阳性植株。对转基因阳性植株进行碘-碘化钾染色检测花粉活性，结果显示多个转基因植株的不育花粉比例为50％左右，这说明首先育性恢复基因起作用将ms8突变体的雄性不育表型恢复，其次花粉致死基因将一半的含有转基因花粉失活。在单株收获的T1代转基因植株种子中，能观察到正常颜色种子与蓝色籽粒种子比例约为1：1，即携带外源转基因的种子因为种子标记基因的缘故而呈现蓝粒表型。上述结果表明本发明中育性恢复基因、花粉失活基因和种子标记基因这三个表达盒均能够正确地行使功能，可以成功应用于小麦新一代杂交育种技术。

Claims (21)

  1. 一种育性相关基因TaMS8，其特征在于所述育性相关基因TaMS8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。
  2. 一种表达盒、表达载体或工程菌，其特征在于所述表达盒、表达载体或工程菌包含权利要求1所述的育性相关基因。
  3. 一种育性相关基因、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相关基因、表达盒、表达载体、工程菌含有如下所示的核苷酸序列之一：
    (a)如SEQ ID NO：1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。
  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相关基因，影响其表达水平，进而调控植物育性，其特征在于：所述育性相关基因TaMS8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。
  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相关基因的核苷酸序 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TaMS8基因的核苷酸序列互补由TaMS8基因突变所导致的雄性不育表型，使ms8雄性不育系恢复成可育。
  8. 根据权利要求7所述的方法，TaMS8基因突变的序列如SEQ ID NO：23或24所示，氨基酸如SEQ ID NO：25所示.
  9. 权利要求4-8之任一所述的方法在调控植物育性中的应用。
  10. 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ms8雄性不育系中转入下述载体，以获得含有下述载体的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复ms8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ms8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ms8不育系植株授粉上，使ms8不育系授粉繁殖出ms8不育系种子。
  11. 根据权利要求10所述的生产或繁殖方法，其中所述的育性相关基因TaMS8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。
  12. 根据权利要求11所述的生产或繁殖方法，其中所述的育性相关基因TaMS8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SEQID NO:10所示。
  13. 根据权利要求10-12之任一所述的生产或繁殖方法，其中所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等，优选的所述花粉失活基因是玉米α淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO：14所示。
  14. 根据权利要求13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。
  15. 根据权利要求10-14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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等，优选的所述筛选基因包括但不限于：蓝粒基因、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。
  16. 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ms8雄性不育系中转入下述载体，即获得了ms8雄性不育系的保持系，所述载体包含：育性相关基因TaMS8，所述育性相关基因TaMS8可以恢复ms8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ms8雄性不育系种子和含载体的保持系种子。
  17. 根据权利要求16所述的生产或繁殖方法，其中所述的育性相关基因TaMS8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。
  18. 根据权利要求17所述的生产或繁殖方法，其中所述的育性恢复基因TaMS8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SEQID NO:10所示。
  19. 根据权利要求16-18之任一所述的生产或繁殖方法，其中所述的花粉失活基因包括但不限于barnase基因、淀粉酶基因、DAM甲基化酶等，优选的所述花粉失活基因是玉米α淀粉酶基因，更优选的其核苷酸序列如SEQ ID NO：14所示。
  20. 根据权利要求19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。
  21. 根据权利要求16-20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蓝粒基因、抗生素抗性基因、抗除草剂基因、荧光蛋白基因等，优选的所述筛选基因包括但不限于：蓝粒基因、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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