WO2022109764A1 - Gène lié à la fertilité et son application dans la sélection d'hybrides - Google Patents

Gène lié à la fertilité et son application dans la sélection d'hybrides 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)
Chinese (zh)
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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 CN202080106639.1A priority Critical patent/CN116529376A/zh
Priority to PCT/CN2020/130982 priority patent/WO2022109764A1/fr
Publication of WO2022109764A1 publication Critical patent/WO2022109764A1/fr

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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

La présente invention concerne un gène lié à la fertilité du blé, TaMS8, et un procédé d'application de celui-ci, relevant du domaine de la biotechnologie. Un matériel stérile mâle commandé par un gène unique récessif est obtenu au moyen d'un criblage phénotypique et d'une analyse génétique d'une banque de mutagenèse EMS, puis, le gène TaMS8 lié à la fertilité du blé est obtenu au moyen d'un procédé Mutmap, et la fertilité de la plante est régulée en ajustant l'expression du gène, ce qui permet de produire et de maintenir des lignées stériles mâles de blé et de préparer des semences hybrides, ce qui a une signification théorique et pratique importante pour établir des techniques efficaces de production de semences hybrides de blé, et étudier le mécanisme de stérilité mâle du blé et l'hétérosis.
PCT/CN2020/130982 2020-11-24 2020-11-24 Gène lié à la fertilité et son application dans la sélection d'hybrides WO2022109764A1 (fr)

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