[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2024174954A1 - 小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用 - Google Patents

小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用 Download PDF

Info

Publication number
WO2024174954A1
WO2024174954A1 PCT/CN2024/077521 CN2024077521W WO2024174954A1 WO 2024174954 A1 WO2024174954 A1 WO 2024174954A1 CN 2024077521 W CN2024077521 W CN 2024077521W WO 2024174954 A1 WO2024174954 A1 WO 2024174954A1
Authority
WO
WIPO (PCT)
Prior art keywords
wtk7
wheat
protein
plant
gene
Prior art date
Application number
PCT/CN2024/077521
Other languages
English (en)
French (fr)
Inventor
刘志勇
李淼淼
张怀志
朱科宇
董玲丽
吴秋红
Original Assignee
中国科学院遗传与发育生物学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院遗传与发育生物学研究所 filed Critical 中国科学院遗传与发育生物学研究所
Publication of WO2024174954A1 publication Critical patent/WO2024174954A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • 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
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • 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
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present application belongs to the field of crop molecular biology and molecular breeding, and specifically relates to the cloning, functional labeling and application of the wheat broad-spectrum powdery mildew resistance gene WTK7-TM.
  • Wheat powdery mildew is a worldwide fungal disease caused by the obligate parasitic Blumeria graminis f.sp.tritici, which seriously harms the yield and quality of wheat. Due to changes in wheat production and farming systems, especially the increase in close planting, irrigation and nitrogen fertilizer use, the occurrence of powdery mildew has become more serious year by year. Production practice shows that compared with chemical control, exploring disease-resistant genes and cultivating disease-resistant varieties are the most economical, effective and safe measures to prevent and control wheat powdery mildew. However, due to the continuous emergence of new pathogenic species of pathogens, the disease resistance of varieties is often at risk of reduction or loss.
  • powdery mildew resistance genes have been discovered from wild emmer wheat, such as Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64 and Pm69, as well as several temporarily named genes, such as MlZec1, MlIW72, PmG16, Ml3D232, MlIW170, PmAs846, PmG3M, MlIW172, MlIW30 and MlIW39.
  • Pm41 and Pm69 genes have been successfully cloned, and many powdery mildew resistance genes from wild emmer wheat are urgently needed to be cloned.
  • wheat powdery mildew resistance genes have been successfully cloned from wheat and its wild species, such as Pm1, Pm2, Pm3, Pm4, Pm5, Pm8, Pm17, Pm21, Pm24, Pm38/Lr34/Yr18/Sr57, Pm41, Pm46/Lr67/Yr46/Sr55, Pm55, Pm60 and Pm69.
  • NLR-type disease resistance proteins are only resistant to a single or a few specific species of pathogens. After a few years of large-scale use in production, they are very likely to lose their disease resistance, causing a powdery mildew epidemic. Therefore, further discovering and utilizing new wheat broad-spectrum powdery mildew resistance genes has important theoretical significance and production application value.
  • the technical problem to be solved by this application is: how to regulate wheat resistance to powdery mildew and/or how to screen, Identification of wheat with resistance to powdery mildew.
  • the present application provides the use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein may be a WTK7-TM protein; A1), use in regulating plant stress resistance; A2), use in preparing a product that regulates plant resistance; A3), use in regulating plant powdery mildew resistance; A4), use in preparing a product that regulates plant powdery mildew resistance; A5), use in plant breeding or plant-assisted breeding;
  • the WTK7-TM protein can be any of the following proteins: a1), a protein whose amino acid sequence is shown in SEQ ID No. 3; a2), a protein that has more than 80% identity with the amino acid sequence shown in a1) and is related to plant stress resistance, obtained by replacing and/or deleting and/or adding amino acid residues in the amino acid sequence shown in a1); a3), a fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2).
  • the evaluation index of plant breeding includes the powdery mildew resistance of the plant.
  • the purpose of plant breeding includes cultivating plants resistant to powdery mildew.
  • the protein is derived from wheat.
  • SEQ ID No. 3 consists of 667 amino acid residues.
  • the above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.
  • the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and/or a SUMO protein tag, etc.
  • connection described in a3) can be via a peptide bond.
  • the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material
  • the biological material can be any of the following: B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the above protein or the activity of the above protein; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant containing the nucleic acid molecule described in B1).
  • B6 transgenic plant tissue containing the nucleic acid molecule described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);
  • B7) transgenic plant organ containing the nucleic acid molecule described in B1), transgenic plant organ containing the expression cassette described in B2), or transgenic plant organ containing the recombinant vector described in B3);
  • B8) nucleic acid molecule encoding the above protein;
  • B9 expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).
  • the expression cassette described in B2 refers to a cassette capable of expressing interference in the host cell.
  • the DNA of the interfering RNA may include not only a promoter for initiating transcription of the interfering RNA gene, but also a terminator for terminating transcription of the interfering RNA gene.
  • the recombinant microorganisms described in B4) can specifically be yeast, bacteria, algae and fungi.
  • the plant tissue described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
  • the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.
  • the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.
  • the expression cassette described in B9) refers to a DNA capable of expressing WTK7-TM protein in a host cell, which DNA may include not only a promoter for initiating transcription of the WTK7-TM protein encoding gene, but also a terminator for terminating transcription of the WTK7-TM protein encoding gene.
  • the expression cassette may further include an enhancer sequence.
  • the recombinant vector described in B9) may contain the DNA molecule for encoding WTK7-TM protein shown in SEQ ID No.2.
  • the nucleic acid molecule in B1) may be an RNA or DNA molecule that expresses the gene targeted in the above application.
  • nucleotide sequence of the target sequence of the nucleic acid molecule described in B1) is SEQ ID No. 4 and/or SEQ ID No. 5.
  • the nucleic acid molecule described in B8) can be a DNA molecule described in any one of the following g1)-g3): g1), a DNA molecule whose coding sequence of the coding chain is SEQ ID No.2; g2), a DNA molecule whose nucleotide sequence of the coding chain is SEQ ID No.1; g3), a DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2), and regulates plant stress resistance.
  • the plant is selected from monocotyledonous plants.
  • the monocotyledonous plant is selected from the Poaceae family.
  • the grass plant is selected from the genus Triticum.
  • the wheat plant is selected from wheat (Triticum aestivum L.).
  • the above-mentioned wheat may be wild emmer wheat (Triticum dicoccoides L.).
  • the application may specifically be any one of the following: A1') A method for improving plant stress resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant, wherein the stress resistance of the target plant is higher than that of the recipient plant.
  • A3') A method for improving plant powdery mildew resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant, wherein the powdery mildew resistance of the target plant is higher than that of the recipient plant.
  • A4' A method for preparing a plant with improved powdery mildew resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant with improved powdery mildew resistance.
  • A5' A method for plant breeding, comprising introducing the nucleic acid molecule described in B1) into a recipient plant Recipient plant, obtain the target plant.
  • identity refers to the identity of an amino acid sequence or a nucleotide sequence.
  • the identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for a pair of amino acid sequences to calculate the identity, then the value of the identity (%) can be obtained.
  • the above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.
  • the 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.
  • the substance that regulates the activity or content of the protein may be a substance that downregulates or reduces or knocks out the gene encoding the protein and/or a substance that downregulates or reduces the expression of the gene encoding the protein.
  • the regulation of plant stress resistance may be reducing plant stress resistance.
  • the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).
  • the regulating gene expression may be inhibiting or reducing the gene expression, and the inhibiting or reducing the gene expression may be achieved by gene knockout or gene silencing.
  • the gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.
  • Gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and position effects. The other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and micro RNA (miRNA). mediated translation inhibition, etc.
  • the substance regulating gene expression may be an agent that inhibits or reduces the expression of the gene.
  • the agent that inhibits or reduces the expression of the gene may be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas9.
  • the agent that inhibits or reduces the expression of the gene may include a polynucleotide that targets the gene, such as siRNA, shRNA, double-stranded RNA, miRNA or antisense RNA.
  • the present application also provides a method for cultivating plants with reduced powdery mildew resistance, the method comprising down-regulating or reducing the expression of the WTK7-TM protein in a recipient plant or down-regulating or reducing the activity or content of the WTK7-TM protein to obtain a target plant with reduced powdery mildew resistance.
  • the recipient plant contains the coding gene of the WTK7-TM protein or the WTK7-TM protein.
  • the present application also provides a method for regulating plant powdery mildew resistance, which comprises regulating plant powdery mildew resistance by regulating the expression of the encoding gene in a plant containing the above-mentioned WTK7-TM protein or by regulating the activity or content of the WTK7-TM protein in a plant containing the above-mentioned WTK7-TM protein.
  • the regulation may be down-regulation or reduction.
  • the regulation of the expression of the encoding gene in a plant containing the encoding gene of the WTK7-TM protein or the regulation of the activity or content of the WTK7-TM protein in a plant containing the WTK7-TM protein can be achieved by silencing the WTK7-TM gene in the receptor plant.
  • the WTK7-TM gene in the silencing recipient plant can be achieved by virus-induced gene silencing.
  • the virus-induced gene silencing includes introducing a recombinant virus targeting the WTK7-TM gene into the recipient plant, and the silencing target of the recombinant virus is SEQ ID No. 4 and/or SEQ ID No. 5.
  • the recombinant virus is a recombinant barley streak mosaic virus (BSMV).
  • BSMV barley streak mosaic virus
  • the preparation method of the recombinant barley streak mosaic virus is:
  • the three plasmids were transfected into Agrobacterium tumefaciens EHA105 to prepare recombinant Agrobacterium tumefaciens bacterial solution, as follows:
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ pCaBS- ⁇ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ .
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ pCaBS- ⁇ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ .
  • pCaBS- ⁇ bLIC was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC.
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI pCaBS- ⁇ bLIC-WTK7-TM KinI was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI .
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII pCaBS- ⁇ bLIC-WTK7-TM KinII was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens Agrobacterium EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII .
  • recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ , recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ , recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC, recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI , and recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII were picked and inoculated into 1mL LB liquid medium, and cultured at 28°C, 220rpm shaking for 24h.
  • the infection solution of EHA105/pCaBS- ⁇ , EHA105/pCaBS- ⁇ , EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII was mixed in a ratio of 1:1:1, and then allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, marked as BSMV:WTK7-TM KinII .
  • the preparation methods of recombinant virus BSMV:WTK7-TM KinI and control group recombinant virus BSMV refer to the recombinant virus BSMV:WTK7-TM KinII , with the only difference that the EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII infection solution is replaced by EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI infection solution and EHA105/pCaBS- ⁇ bLIC infection solution, respectively.
  • the plants are selected from monocotyledons.
  • the monocotyledonous plant is selected from the Poaceae family.
  • the grass plant is selected from the genus Triticum;
  • Triticum plant is selected from wheat (Triticum aestivum L.).
  • the expression of WTK7-TM gene in wheat is downregulated or reduced by virus-induced gene silencing, thereby reducing the powdery mildew resistance of wheat.
  • This embodiment demonstrates the role of WTK7-TM gene or its encoded protein in regulating wheat powdery mildew resistance.
  • the wheat may be wheat line 3D232.
  • the present application also provides the protein in the above application and/or the biological material in the above application.
  • the present application also provides the use of WTK7-TM molecular markers and/or substances for detecting WTK7-TM molecular markers in any of the following:
  • the WTK7-TM molecular marker is a fragment in the wheat genome, and its nucleotide sequence is SEQ ID No.6;
  • the substance for detecting the WTK7-TM molecular marker may be any of the following:
  • PCR primer composition for amplifying a wheat genomic DNA fragment including the WTK7-TM molecular marker
  • E3 A kit containing the PCR primer composition described in E1) or the PCR reagent described in E2).
  • the application may specifically be any of the following:
  • the PCR primer composition includes a single-stranded DNA whose nucleotide sequence is SEQ ID No.7 and a single-stranded DNA whose nucleotide sequence is SEQ ID No.8.
  • the present application also provides a DNA molecule whose nucleotide sequence is SEQ ID No. 6 or/and the above-mentioned primer composition or/and the above-mentioned reagent or/and the above-mentioned kit.
  • the present application also provides a method for identifying or assisting in identifying the powdery mildew resistance trait of wheat, the method comprising detecting the WTK7-TM molecular marker in the wheat to be tested, and identifying or assisting in identifying the powdery mildew resistance of wheat based on whether the wheat to be tested contains the WTK7-TM molecular marker, the powdery mildew resistance of the wheat to be tested containing the WTK7-TM molecular marker being higher or having a potential to be higher than the powdery mildew resistance of the wheat to be tested that does not contain the WTK7-TM molecular marker.
  • nucleotide sequence of the WTK7-TM molecular marker is SEQ ID No. 6.
  • SEQ ID No. 6 consists of 1198 nucleotides.
  • the wheat to be tested that contains the WTK7-TM molecular marker can use the above primer combination to amplify the DNA fragment of the WTK7-TM molecular marker, while the wheat to be tested that does not contain the WTK7-TM molecular marker cannot use the above primer combination to amplify the DNA fragment of the WTK7-TM molecular marker.
  • the method for detecting the WTK7-TM molecular marker in the wheat to be tested includes using the genomic DNA of the wheat to be tested as a template, amplifying using the above-mentioned PCR primer combination to obtain a PCR product, and performing agarose gel electrophoresis or sequencing on the PCR product.
  • the present application also provides a method for wheat breeding, which comprises selecting wheat containing the WTK7-TM molecular marker as a parent for breeding.
  • the breeding target of the breeding includes wheat powdery mildew resistance.
  • the purpose of the breeding includes cultivating wheat resistant to powdery mildew ((wheat powdery mildew resistance is higher than that of the parent)).
  • the powdery mildew may be a fungal disease caused by the obligate parasitic Blumeria graminis f.sp.tritici (commonly referred to as powdery mildew).
  • the disease resistance or powdery mildew resistance described in the present application may be resistance to diseases caused by Blumeria graminis f.sp.tritici.
  • the powdery mildew described in the powdery mildew resistance experiment may specifically be powdery mildew physiological race E09.
  • the present application provides a method for identifying the gene localization, map-based cloning and biological function of wheat broad-spectrum powdery mildew resistance gene WTK7-TM.
  • the wheat broad-spectrum powdery mildew resistance gene WTK7-TM can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, It plays an important role in improving and modifying the germplasm resources of crops such as wheat.
  • the functional marker WTK-TM-FM provided in the present application and the primer composition for amplification thereof can be used for auxiliary screening or identification of powdery mildew-resistant wheat and wheat breeding.
  • Figure 1 shows the identification of multiple races of powdery mildew fungus in common wheat line 3D232.
  • Figure 2 shows the positional cloning of the wheat powdery mildew resistance gene WTK7-TM.
  • Figure 3 shows the gene function verification of CYB561-Domon and TM9SF4 transgenes.
  • FIG. 4 shows the splicing pattern of WTK7-TM gene.
  • Figure 5 shows the verification of WTK7-TM's powdery mildew resistance function by BSMV-VIGS and EMS mutants.
  • FIG6 is a flow chart of the construction of a silencing vector.
  • FIG. 7 shows the development and application of WTK7-TM gene functional markers.
  • the wheat line 5BIL-29 described in the following examples was kindly donated by Dr. Antonio Blanco of the University of Bari, Italy. According to the document "Blanco, A. et al. Molecular mapping of the novel powdery mildew resistance gene Pm36 introduced from Triticum turgidum var. dicoccoides in durum wheat. Theor. Appl. Genet. 117, 135-142 (2008)", the public can obtain the above biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.
  • the powdery mildew strain E09 described in the following examples is preserved by this laboratory and is described in the document "Li, M. et al. A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 228, 1027-1037 (2020).
  • the public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.
  • the gene silencing vectors pCaBS- ⁇ , pCaBS- ⁇ and pCaBS- ⁇ bLIC described in the following examples were kindly donated by Professor Li Dawei of China Agricultural University. Stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One 6, e26468 (2011). "The public can obtain the above biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.
  • reagent AS represents acetosyringone.
  • MES 2-Morpholinoethanesulphonic acid, which is a zwitterionic buffer.
  • Example 1 Powdery mildew resistance identification of common wheat 3D232
  • the common wheat line 3D232 is a hybrid of wild emmer wheat I222 (kindly donated by Dr. Gerechter-Amitai of the Volcani Center, Agricultural Research Organization, Israel) and common wheat line 87-1, and then backcrossed with common wheat Jing 411.
  • the pedigree is 87-1/I222//Jing 411*7.
  • 104 powdery mildew species collected from different ecological zones across the country were inoculated in vitro on leaves of 3D232 at the seedling stage. It was found that it was immune or highly resistant to all powdery mildew species, and is an excellent broad-spectrum powdery mildew resistant material (Figure 1).
  • the seedling resistance of F1 , F2 segregating populations and F3 families of common wheat lines 3D232, Xuezao and Xuezao ⁇ 3D232 was identified using powdery mildew race E09, which is prevalent in Beijing.
  • the identification of 630 F2 segregating populations of Xuezao/3D232 showed that 477 of them showed resistance and 153 showed susceptible.
  • the chi-square test was consistent with the 3:1 segregation ratio controlled by a single gene.
  • the wheat powdery mildew resistance gene M13D232 was located in the 0.14 cM genetic interval between the near-terminal molecular markers XBD37670 and XBD37760 on chromosome 5BL by developing molecular markers (D Zhang, S H Ouyang, L L Wang, Y Cui, Q H Wu, Y Liang, Z Z Wang, J Z Xie, D Y Zhang, Y Wang, Y X Chen, Z Y Liu. Comparative genetic mapping revealed powdery mildew resistance gene MIWE4 derived from wild emmer is located in the same genomic region of Pm36 and MI3D232 on chromosome 5BL. Journal of Integrative Agriculture, 4 (2015), pp. 603-609).
  • Xue Zao and 3D232 hybrids to construct a genetic segregation population containing 15,893 F2 individual plants.
  • the genotypes of the F2 genetic segregation population were identified using the molecular markers XBD37670 and XBD37760, which were closest to the MI3D232 gene localization interval, and the key recombinant exchange plants were screened.
  • the powdery mildew resistance of the F2 :3 family of the key recombinant exchange plants screened by the molecular markers XBD37670 and XBD37760 was identified using the powdery mildew physiological race E09.
  • the MI3D232 gene was finely positioned in the genetic interval of 0.021 cM between molecular markers WGGBM2 and WGGBM7, among which molecular markers WGGBM3-WGGBM6 were co-segregated with the MI3D232 gene ( Figure 2).
  • MI3D232 gene is the same gene or allele as the officially named wheat powdery mildew resistance gene Pm36 (D Zhang, S H Ouyang, L L Wang, Y Cui, Q H Wu, Y Liang, Z Z Wang, J Z Xie, D Y Zhang, Y Wang, Y X Chen, Z Y Liu. Comparative genetic mapping revealed powdery mildew resistance gene MIWE4 derived from wild emmer is located in the same genomic region of Pm36 and MI3D232 on chromosome 5BL. Journal of Integrative Agriculture, 4 (2015), pp. 603-609).
  • One of the contigs can completely cover the Ml3D232 localization interval, of which the physical distance between the nearest molecular markers WGGBM2 and WGGBM7 on both sides of the Ml3D232 gene localization interval is about 1.17Mb, while the physical interval of the reference genome of the Chinese Spring is about 275Kb, indicating that there is a large genomic structural variation in this interval in different species.
  • 1.17Mb contig a total of 11 genes were obtained, including CYB561-Domon, three F-boxes, five Serpins, TM9SF4, and WTK7-TM ( Figure 2 and Table 3).
  • RNA-seq analysis showed that CYB561-Domon, TM9SF4, and WTK7-TM were expressed in the leaves of 3D232 after inoculation with powdery mildew E09, while no expression of other genes was detected.
  • the WTK7-TM gene By designing specific primers to amplify and sequence the WTK7-TM gene, it was found that its gene length was 3277bp. Further RACE experiments revealed that the WTK7-TM gene contains 9 different splicing forms (TV1-TV9). Among them, TV1 is the main splicing mode, with a gene coding region length of 2004bp, which can encode 667 amino acids ( Figure 4). By analyzing the WTK7-TM protein, it was found that it contains two tandem kinases and a C-terminal transmembrane domain ( Figure 2). Sequence analysis showed that the WTK7-TM gene is exactly the same in common wheat 3D232 and 5BIL-29, but is missing in the susceptible parents Xue Zao and Chinese Spring.
  • the wheat broad-spectrum powdery mildew resistance gene WTK7-TM described in the present application is located on the wheat chromosome 5BL, the nucleotide sequence of its genomic sequence is SEQ ID No. 1, the nucleotide sequence of the cDNA sequence (coding sequence) is SEQ ID No. 2, and the amino acid sequence of the encoded WTK7-TM protein is SEQ ID No. 3.
  • Example 3 Silencing of WTK7-TM gene by BSMV-VIGS technology
  • the gene silencing vectors were pCaBS- ⁇ , pCaBS- ⁇ , and pCaBS- ⁇ bLIC, which were provided by Li Da from China Agricultural University.
  • the plasmid containing the full-length WTK7-TM gene was amplified by VIGS-1F/VIGS-1R and VIGS-2F/VIGS-2R (Table 1) to obtain two target fragments for constructing the silencing vector.
  • the target fragments amplified by VIGS-1F/VIGS-1R and VIGS-2F/VIGS-2R primers correspond to the kinase 1 (KinI) and kinase 2 (KinII) domains of the WTK7-TM gene, respectively.
  • the pCaBS- ⁇ bLIC vector was digested with restriction endonuclease ApaI and the linearized product was recovered.
  • pCaBS- ⁇ bLIC-WTK7-TM KinI was a recombinant vector obtained by replacing the fragment between LIC1 and LIC2 on pCaBS- ⁇ bLIC with a DNA molecule having a nucleotide sequence of SEQ ID No.4, while keeping the other nucleotides of pCaBS- ⁇ bLIC unchanged.
  • pCaBS- ⁇ bLIC-WTK7-TM KinII was a recombinant vector obtained by replacing the fragment between LIC1 and LIC2 on pCaBS- ⁇ bLIC with a DNA molecule having a nucleotide sequence of SEQ ID No.5, while keeping the other nucleotides of pCaBS- ⁇ bLIC unchanged.
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ pCaBS- ⁇ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ .
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ pCaBS- ⁇ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ .
  • pCaBS- ⁇ bLIC was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC.
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI pCaBS- ⁇ bLIC-WTK7-TM KinI was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI .
  • Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII pCaBS- ⁇ bLIC-WTK7-TM KinII was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII .
  • the cells were divided into three groups according to the different infection plasmids:
  • EHA105/pCaBS- ⁇ , EHA105/pCaBS- ⁇ , and EHA105/pCaBS- ⁇ bLIC infection solutions were mixed at a ratio of 1:1:1, allowed to stand at 28°C for 3-5 hours, and then injected into the expanded leaves of Nicotiana benthamiana at the 6-8 leaf stage, marked as BSMV:00;
  • EHA105/pCaBS- ⁇ , EHA105/pCaBS- ⁇ , and EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinI were mixed in a ratio of 1:1:1, and allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, and were labeled as BSMV:WTK7-TM KinI ;
  • EHA105/pCaBS- ⁇ , EHA105/pCaBS- ⁇ , and EHA105/pCaBS- ⁇ bLIC-WTK7-TM KinII were mixed in a ratio of 1:1:1, and allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, and were labeled as BSMV:WTK7-TM KinII .
  • Example 4 EMS mutants verify the powdery mildew resistance function of WTK7-TM
  • WTK7-TM-FMF and WTK-TM-FMR in Table 1 a primer combination for specific amplification of the functional marker WTK-TM-FM (WTK-TM-FMF and WTK-TM-FMR in Table 1) was developed based on the WTK7-TM gene sequence, and the genomic DNA of the 3D232 material was used as a template for amplification.
  • the functional marker WTK-TM-FM is a DNA molecule with a nucleotide sequence of SEQ ID No.6.
  • the amplification procedure is:
  • PCR reaction system (10 ⁇ L): 2 ⁇ L of wheat leaf genomic DNA (25 ng/ ⁇ L), 5 ⁇ L of 2 ⁇ PCR Mix, 1 ⁇ L of primer WTK-TM-FMF aqueous solution (concentration 10 ⁇ mol/L), 1 ⁇ L of primer WTK-TM-FMR aqueous solution (concentration 10 ⁇ mol/L), and 1 ⁇ L of ddH 2 O.
  • PCR reaction conditions 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 7 min.
  • the amplified product was detected by 1% agarose gel electrophoresis and the band size was 1198bp.
  • the amplified product was sequenced and found to be completely matched with the WTK7-TM gene sequence, indicating that WTK-TM-FMF and WTK-TM-FMR can specifically amplify the functional marker WTK-TM-FM from the wheat genome. If the functional marker WTK-TM-FM is present, a 1198bp band can be amplified, but if the functional marker WTK-TM-FM is not present, a band of the same size cannot be amplified.
  • the functional marker WTK7-TM-FM was used to construct an F2 segregation population (the female parent was common wheat Xue Zao and the male parent was common wheat 3D232) by hybridization of the disease-resistant material common wheat 3D232 and the disease-susceptible material common wheat Xue Zao. 25 disease-resistant and 25 susceptible materials (the method for identifying powdery mildew resistance refers to Example 3) were tested and verified. It was found that the detection results of the functional marker WTK7-TM-FM were co-segregated with the phenotype, that is, the materials with amplified 1198bp bands all showed disease resistance, while the materials without amplified 1198bp bands all showed high sensitivity (Table 5).
  • R indicates that the phenotype of the tested wheat is resistant to powdery mildew
  • S indicates that the phenotype of the tested wheat is susceptible to powdery mildew
  • amplification means that a target band of 1198 bp can be amplified
  • no amplification means that no band is amplified.
  • the present application provides a method for identifying the gene localization, map-based cloning and biological function of wheat broad-spectrum powdery mildew resistance gene WTK7-TM.
  • the wheat broad-spectrum powdery mildew resistance gene WTK7-TM can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and modifying the germplasm resources of crops such as wheat.
  • the functional marker WTK-TM-FM provided in the present application and the primer composition for amplification thereof can be used for auxiliary screening or identification of powdery mildew-resistant wheat.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Botany (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Physiology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

一种小麦广谱抗白粉病基因WTK7-TM克隆、功能标记及其应用。提供WTK7-TM蛋白质或调控其编码基因表达的物质或调控所述蛋白质活性或含量的物质在调控植物白粉病抗性中的应用，所述WTK7-TM蛋白可为氨基酸序列是SEQ ID No.3的蛋白质，所述植物可为小麦。提供了小麦广谱抗白粉病基因WTK7-TM的基因定位、图位克隆以及抗白粉病生物学功能鉴定方法。小麦广谱抗白粉病基因WTK7-TM可以广泛应用于小麦抗病遗传育种、种质资源改良、转基因和基因组编辑育种等领域，对改进和改良小麦等作物的种质资源具有重要作用。

Description

小麦广谱抗白粉病基因WTK7-TM克隆、功能标记及其应用
相关申请的交叉引用
本申请要求申请日为2023年2月20日的中国专利申请申请号为202310138434.7的优先权，该专利申请的全部内容在此被援引加入本文。
技术领域
本申请属于作物分子生物学和分子育种领域，具体涉及小麦广谱抗白粉病基因WTK7-TM克隆、功能标记及其应用。
背景技术
小麦白粉病是由专性寄生的布氏白粉菌(Blumeria graminis f.sp.tritici)所引起的世界性真菌病害，严重危害小麦的产量和品质。由于小麦生产耕作制度的变化，特别是密植、灌溉和氮肥使用量的增加，白粉病发生逐年加重。生产实践表明，与化学防治相比，挖掘抗病基因，培育抗病品种是防治小麦白粉病最为经济、有效、安全的措施。然而，由于病原菌新的致病小种的不断出现，品种的抗病性常常面临降低或丧失的风险。
野生二粒小麦(Triticum dicoccoides,2n＝4x＝28,AABB)是现代栽培四倍体和六倍体小麦的直接祖先，起源于中东“新月沃土”地区，广泛分布于以色列、叙利亚、黎巴嫩、和土耳其等地区。野生二粒小麦经历长期复杂的环境演变，积累丰富的遗传多样性，对小麦白粉病具有良好抗性，使其成为现代小麦抗病遗传改良的重要基因资源。截止目前，已经从野生二粒小麦中发掘出8个正式命名的抗白粉病基因，如Pm16、Pm26、Pm30、Pm36、Pm41、Pm42、Pm64和Pm69，以及多个临时命名的基因，如MlZec1、MlIW72、PmG16、Ml3D232、MlIW170、PmAs846、PmG3M、MlIW172、MlIW30和MlIW39等，然而其中仅有Pm41和Pm69基因被成功克隆，还有许多野生二粒小麦来源的抗白粉病基因亟待克隆。
目前，已经从小麦及其野生种中成功克隆多个小麦抗白粉病基因，如Pm1，Pm2，Pm3，Pm4,Pm5，Pm8，Pm17，Pm21，Pm24，Pm38/Lr34/Yr18/Sr57，Pm41，Pm46/Lr67/Yr46/Sr55,Pm55,Pm60和Pm69。其中除了具有广谱抗病性的Pm24(串联激酶蛋白)，Pm38/Lr34/Yr18/Sr57(ABC转运蛋白)和Pm46/Lr67/Yr46/Sr55(糖转运蛋白)外，其余已克隆的抗白粉病基因均编码典型的NLR类型抗病蛋白。NLR类型抗病蛋白仅对单一或少数病原菌的特异小种具有抗病性，生产上大规模使用几年后非常容易丧失抗病性，而造成白粉病大流行。因此，进一步发掘与利用新的小麦广谱抗白粉病基因具有重要的理论意义和生产应用价值。
发明公开
本申请要解决的技术问题是：如何调控小麦对白粉病的抗性和/或如何筛选、 鉴定具有白粉病抗性的小麦。
为解决该技术问题，本申请提供蛋白质或调控基因表达的物质或调控所述蛋白质活性或含量的物质在下述任一项中的应用，所述基因编码所述蛋白质，所述蛋白质可为WTK7-TM蛋白；A1)、在调控植物抗逆性中的应用；A2)、在制备调控植物抗性的产品中的应用；A3)、在调控植物白粉病抗性中的应用；A4)、在制备调控植物白粉病抗性的产品中的应用；A5)、在植物育种或植物辅助育种中应用；
所述WTK7-TM蛋白可为下述任一种蛋白质：a1)、氨基酸序列是SEQ ID No.3所示的蛋白质；a2)、将a1)所示的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与a1)所示的氨基酸序列具有80％以上同一性，且与植物抗逆性相关的蛋白质；a3)、在a1)或a2)的N端或/和C端连接标签得到的融合蛋白质。
进一步地，上述的应用中，所述植物育种的考察指标包括植物的白粉病抗性。
进一步地，上述的应用中，所述植物育种的目的包括培育抗白粉病的植物。
进一步地，上述的应用中，所述蛋白质来源于小麦。
本申请中，SEQ ID No.3由667个氨基酸残基组成。
上述蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
所述蛋白标签(protein-tag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag蛋白标签、His蛋白标签、MBP蛋白标签、HA蛋白标签、myc蛋白标签、GST蛋白标签和/或SUMO蛋白标签等。
进一步地，a3)所述连接可通过肽键链接。
进一步地，上述的应用中，所述调控基因表达的物质或调控所述蛋白质活性或含量的物质为生物材料，所述生物材料可为下述任一种：B1)、抑制或降低上述蛋白质的编码基因的表达或上述蛋白质的活性的核酸分子；B2)、含有B1)所述核酸分子的表达盒；B3)、含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；B4)、含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；B5)、含有B1)所述核酸分子的转基因植物细胞系或含有B2)所述表达盒的转基因植物细胞系或含有B3)所述重组载体的转基因植物细胞系；B6)、含有B1)所述核酸分子的转基因植物组织或含有B2)所述表达盒的转基因植物组织或含有B3)所述重组载体的转基因植物组织；B7)、含有B1)所述核酸分子的转基因植物器官或含有B2)所述表达盒的转基因植物器官或含有B3)所述重组载体的转基因植物器官；B8)、编码上述蛋白质的核酸分子；B9)、含有B8)所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系。
上述相关生物材料中，B2)所述的表达盒是指能够在宿主细胞中表达干扰 RNA的DNA，该DNA不但可包括启动干扰RNA基因转录的启动子，还可包括终止干扰RNA基因转录的终止子。
上述相关生物材料中，B4)所述重组微生物具体可为酵母、细菌、藻和真菌。
上述相关生物材料中，B6)所述植物组织可来源于根、茎、叶、花、果实、种子、花粉、胚和花药。
上述相关生物材料中，B7)所述转基因植物器官可为转基因植物的根、茎、叶、花、果实和种子。
上述相关生物材料中，所述转基因植物细胞系、转基因植物组织和转基因植物器官可包括繁殖材料，也可不包括繁殖材料。
上述相关生物材料中，B9)所述的表达盒是指能够在宿主细胞中表达WTK7-TM蛋白的DNA，该DNA不但可包括启动WTK7-TM蛋白编码基因转录的启动子，还可包括终止WTK7-TM蛋白编码基因转录的终止子。
进一步，B9)所述表达盒还可包括增强子序列。
上述相关生物材料中，B9)所述重组载体可含有SEQ ID No.2所示的用于编码WTK7-TM蛋白的DNA分子。
进一步地，上述的应用中，B1)所述核酸分子可为表达靶向上述应用中的所述基因的RNA或DNA分子。
进一步地，上述的应用中，B1)所述核酸分子的靶标序列的核苷酸序列是SEQ ID No.4或/和SEQ ID No.5位。
进一步地，上述的应用中，B8)所述核酸分子可为如下g1)-g3)任一项所述的DNA分子：g1)、编码链的编码序列是SEQ ID No.2的DNA分子；g2)、编码链的核苷酸序列是SEQ ID No.1的DNA分子；g3)、与g1)或g2)所述DNA分子具有80％以上的同一性，且调控植物抗逆性的DNA分子。
进一步地，上述的应用中，所述植物选自单子叶植物。
进一步地，上述的应用中，所述单子叶植物选自禾本科植物。
进一步地，上述的应用中，所述禾本科植物选自小麦属植物。
进一步地，上述的应用中，所述小麦属植物选自小麦(Triticum aestivum L.)。
进一步地，上述小麦可为野生二粒小麦(Triticum dicoccoides L.)。
进一步，所述的应用中，所述应用具体可为如下任一种：A1’)提高植物抗逆性的方法，包括将B1)所述核酸分子导入受体植物得到目的植物，所述目的植物的抗逆性高于所述受体植物。A2’)制备抗逆性提高的植物的方法，所述方法包括将B1)所述核酸分子导入受体植物，得到抗逆性提高的目的植物。A3’)提高植物白粉病抗性的方法，包括将B1)所述核酸分子导入受体植物得到目的植物，所述目的植物的白粉病抗性高于所述受体植物。A4’)制备白粉病抗性提高的植物的方法，所述方法包括将B1)所述核酸分子导入受体植物，得到白粉病抗性提高的目的植物。A5’)植物育种的方法，所述方法包括将B1)所述核酸分子导入 受体植物，得到目的植物。
本申请中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列(或核苷酸序列)的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述80％或80％以上同一性，可为80％、85％、90％或95％以上的同一性。
所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述85％以上的同一性可为至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述90％以上的同一性可为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述95％以上的同一性可为至少95％、96％、97％、98％或99％的同一性。
本申请中，调控所述蛋白质活性或含量的物质可为下调或降低或敲除所述蛋白质的编码基因的物质和/或下调或降低所述蛋白质的编码基因表达的物质。所述调控植物抗逆性可为降低植物抗逆性。
本申请中，所述调控基因表达的物质可为进行如下6种调控中至少一种调控的物质：1)在所述基因转录水平上进行的调控；2)在所述基因转录后进行的调控(也就是对所述基因的初级转录物的剪接或加工进行的调控)；3)对所述基因的RNA转运进行的调控(也就是对所述基因的mRNA由细胞核向细胞质转运进行的调控)；4)对所述基因的翻译进行的调控；5)对所述基因的mRNA降解进行的调控；6)对所述基因的翻译后的调控(也就是对所述基因翻译的蛋白质的活性进行调控)。
本申请中，所述调控基因表达可为抑制或降低所述基因表达，所述抑制或降低所述基因表达可通过基因敲除实现或通过基因沉默实现。
所述基因敲除(gene knock out)是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。
所述基因沉默是指在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默以不改变DNA序列为前提，使基因不表达或低表达。基因沉默可发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，另一种是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活，包括反义RNA、共抑制(co-suppression)、基因压抑(quelling)、RNA干扰(RNAi)和微小RNA(miRNA) 介导的翻译抑制等。
上述应用中，所述调控基因表达的物质可为抑制或降低所述基因表达的试剂。所述抑制或降低所述基因表达的试剂可为敲除所述基因的试剂，如通过同源重组敲除所述基因的试剂，或通过CRISPR-Cas9敲除所述基因的试剂。所述抑制或降低所述基因表达的试剂可以包含靶向所述基因的多核苷酸，例如siRNA、shRNA、双链RNA、miRNA或反义RNA。
本申请还提供一种培育植物白粉病抗性降低植物的方法，所述方法包括下调或降低受体植物中所述WTK7-TM蛋白的表达或下调或降低所述WTK7-TM蛋白的活性或含量，得到白粉病抗性降低的目的植物。
进一步地，所述受体植物含有所述WTK7-TM蛋白的编码基因或所述WTK7-TM蛋白。
本申请还提供调控植物白粉病抗性的方法，所述方法包括通过调控含有上述WTK7-TM蛋白的编码基因的植物中的所述编码基因的表达或通过调控含有上述WTK7-TM蛋白的植物中所述WTK7-TM蛋白的活性或含量，来调控植物白粉病抗性。
所述方法中，所述调控可为下调或降低。
上述培育植物白粉病抗性降低植物的方法和调控植物白粉病抗性的方法中，所述调控含有所述WTK7-TM蛋白的编码基因的植物中的所述编码基因的表达或通过调控含有所述WTK7-TM蛋白的植物中所述WTK7-TM蛋白的活性或含量，可通过沉默受体植物中所述WTK7-TM基因实现。
所述沉默受体植物中所述WTK7-TM基因可通过病毒诱导的基因沉默实现。
所述病毒诱导的基因沉默包括将靶向WTK7-TM基因的重组病毒导入所述受体植物，所述重组病毒的沉默靶点为SEQ ID No.4或/和SEQ ID No.5。
在本申请的一些实施例中，所述重组病毒为重组大麦条纹花叶病毒(BSMV)。所述重组大麦条纹花叶病毒的制备方法为：
将三质粒分别转染根癌农杆菌EHA105制备重组根癌农杆菌菌液，具体如下：
重组根癌农杆菌EHA105/pCaBS-α的制备：将pCaBS-α采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-α。
重组根癌农杆菌EHA105/pCaBS-β的制备：将pCaBS-β采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-β。
重组根癌农杆菌EHA105/pCaBS-γbLIC的制备：将pCaBS-γbLIC采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-γbLIC。
重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ的制备：将pCaBS-γbLIC-WTK7-TMKinⅠ采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ。
重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ的制备：将pCaBS-γbLIC-WTK7-TMKinⅡ采用冻融法转化入根癌农杆菌EHA105，得到重组根癌 农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ。
重组根癌农杆菌EHA105/pCaBS-α、重组根癌农杆菌EHA105/pCaBS-β、重组根癌农杆菌EHA105/pCaBS-γbLIC、重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ、重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ分别活化处理36-48h后，挑取单克隆接种于1mL LB液体培养基中，28℃，220rmp摇床培养24h。按照1:100比例接种于10mL LB液体培养基中，28℃，200pm摇床培养12h获得菌液。将菌液6000rpm离心5min收集菌体，用等体积的烟草侵染液(10mM MgCl2,10mM MES,pH＝5.2,0.1Mm AS)分别重悬菌体获得侵染菌液。
以重组病毒BSMV:WTK7-TMKinⅡ为例，将EHA105/pCaBS-α，EHA105/pCaBS-β，EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ侵染菌液按照1:1:1比例混合，28℃静置3-5h后分别注射6-8叶期本生烟展开叶，标记为BSMV:WTK7-TMKinⅡ。注射7-12天后采集注射后的本生烟叶片及其上部第一片叶，在PBS(pH＝7.2)缓冲液中充分研磨获得含有重组病毒BSMV:WTK7-TMKinⅡ的汁液。
重组病毒BSMV:WTK7-TM KinⅠ和对照组重组病毒BSMV的制备方法参照重组病毒BSMV:WTK7-TMKinⅡ，其区别仅在于：将EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ侵染菌液分别替换为EHA105/pCaBS-γbLIC-WTK7-TM KinⅠ侵染菌液、EHA105/pCaBS-γbLIC侵染菌液。
进一步地，上述培育植物白粉病抗性降低植物的方法和调控植物白粉病抗性的方法中，所述植物选自单子叶植物。
进一步地，所述单子叶植物选自禾本科植物。
进一步地，所述禾本科植物选自小麦属植物；
进一步地，所述小麦属植物选自小麦(Triticum aestivum L.)。
在本申请的一个实施例中，通过病毒诱导的基因沉默下调或降低小麦中WTK7-TM基因的表达，从而降低了小麦的白粉病抗性。该实施例证明了WTK7-TM基因或其编码的蛋白质在小麦白粉病抗性调控中的作用。
具体地，在本申请的实施例中，所述小麦可为小麦品系3D232。
本申请还提供上述应用中的所述蛋白质或/和上述应用中的所述生物材料。
本申请还提供WTK7-TM分子标记和/或检测WTK7-TM分子标记的物质在下述任一种中的应用：
D1)、鉴定或辅助鉴定小麦白粉病抗性；
D2)、筛选或辅助筛选具有白粉病抗性的小麦单株或株系或品系或品种；
D3)、小麦辅助育种；
所述WTK7-TM分子标记是小麦基因组中的一个片段，其核苷酸序列是SEQ ID No.6；
所述检测WTK7-TM分子标记的物质可为下述任一种：
E1)、扩增包括所述WTK7-TM分子标记在内的小麦基因组DNA片段的PCR引物组合物；
E2)、含有E1)所述PCR引物组合物的PCR试剂；
E3)、含有E1)所述PCR引物组合物或E2)所述PCR试剂的试剂盒。
进一步地，所述的应用具体可为下述任一种：
D1’)、使用WTK7-TM分子标记及检测WTK7-TM分子标记的物质鉴定或辅助鉴定小麦白粉病抗性。
D2’)、使用WTK7-TM分子标记及检测WTK7-TM分子标记的物质筛选或辅助筛选具有白粉病抗性的小麦单株或株系或品系或品种。
D3’)、使用含有WTK7-TM分子标记的小麦用于小麦育种或小麦辅助育种。
进一步地，所述的应用中，所述PCR引物组合物包括核苷酸序列是SEQ ID No.7的单链DNA和核苷酸序列是SEQ ID No.8的单链DNA。
本申请还提供核苷酸序列是SEQ ID No.6的DNA分子或/和上述的引物组合物或/和上述的试剂或/和上述的试剂盒。
本申请还提供鉴定或辅助鉴定小麦白粉病抗性性状的方法，所述方法包括检测待测小麦中所述WTK7-TM分子标记，根据待测小麦是否含有所述WTK7-TM分子标记鉴定或辅助鉴定小麦白粉病抗性，含有所述WTK7-TM分子标记的待测小麦的白粉病抗性高于或候选高于不含有WTK7-TM分子标记的待测小麦。
其中，所述WTK7-TM分子标记的核苷酸序列是SEQ ID No.6。SEQ ID No.6由1198个核苷酸组成。
含有所述WTK7-TM分子标记的待测小麦可以用上述引物组合物扩增出所述WTK7-TM分子标记的DNA片段，不含有所述WTK7-TM分子标记的待测小麦用上述引物组合物扩增不出所述WTK7-TM分子标记的DNA片段。
进一步地，所述的方法中，所述检测待测小麦中所述WTK7-TM分子标记方法包括以待测小麦基因组DNA为模板，利用上述的PCR引物组合物进行扩增，得到PCR产物，对所述PCR产物进行琼脂糖凝胶电泳或者测序。
本申请还提供小麦育种的方法，所述方法包括选择含有所述WTK7-TM分子标记的小麦作为亲本进行育种。
进一步地，上述方法中，所述育种的育种目标包括小麦白粉病抗性。所述育种的目的包括培育抗白粉病的小麦((小麦白粉病抗性高于亲本))。
本申请中，所述白粉病可为专性寄生的布氏白粉菌(Blumeria graminis f.sp.tritici，一般称为白粉菌)所引起的真菌病害。本申请中所述的抗病性或白粉病抗性可为抗布氏白粉菌引起的病害。
在本申请的实施例中，白粉病抗性实验中所述的白粉菌具体可为白粉菌生理小种E09。
本申请所取得的有益技术效果如下：
本申请提供了小麦广谱抗白粉病基因WTK7-TM的基因定位、图位克隆以及抗白粉病生物学功能鉴定方法。小麦广谱抗白粉病基因WTK7-TM可以广泛应用于小麦抗病遗传育种、种质资源改良、转基因和基因组编辑育种等植物领域， 对改进和改良小麦等作物的种质资源具有重要作用。
本申请提供的功能标记WTK-TM-FM及其扩增的引物组合物可用于白粉病抗性小麦的辅助筛选或鉴定以及小麦育种。
附图说明
图1为普通小麦品系3D232白粉病菌多小种鉴定。
图2为小麦抗白粉病基因WTK7-TM图位克隆。
图3为CYB561-Domon和TM9SF4转基因验证基因功能。
图4为WTK7-TM基因剪切方式。
图5为BSMV-VIGS和EMS突变体验证WTK7-TM抗白粉病功能。
图6为沉默载体的构建流程图。
图7为WTK7-TM基因功能标记开发和应用。
实施发明的最佳方式
下面结合具体实施方式对本申请进行进一步的详细描述，给出的实施例仅为了阐明本申请，而不是为了限制本申请的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本申请的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量试验，如无特殊说明均设置三次重复，结果取平均值。
下述实施例中所述普通小麦品系3D232和普通小麦薛早为本实验室保存，在文献“Zhang,H.et al.Genetic and comparative genomics mapping reveals that a powdery mildew resistance gene Ml3D232originating from wild emmer co-segregates with an NBS-LRR analog in common wheat(Triticum aestivum L.).Theor.Appl.Genet.121,1613-1621(2010)，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本申请的实验所用，不可作为其它用途使用。
下述实施例中所述小麦品系5BIL-29为意大利巴里大学Antonio Blanco博士惠赠，在文献“Blanco,A.et al.Molecular mapping of the novel powdery mildew resistance gene Pm36introgressed from Triticum turgidum var.dicoccoides in durum wheat.Theor.Appl.Genet.117,135-142(2008)”，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本申请的实验所用，不可作为其它用途使用。
下述实施例中所述白粉菌菌株E09由本实验室保存，在文献“Li,M.et al.A CNL protein in wild emmer wheat confers powdery mildew resistance.New Phytol.228,1027-1037(2020)，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本申请的实验所用，不可作为其它用途使用。
下述实施例中所述基因沉默载体pCaBS-α、pCaBS-β和pCaBS-γbLIC由中国农业大学李大伟教授惠赠，在文献“Yuan,C.et al.A high throughput barley  stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.PLoS One 6,e26468(2011).”，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本申请的实验所用，不可作为其它用途使用。
下述实施例中试剂AS表示乙酰丁香酮。
下述实施例中MES表示2-Morpholinoethanesulphonic acid，是一种两性离子缓冲液。
下述实施例采用SPSS11.5统计软件对数据进行处理，实验结果以平均值±标准偏差表示，采用One-way ANOVA检验，*表示具有显著性差异(P＜0.05)，**表示具有极显著性差异(P＜0.01)，***表示具有极显著性差异(P＜0.001)。
表1本研究的引物序列

实施例1、普通小麦3D232白粉病抗性鉴定
普通小麦品系3D232是由野生二粒小麦I222(由以色列Agricultural Research Organization,The Volcani Center的Gerechter-Amitai博士惠赠)与普通小麦品系87-1杂交，后与普通小麦京411回交后代选系而来，系谱为87-1/I222//京411*7。利用从全国不同生态区采集的104种白粉菌生理小种在苗期离体接种3D232叶片，发现其对所有白粉菌生理小种均表现免疫或高抗，是一个优异的广谱抗白粉病材料(图1)。
实施例2、小麦广谱抗白粉病基因Ml3D232精细定位
利用北京地区流行白粉病菌小种E09对普通小麦品系3D232、薛早和薛早×3D232的F1代、F2代分离群体和F3代家系进行苗期抗病性鉴定。鉴定结果表明，3D232表现免疫反应型(IT＝0)，薛早表现高感反应型(IT＝4)。薛早/3D232的F1代表现为免疫反应型(IT＝0)，与抗病亲本表现一致。鉴定630株薛早/3D232的F2分离群体发现，其中477株表现抗病，153株表现感病，卡方测验符合单基因控制的3:1的分离比。薛早/3D232的630个F3家系中，有157个家系表现为纯合抗病，320个家系表现为抗感分离，153个家系表现为纯合感病，卡方测验其比例符合单基因控制的1:2:1的分离比(表2)。以上遗传学分析表明普通小麦3D232的白粉病抗性由一个显性单基因控制，暂命名为Ml3D232。
之前通过开发分子标记将小麦抗白粉病基因Ml3D232定位在5BL染色体近末端分子标记XBD37670和XBD37760之间0.14cM的遗传区间(D Zhang,S H Ouyang,L L Wang,Y Cui,Q H Wu,Y Liang,Z Z Wang,J Z Xie,D Y Zhang,Y  Wang,Y X Chen,Z Y Liu.Comparative genetic mapping revealed powdery mildew resistance gene MIWE4derived from wild emmer is located in same genomic region of Pm36and MI3D232on chromosome 5BL.Journal of Integrative Agriculture,4(2015),pp.603-609)。为了进一步精细定位MI3D232基因，我们利用薛早和3D232杂交构建了含有15,893个F2单株遗传分离群体。利用MI3D232基因定位区间两侧最近的分子标记XBD37670和XBD37760对该F2遗传分离群体进行基因型鉴定，筛选获得关键的重组交换单株。在苗期利用白粉菌生理小种E09对分子标记XBD37670和XBD37760筛选的关键重组交换单株的F2:3家系进行白粉病抗性鉴定。同时，根据中国春和野生二粒小麦Zavitan的参考基因组序列在分子标记XBD37670和XBD37760之间开发新的与MI3D232基因紧密连锁及共分离的分子标记，共开发出WGGBM1-WGGBM9共9个分子标记与MI3D232紧密连锁(表1)。利用这些新开发的分子标记WGGBM1-WGGBM9对分子标记XBD37670和XBD37760筛选的关键重组交换单株的F2:3家系进行基因型鉴定从而对MI3D232基因进行精细定位。最终将MI3D232基因精细定位于分子标记WGGBM2和WGGBM7之间0.021cM的遗传区间，其中分子标记WGGBM3-WGGBM6与MI3D232基因共分离(图2)。
之前研究发现MI3D232基因与正式命名的小麦抗白粉病基因Pm36是同一个基因或者等位基因(D Zhang,S H Ouyang,L L Wang,Y Cui,Q H Wu,Y Liang,Z Z Wang,J Z Xie,D Y Zhang,Y Wang,Y X Chen,Z Y Liu.Comparative genetic mapping revealed powdery mildew resistance gene MIWE4derived from wild emmer is located in same genomic region of Pm36and MI3D232on chromosome 5BL.Journal of Integrative Agriculture,4(2015),pp.603-609)。为了克隆MI3D232基因，我们采用PacBio SMRT long-read基因组测序的策略对携带有Pm36/MI3D232基因的硬粒小麦材料5BIL-29(意大利巴里大学Antonio Blanco博士惠赠)进行基因组重测序。通过质量控制，共计获得172.53Gb HiFi reads，约17×基因组覆盖度。采用hifiasm 0.16.1-r375软件进行数据组装，共获得15,347contigs，其中N50长度为3.2Mb，N90长度为0.69Mb,最长的contig长度为25Mb,基因组总长度约10.7Gb。其中一个contig utg0040681(1.69Mb)能够完全覆盖Ml3D232的定位区间，其中Ml3D232基因定位区间两侧最近分子标记WGGBM2和WGGBM7之间对应的物理距离约1.17Mb，而对应中国春的参考基因组物理区间约为275Kb，表明该区间在不同物种中存在较大的基因组结构变异。通过将1.17Mb的contig进行基因注释共获得11个基因，包括CYB561-Domon,三个F-box,五个Serpin,TM9SF4和WTK7-TM(图2和表3)。RNA-seq分析显示在接种白粉菌E09后的3D232叶片中CYB561-Domon,TM9SF4和WTK7-TM三个基因有表达，其他基因没有检测到表达。我们将CYB561-Domon和TM9SF4基因分别在高感白粉病的小麦Fielder中进行农杆菌介导的稳定遗传转化获得转基因植株。通过在苗期对CYB561-Domon和TM9SF4 基因的T2代转基因阳性植株进行白粉病抗性鉴定，结果发现两个基因的转基因植株均高感白粉病(图3)，说明CYB561-Domon和TM9SF4基因不具有抗白粉病的功能。因此，WTK7-TM基因就是Ml3D232基因最有可能的候选基因。
通过设计特异引物对WTK7-TM基因进行扩增测序，发现其基因长度为3277bp。进一步通过RACE实验发现WTK7-TM基因共含有9种不同的剪切体(TV1-TV9)。其中TV1是最主要的剪切方式，基因编码区长度为2004bp，能够编码667个氨基酸(图4)。通过分析WTK7-TM蛋白发现其含有两个串联激酶和一个C端的跨膜结构域(图2)。序列分析显示WTK7-TM基因在普通小麦3D232和5BIL-29中完全一样，但在感病亲本薛早和中国春中均缺失。
具体地，本申请所述的小麦广谱抗白粉病基因WTK7-TM位于小麦5BL染色体上，其基因组序列的核苷酸序列是SEQ ID No.1，cDNA序列(编码序列)的核苷酸序列是SEQ ID No.2，编码的WTK7-TM蛋白的氨基酸序列是SEQ ID No.3。
表2小麦抗白粉病基因Ml3D232的遗传分析
表3 Ml3D232定位区间候选基因
实施例3、BSMV-VIGS技术沉默WTK7-TM基因
为了验证WTK7-TM是否具有抗白粉病功能，利用之前报道的BSMV-VIGS技术沉默小麦中内源基因的体系(Yuan C,Li C,Yan L,et al.A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.PLoS One.2011；6(10):e26468)对3D232中的WTK7-TM基因进行沉默。
基因沉默载体为pCaBS-α、pCaBS-β、pCaBS-γbLIC，由中国农业大学李大 伟教授惠赠。白粉菌菌株E09由本实验室保存。首先用VIGS-1F/VIGS-1R和VIGS-2F/VIGS-2R(表1)扩增含有全长WTK7-TM基因的质粒分别获得两个目的片段用于构建沉默载体。VIGS-1F/VIGS-1R和VIGS-2F/VIGS-2R引物扩增获得的目的片段(含有核苷酸序列分别为SEQ ID No.4和SEQ ID No.5的沉默片段)分别对应WTK7-TM基因的激酶1(KinⅠ)和激酶2(KinⅡ)结构域。同时用限制性内切酶ApaⅠ对pCaBS-γbLIC载体进行酶切并回收线性化产物。然后利用LIC位点连接原理，将二者进行连接，分别构建成pCaBS-γbLIC-WTK7-TMKinⅠ和pCaBS-γbLIC-WTK7-TMKinⅡ，具体操作如图6所示。
测序结果表明，pCaBS-γbLIC-WTK7-TMKinⅠ的结构为：将pCaBS-γbLIC上的LIC1和LIC2之间的片段替换为核苷酸序列是SEQ ID No.4的DNA分子，保持pCaBS-γbLIC的其它核苷酸不变得到的重组载体。pCaBS-γbLIC-WTK7-TMKinⅡ的结构为：将pCaBS-γbLIC上的LIC1和LIC2之间的片段替换为核苷酸序列是SEQ ID No.5的DNA分子，保持pCaBS-γbLIC的其它核苷酸不变得到的重组载体。
重组根癌农杆菌EHA105/pCaBS-α的制备：将pCaBS-α采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-α。
重组根癌农杆菌EHA105/pCaBS-β的制备：将pCaBS-β采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-β。
重组根癌农杆菌EHA105/pCaBS-γbLIC的制备：将pCaBS-γbLIC采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-γbLIC。
重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ的制备：将pCaBS-γbLIC-WTK7-TMKinⅠ采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ。
重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ的制备：将pCaBS-γbLIC-WTK7-TMKinⅡ采用冻融法转化入根癌农杆菌EHA105，得到重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ。
BSMV-VIGS诱导WTK7-TM基因沉默程序如下：
1、将本生烟点播后置于20-22℃的培养间培养，16h光照，8h黑暗。待烟草长至6-8叶期用于BSMV-VIGS实验；
2、将重组根癌农杆菌EHA105/pCaBS-α、重组根癌农杆菌EHA105/pCaBS-β、重组根癌农杆菌EHA105/pCaBS-γbLIC、重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ、重组根癌农杆菌EHA105/pCaBS-γbLIC-WTK7-TMKinⅡ分别活化处理36-48h后，挑取单克隆接种于1mL LB液体培养基(Kan+Rif)中，28℃，220rmp摇床培养24h获得种子液；
3、按照1:100比例接种于10mL LB液体培养基(Kan+Rif)，含有20μM AS和100μM MES)中，28℃，200pm摇床培养12h获得菌液；
4、将菌液6000rpm离心5min收集菌体，用等体积的烟草侵染液(10mM MgCl2,10mM MES,pH＝5.2,0.1Mm AS)重悬菌体获得侵染菌液；
5、将侵染菌液浓度调至OD600＝0.7后，根据感染质粒的不同分为三组：
第一组：将EHA105/pCaBS-α、EHA105/pCaBS-β、EHA105/pCaBS-γbLIC侵染菌液按照1:1:1比例混合，28℃静置3-5h后注射6-8叶期本生烟展开叶，标记为BSMV:00；
第二组：将EHA105/pCaBS-α、EHA105/pCaBS-β、EHA105/pCaBS-γbLIC-WTK7-TMKinⅠ侵染菌液按照1:1:1比例混合，28℃静置3-5h后注射6-8叶期本生烟展开叶，标记为BSMV:WTK7-TMKinⅠ；
第三组：将EHA105/pCaBS-α、EHA105/pCaBS-β、EHA105/pCaBS-γbLIC-WTK7-TM KinⅡ侵染菌液按照1:1:1比例混合，28℃静置3-5h后注射6-8叶期本生烟展开叶，标记为BSMV:WTK7-TMKinⅡ；
6、注射7-12天后，分组采集注射后的本生烟叶片及其上部第一片叶，在PBS(pH＝7.2)缓冲液中充分研磨获得汁液，按照步骤5的分组分别将汁液摩擦接种至一叶一心3D232和5BIL-29小麦的第一片叶，实验设置3次重复，每次重复设置5株小麦/组；
7、在接种3天后，观察病毒在小麦叶片的扩展症状，同时接种白粉菌E09进行小麦苗期白粉病的抗病性鉴定。小麦苗期白粉病抗性鉴定的具体操作是：首先利用高感白粉菌生理小种E09的薛早作为白粉菌繁殖寄主进行繁殖白粉菌。在待测材料(对照组BSMV:00、接种BSMV:WTK7-TMKinⅠ、BSMV:WTK7-TMKinⅡ的3D232和5BIL-29)四周放置充分感染白粉病的薛早，用鸡毛毯轻轻将繁菌盆中的白粉菌孢子轻轻拂掸的方式进行接种，每天早上、中午和晚上拂掸接种三次。根据小麦叶片白粉病菌的病斑大小、厚薄和多少，以及是否有过敏性坏死等反应型共分为6级：0(免疫)、0；(过敏性坏死)、1(高度抗病)、2(中度抗病)、3(中度感病)和4(高度感病)，其中0-2级是抗病反应类型，3-4级是感病反应类型(表4)。
表4小麦苗期白粉病反应型分级标准
结果发现，在接种白粉菌E09后第10天，与对照组BSMV:00相比(对照组的小麦叶片没有感染白粉病)，无论是在接种BSMV:WTK7-TMKinⅠ还是BSMV:WTK7-TMKinⅡ的3D232和5BIL-29的叶片均表现感白粉病，具体表现为小麦叶片高感白粉病(图5中b)。同时，利用qRT-PCR检测发现，出现感白粉病的3D232和5BIL-29植株中，与对照BSMV:00相比(对照组小麦的WTK7-TM基因没有明显下调)，接种BSMV:WTK7-TMKinⅠ和BSMV:WTK7-TMKinⅡ均能 引起WTK7-TM基因明显下调表达(图5中a)。以上结果表明，在3D232和5BIL-29中沉默WTK7-TM基因的表达能使植株感白粉病，更进一步说明WTK7-TM是Ml3D232基因。qRT-PCR以ACTIN基因为内参，其中qRT-PCR的引物核苷酸序列见表1。
实施例4、EMS突变体验证WTK7-TM抗白粉病功能
为了进一步证实WTK7-TM的抗白粉病功能，我们利用EMS诱变创制了3D232的感白粉病突变体。首先，选取约10,000粒饱满的抗病亲本3D232种子，利用0.6％的甲基磺酸乙酯(EMS)诱变剂进行处理，播种于试验田，最终共收获3,360份M2代材料。在温室对收获的M2代材料参照实施例3的方法进行苗期白粉病抗性鉴定，共获得23个感病突变体家系。然后，我们对所有的感病突变体中的WTK7-TM基因进行扩增和分析，结果发现17个感病突变体(M1-M17)(图5中c)在WTK7-TM基因上均有突变，其中有4个是提前终止突变，1个可变剪切改变和12个错义突变(图5中d)。以上突变体分析结果进一步证实，WTK7-TM就是Ml3D232基因。
实施例5、WTK7-TM基因功能标记开发与应用
为了利用WTK7-TM基因进行小麦抗白粉病品种培育，基于WTK7-TM基因序列开发了特异扩增的功能标记WTK-TM-FM的引物组合物(表1中WTK-TM-FMF和WTK-TM-FMR)，以3D232材料的基因组DNA为模板进行扩增。功能标记WTK-TM-FM为核苷酸序列是SEQ ID No.6的DNA分子。
扩增程序为：
PCR反应体系(10μL)：小麦叶片的基因组DNA(25ng/μL)2μL、2×PCR Mix 5μL、引物WTK-TM-FMF的水溶液(浓度为10μmol/L)1μL、引物WTK-TM-FMR的水溶液(浓度为10μmol/L)1μL和ddH2O 1μL。
PCR反应条件：94℃5min；94℃30s，58℃30s，72℃30s，35个循环；72℃7min。
其扩增产物经过1％琼脂糖凝胶电泳检测条带大小为1198bp。扩增产物经过测序发现其与WTK7-TM基因序列能够完全匹配，说明WTK-TM-FMF和WTK-TM-FMR能够从小麦基因组中特异扩增功能标记WTK-TM-FM。如果含有功能标记WTK-TM-FM则能扩增出1198bp条带，如果不含功能标记WTK-TM-FM则扩增不出来相同大小条带。
利用功能标记WTK7-TM-FM对抗病材料普通小麦3D232和感病材料普通小麦薛早杂交构建的F2分离群体(母本为普通小麦薛早，父本为普通小麦3D232)中的25个抗病25个感病材料(白粉病抗性鉴定方法参照实施例3)检测验证发现，功能标记WTK7-TM-FM检测结果与表型共分离，即扩增出来1198bp条带的材料均表现抗病，而扩增不出来1198bp条带的材料均表现高感(表5)。
进一步选用442份野生二粒小麦，124份栽培二粒小麦，230份硬粒小麦，262份中国小麦微核心种质，和394份国外地方品种(Li,M.et al.A CNL protein  in wild emmer wheat confers powdery mildew resistance.New Phytol.228,1027-1037(2020))，用WTK7-TM-FM进行扩增，结果发现仅有38份野生二粒小麦中含有WTK7-TM，而在栽培二粒小麦，硬粒小麦和普通小麦中均没有WTK7-TM基因(图7)。
表5功能标记WTK7-TM-FM检测薛早×3D232F2群体
表5中，R表示待测小麦的表型为抗白粉病，S表示待测小麦的表型为白粉病敏感型；有扩增是指能扩增出来1198bp大小的目的条带；无扩增是指没有扩增出条带。
本申请的研发过程中，从广谱抗白粉病材料3D232创制到克隆出Ml3D232基因花费了超过20年的时间。期间数名博士研究生参与到了课题的公关，通过筛选大量的作图群体对Ml3D232基因进行了精细定位。在没有参考基因组的情况下，通过筛选野生二粒小麦IW2的基因组BAC文库，获得了覆盖Ml3D232基因的物理图谱。通过对候选区间的基因分析发现，仅有CYB561-Domon和 TM9SF4表达。然而，通过对这两个基因进行稳定的遗传转化高感白粉病的小麦品种Fielder后发现，两个基因均不具有抗白粉病的功能，说明Ml3D232基因不存在目前已知的参考基因组。为了克服这个困难，对含有Ml3D232的硬粒小麦5BIL-29进行基因组重测序。通过组装和基因注释，发现5BIL-29中Ml3D232区间的物理区间为1.17Mb物理距离，共注释出11个基因，其中包括一个编码具有跨膜结构域的串联激酶蛋白WTK7-TM。进一步通过BSMV-VIGS和EMS感病突变体实验验证WTK7-TM基因就是Ml3D232。
表6本申请的部分序列


工业应用
本申请提供了小麦广谱抗白粉病基因WTK7-TM的基因定位、图位克隆以及抗白粉病生物学功能鉴定方法。小麦广谱抗白粉病基因WTK7-TM可以广泛应用于小麦抗病遗传育种、种质资源改良、转基因和基因组编辑育种等植物领域，对改进和改良小麦等作物的种质资源具有重要作用。
本申请提供的功能标记WTK-TM-FM及其扩增的引物组合物可用于白粉病抗性小麦的辅助筛选或鉴定。
以上对本申请进行了详述。对于本领域技术人员来说，在不脱离本申请的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本申请。虽然本申请给出了特殊的实施例，应该理解为，可以对本申请作进一步的改进。总之，按本申请的原理，本申请欲包括任何变更、用途或对本申请的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。

Claims (17)

  1. 蛋白质或调控基因表达的物质或调控所述蛋白质活性或含量的物质在下述任一项中的应用，所述基因编码所述蛋白质，所述蛋白质为WTK7-TM蛋白；
    A1)、在调控植物抗逆性中的应用；A2)、在制备调控植物抗逆性的产品中的应用；A3)、在调控植物白粉病抗性中的应用；A4)、在制备调控植物白粉病抗性的产品中的应用；A5)、在植物育种或植物辅助育种中应用；
    所述WTK7-TM蛋白是下述任一种蛋白质：a1)、氨基酸序列是SEQ ID No.3所示的蛋白质；a2)、将a1)所示的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与a1)所示的氨基酸序列具有80％以上同一性，且与植物抗逆性相关的蛋白质；a3)、在a1)或a2)的N端或/和C端连接标签得到的融合蛋白质。
  2. 根据权利要求1所述的应用，其特征在于：所述调控基因表达的物质或调控所述蛋白质活性或含量的物质为生物材料，所述生物材料为下述任一种：
    B1)、抑制或降低权利要求1中所述蛋白质的编码基因的表达或权利要求1中所述蛋白质的活性的核酸分子；B2)、含有B1)所述核酸分子的表达盒；B3)、含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；B4)、含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；B5)、含有B1)所述核酸分子的转基因植物细胞系或含有B2)所述表达盒的转基因植物细胞系或含有B3)所述重组载体的转基因植物细胞系；B6)、含有B1)所述核酸分子的转基因植物组织或含有B2)所述表达盒的转基因植物组织或含有B3)所述重组载体的转基因植物组织；B7)、含有B1)所述核酸分子的转基因植物器官或含有B2)所述表达盒的转基因植物器官或含有B3)所述重组载体的转基因植物器官；B8)、编码权利要求1中所述蛋白质的核酸分子；B9)、含有B8)所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系。
  3. 根据权利要求2所述的应用，其特征在于：
    B1)所述核酸分子为表达靶向权利要求1所述应用中的所述基因的RNA或DNA分子；
    B8)所述核酸分子为如下g1)-g3)任一项所述的DNA分子：g1)、编码链的编码序列是SEQ ID No.2的DNA分子；g2)、编码链的核苷酸序列是SEQ ID No.1的DNA分子；g3)、与g1)或g2)所述DNA分子具有80％以上的同一性，且调控植物抗逆性的DNA分子。
  4. 根据权利要求3所述的应用，其特征在于：B1)所述核酸分子的靶标序列的核苷酸序列是SEQ ID No.4或/和SEQ ID No.5。
  5. 根据权利要求1-4中任一项所述的应用，其特征在于：所述植物选自单子叶植物。
  6. 根据权利要求5所述的应用，其特征在于：所述单子叶植物选自禾本科 植物。
  7. 根据权利要求6所述的应用，其特征在于：所述禾本科植物选自小麦属植物。
  8. 根据权利要求7所述的应用，其特征在于：所述小麦属植物选自小麦(Triticum aestivum L.)。
  9. 一种培育植物白粉病抗性降低植物的方法，其特征在于：所述方法包括下调或降低受体植物中所述WTK7-TM蛋白的表达或下调或降低所述WTK7-TM蛋白的活性或含量，得到白粉病抗性降低的目的植物。
  10. 权利要求1-8中任一项所述应用中的所述蛋白质。
  11. 权利要求2-8中任一项所述应用中的所述生物材料。
  12. WTK7-TM分子标记和/或检测WTK7-TM分子标记的物质在下述任一种中的应用：D1)、鉴定或辅助鉴定小麦白粉病抗性；D2)、筛选或辅助筛选具有白粉病抗性的小麦单株或株系或品系或品种；D3)、小麦辅助育种；
    所述WTK7-TM分子标记是WTK7-TM基因的一段DNA片段，该DNA片段的核苷酸序列是SEQ ID No.6；
    所述检测WTK7-TM分子标记的物质为下述任一种：E1)、扩增包括所述WTK7-TM分子标记在内的小麦基因组DNA片段的PCR引物组合物；E2)、含有E1)所述PCR引物组合物的PCR试剂；E3)、含有E1)所述PCR引物组合物或E2)所述PCR试剂的试剂盒。
  13. 根据权利要求12所述的应用，其特征在于：所述PCR引物组合物包括核苷酸序列是SEQ ID No.7的单链DNA和核苷酸序列是SEQ ID No.8的单链DNA。
  14. 核苷酸序列是SEQ ID No.6的DNA分子或/和权利要求12或13所述的引物组合物或/和权利要求12或13所述的试剂或/和权利要求8或9所述的试剂盒。
  15. 鉴定或辅助鉴定小麦白粉病抗性的方法，所述方法包括检测待测小麦中权利要求12中所述WTK7-TM分子标记，根据待测小麦是否含有所述WTK7-TM分子标记鉴定或辅助鉴定小麦白粉病抗性，含有所述WTK7-TM分子标记的待测小麦的白粉病抗性高于或候选高于不含有WTK7-TM分子标记的待测小麦。
  16. 小麦育种的方法，其特征在于：所述方法包括选择含有权利要求12中所述WTK7-TM分子标记的小麦作为亲本进行育种。
  17. 调控植物白粉病抗性的方法，其特征在于：所述方法包括通过调控含有权利要求1中所述WTK7-TM蛋白的编码基因的植物中的所述编码基因的表达或通过调控含有权利要求1中所述WTK7-TM蛋白的植物中所述WTK7-TM蛋白的活性或含量，来调控植物白粉病抗性。
PCT/CN2024/077521 2023-02-20 2024-02-19 小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用 WO2024174954A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310138434.7A CN118516393A (zh) 2023-02-20 2023-02-20 小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用
CN202310138434.7 2023-02-20

Publications (1)

Publication Number Publication Date
WO2024174954A1 true WO2024174954A1 (zh) 2024-08-29

Family

ID=92274940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/077521 WO2024174954A1 (zh) 2023-02-20 2024-02-19 小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用

Country Status (2)

Country Link
CN (1) CN118516393A (zh)
WO (1) WO2024174954A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101736076A (zh) * 2008-11-19 2010-06-16 朱玉丽 小麦抗白粉病基因的分子标记定位研究进展
US20190367942A1 (en) * 2016-12-20 2019-12-05 Nanjing Agricultural University Broad-spectrum high-resistancegene pm21 resistant to wheat powdery mildew as well as expression vector and use thereof
CN111732644A (zh) * 2019-03-20 2020-10-02 中国科学院遗传与发育生物学研究所 抗白粉病相关蛋白Pm41及其编码基因与应用
CN112176083A (zh) * 2019-07-05 2021-01-05 中国科学院遗传与发育生物学研究所 小麦抗白粉病相关基因Pm41的功能型分子标记及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101736076A (zh) * 2008-11-19 2010-06-16 朱玉丽 小麦抗白粉病基因的分子标记定位研究进展
US20190367942A1 (en) * 2016-12-20 2019-12-05 Nanjing Agricultural University Broad-spectrum high-resistancegene pm21 resistant to wheat powdery mildew as well as expression vector and use thereof
CN111732644A (zh) * 2019-03-20 2020-10-02 中国科学院遗传与发育生物学研究所 抗白粉病相关蛋白Pm41及其编码基因与应用
CN112176083A (zh) * 2019-07-05 2021-01-05 中国科学院遗传与发育生物学研究所 小麦抗白粉病相关基因Pm41的功能型分子标记及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 20 March 2023 (2023-03-20), ANONYMOUS: "wheat tandem kinase WTK-TM [Triticum dicoccoides]", XP093202830, retrieved from Genbank Database accession no. WEM02089 *
ZHANG, DONG: "Map-based Cloning of Powdery Mildew Resistance Gene Ml3D232 in Wheat （Triticum aestivum L.）", DOCTORAL DISSERTATION, 1 May 2014 (2014-05-01), China, pages 1 - 91, XP009556933 *

Also Published As

Publication number Publication date
CN118516393A (zh) 2024-08-20

Similar Documents

Publication Publication Date Title
AU2019276382B2 (en) Use of Yr4DS gene of Aegilops tauschii in stripe rust resistance breeding of Triticeae plants
US9663794B2 (en) Heat-resistance rice gene OsZFP, screening marker and separation method thereof
US11041165B2 (en) Identification of a Xanthomonas euvesicatoria resistance gene from pepper (Capsicum annuum) and method for generating plants with resistance
ES2382898T3 (es) Método para aumentar la resistencia contra la roya de la soja en plantas transgénicas
Fekih et al. The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response
CN111902547B (zh) 鉴定、选择和产生疾病抗性作物的方法
WO2020207125A1 (zh) 用于检测玉米植物dbn9501的核酸序列及其检测方法
CN113121664A (zh) 鉴定、选择和产生疾病抗性作物的方法
JP7375028B2 (ja) 植物病害に対する抵抗性の遺伝子
CN113677696A (zh) 一种培育抗灰斑病植物的方法
CN113490683A (zh) 灰斑病抗性相关蛋白ZmWAK-RLK及其编码基因和应用
WO2017059582A1 (zh) 一种抗烟草花叶病毒的N'au基因及其克隆方法和应用
Zhang et al. Tomato protein Rx4 mediates the hypersensitive response to Xanthomonas euvesicatoria pv. perforans race T3
EP3696188A1 (en) Gene for resistance to plant disease
JP2023538571A (ja) 植物の抵抗性遺伝子およびその同定手段
US20210032653A1 (en) Compositions and methods for improving crop yields through trait stacking
CN114807174A (zh) 一种逆向调控水稻对稻瘟病菌抗性的遗传位点及其应用
CN116096901A (zh) 植物病原体效应子和疾病抗性基因鉴定、组合物和使用方法
CN117987458A (zh) 调控玉米光周期敏感性的基因及其编码蛋白与应用
Huang et al. Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism
WO2024174954A1 (zh) 小麦广谱抗白粉病基因wtk7-tm克隆、功能标记及其应用
WO2023241004A1 (zh) 一种通过基因组编辑提高小麦赤霉病抗性的方法
WO2012006866A1 (zh) 植物黄矮病抗性蛋白tistk1及其编码基因与应用
EP3193586B1 (en) New phytophthora resistance gene
CN115044592A (zh) 一种调控玉米株型和瘤黑粉病抗性的基因ZmADT2及其编码蛋白和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24759631

Country of ref document: EP

Kind code of ref document: A1