WO2021218961A1 - 获得具有增加的白粉病抗性的小麦的方法 - Google Patents

获得具有增加的白粉病抗性的小麦的方法 Download PDF

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WO2021218961A1
WO2021218961A1 PCT/CN2021/090236 CN2021090236W WO2021218961A1 WO 2021218961 A1 WO2021218961 A1 WO 2021218961A1 CN 2021090236 W CN2021090236 W CN 2021090236W WO 2021218961 A1 WO2021218961 A1 WO 2021218961A1
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tamlo
wheat
wheat plant
protein
plant
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PCT/CN2021/090236
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French (fr)
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邱金龙
李盛楠
高彩霞
王延鹏
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中国科学院微生物研究所
中国科学院遗传与发育生物学研究所
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Priority to CN202180032007.XA priority Critical patent/CN115843314B/zh
Priority to US17/921,375 priority patent/US20230193309A1/en
Priority to EP21796151.5A priority patent/EP4144849A4/en
Priority to CN202311695258.3A priority patent/CN117867009A/zh
Priority to AU2021264887A priority patent/AU2021264887A1/en
Priority to BR112022021808A priority patent/BR112022021808A2/pt
Publication of WO2021218961A1 publication Critical patent/WO2021218961A1/zh

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    • 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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to a method for obtaining wheat with increased resistance to powdery mildew, wherein the wheat has a yield comparable to that of the wild type or even increased.
  • Powdery mildew (Powdery mildew, Pm) is one of the most important cereal diseases in the world. Wheat powdery mildew is a worldwide fungal disease caused by the obligate parasitic fungus Blumeria graminis f.sp tritici. Wheat yield loss caused by powdery mildew is generally 13-34%, and can reach 50% in severe cases.
  • the cultivation of disease-resistant wheat varieties is an economical, effective and environmentally friendly strategy for the prevention and control of powdery mildew.
  • the breeding of powdery mildew-resistant wheat mainly involves the introduction of resistance genes from wheat related species and wild species into the main wheat varieties through cross-breeding.
  • MLO gene Mockout of susceptibility genes (S-genes) in plants to obtain functional loss-of-function mutants of the susceptibility genes is an effective way for plants to acquire disease resistance.
  • the MLO gene Moildew resistance Locus O, MLO
  • MLO Mildew resistance Locus O
  • barley is a typical susceptible gene, which has a negative regulatory effect against powdery mildew (Acevedo-Garcia et al., 2014).
  • the loss-of-function mutants of the MLO gene showed durable, broad-spectrum resistance to almost all races of Barley powdery mildew (Piffanelli et al., 2004).
  • the loss-of-function mutants of the MLO gene have been found in many species such as tomatoes, potatoes, peas, peppers and grapes.
  • hexaploid wheat there are three copies of genes with up to 80% homology to the MLO gene of barley, distributed on 5AL, 4BL and 4DL, named TaMLO-A1, TaMLO-B1 and TaMLO-D1, respectively.
  • TaMLO-A1 genes with up to 80% homology to the MLO gene of barley, distributed on 5AL, 4BL and 4DL, named TaMLO-A1, TaMLO-B1 and TaMLO-D1, respectively.
  • the similarity of these three homologous genes at the nucleic acid level and the amino acid level reached 98% and 99%, respectively.
  • the expression of the wheat TaMLO-B1 gene in the barley mlo mutant can restore the mutant’s susceptibility to powdery mildew (Elliott et al., 2002).
  • the immunity and growth and development of plants are often antagonistic to each other, which is also a long-standing challenge in the process of crop resistance breeding.
  • the mlo mutant has been used in the breeding and agricultural production of barley, tomato, peas and other plants for resistance to powdery mildew
  • the mlo mutant still has certain limitations in its application in plant disease resistance breeding (Acevedo-Garcia ,et al.,2014).
  • MLO gene mutations also lead to a variety of other phenotypes.
  • a follow-up study on the KN199 wheat mlo three-growth plant found that the mutant plant showed premature leaf senescence in the later stage of growth. This premature senescence led to a decline in yield, which would affect its application in breeding and production.
  • the present invention provides a method of producing an engineered wheat plant, the method comprising:
  • modified wheat plant which has an increased resistance to powdery mildew and a comparable or preferably increased yield relative to the corresponding wild-type wheat plant.
  • the method knocks out or knocks down TaMLO-A1, TaMLO-B1, and/or TaMLO-D1 genes into wheat plants by introducing antisense RNA, amiRNA, siRNA, or shRNA into wheat plants.
  • the method leads to TaMLO-A1, TaMLO- Mutations in the B1 and/or TaMLO-D1 genes, thereby knocking out or knocking down the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes in wheat plants.
  • the method further includes the step of obtaining a wheat plant that is homozygous for the mutation in the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 gene.
  • the method knocks out TaMLO-A1, TaMLO-B1, and TaMLO-D1 genes in wheat plants.
  • the method increases the expression of TaTMT3 protein by introducing an expression construct comprising a nucleotide sequence encoding TaTMT3 protein into a wheat plant, wherein the nucleotide sequence encoding TaTMT3 protein and expression control elements can be Operationally connected.
  • the present invention provides a method of producing an engineered wheat plant, the method comprising:
  • TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes are knocked down and/or knocked out;
  • modified wheat plant has increased powdery mildew resistance and a comparable or preferably increased yield relative to the corresponding wild-type wheat plant.
  • the first wheat plant has increased powdery mildew resistance relative to the corresponding wild-type wheat plant.
  • the second plant has increased yield relative to the corresponding wild-type wheat plant.
  • the present invention is a method for producing a modified wheat plant, the method comprising increasing the expression of TaTMT3 protein in a wheat plant, thereby obtaining a modified wheat plant, which has an increase relative to the corresponding wild-type wheat plant The output.
  • the expression of TaTMT3 protein is increased by introducing into wheat plants an expression construct comprising a nucleotide sequence encoding TaTMT3 protein, wherein the nucleotide sequence encoding TaTMT3 protein is operably linked to an expression control element .
  • the modified wheat plant of the present invention or its progeny or part, wherein the wheat plant can be produced by or by the method of the present invention.
  • the modified wheat plant of the present invention or its progeny or part, in which the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes are knocked down and/or knocked out, and TaTMT3 With increased protein expression, the modified wheat plant has increased powdery mildew resistance and comparable or preferably increased yield relative to wild-type wheat plants.
  • the modified wheat plant of the present invention has increased TaTMT3 protein expression, and has increased yield relative to wild-type wheat plants.
  • the TaMLO-A1 gene encodes the amino acid sequence shown in SEQ ID NO: 2
  • the TaMLO-B1 gene encodes the amino acid sequence shown in SEQ ID NO: 4
  • the TaMLO- The D1 gene encodes the amino acid sequence shown in SEQ ID NO: 6.
  • the TaTMT3 protein is TaTMT3B protein.
  • the TaTMT3B protein comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the wheat plant is selected from the group consisting of Triticum aestivum, T. aethiopicum, T. araraticum, T. boeoticum ), Persian wheat (T.carthlicum), dense ear wheat (T.compactum), wild two-corn wheat (T.dicoccoides), cultivated two-corn wheat (T.dicoccum), durum wheat (T.durum), Yis T.ispahanicum, T.karamyschevii, T.macha, T.militinae, T.monococcum, Polish wheat (T.polonicum) , T.repens, T.spelta, T.sphaerococcum, T.timopheevii, T.turanicum, T.turgidum ), T.urartu, T.vavilovii and T.zhukovskyi.
  • the wheat plant is common wheat, especially the cultivar Bobwhite .
  • Figure 1 shows the phenotype of the KN199 wheat mlo trigonal plant.
  • Figure 2 shows the phenotype of mutant R33.
  • Figure 3 shows the genotype identification of mutant R33.
  • A Agarose gel electrophoresis to amplify the target band;
  • B Mutagenesis of TaMLO-A1 and TaMLO-D1 in mutant R33.
  • FIG. 4 Deletion mutations occurred in the B genome of mutant R33.
  • A The B genome of mutant R33 has a fragment of 304KB missing;
  • B Two talen target sequences of the missing fragment, red is the talen recognition site, blue is the mismatch site, and green is the detection site AvaII.
  • TaTMT3B overexpression restored the growth-deficient phenotype of the mlo mutant.
  • the term “and/or” encompasses all combinations of items connected by the term, and should be treated as if each combination has been individually listed herein.
  • “A and/or B” encompasses “A”, “A and B”, and “B”.
  • “A, B, and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
  • plant includes the whole plant and any progeny, cells, tissues, or parts of the plant.
  • plant part includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; Plant cell culture; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and related explants).
  • Plant tissues or plant organs can be seeds, callus, or any other plant cell population organized into structural or functional units.
  • the plant cell or tissue culture can regenerate a plant having the physiological and morphological characteristics of the plant from which the cell or tissue is derived, and can regenerate a plant having substantially the same genotype as the plant. In contrast, some plant cells cannot regenerate plants.
  • the regenerable cells in plant cells or tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silks, flowers, nuts, ears, cobs, shells , Or stem.
  • Plant of a plant includes any subsequent generations of the plant.
  • Modified plants include plants that contain exogenous polynucleotides or contain modified genes or expression control sequences in their genome.
  • exogenous polynucleotides can be stably integrated into the genome and inherited for successive generations.
  • the exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct.
  • the modified gene or expression control sequence includes single or multiple deoxynucleotide substitutions, deletions and additions in the plant genome.
  • nucleic acid sequence is used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural Or changed nucleotide bases.
  • Polypeptide “peptide”, and “protein” are used interchangeably in the present invention and refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are corresponding artificial chemical analogs of naturally occurring amino acids, as well as to naturally occurring amino acid polymers.
  • the terms "polypeptide”, “peptide”, “amino acid sequence” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxyl And ADP-ribosylation.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid. Glycolic acid, but still has the activity described in the present invention.
  • methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical conditions (for example, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
  • sequence identity has the art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • Suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule.
  • those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub .co.,p.224).
  • expression construct refers to a vector suitable for expression of a nucleotide sequence of interest in a plant, such as a recombinant vector. "Expression” refers to the production of a functional product.
  • the expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and/or the translation of RNA into a precursor or mature protein.
  • the "expression construct” of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be an RNA (such as mRNA) that can be translated.
  • the "expression construct" of the present invention may contain expression control sequences and nucleotide sequences of interest from different sources, or expression control sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those normally occurring in nature. .
  • Expression control sequence and “expression control element” are used interchangeably and refer to the upstream (5' non-coding sequence), middle or downstream (3' non-coding sequence) of the coding sequence, and affect the transcription and RNA of the related coding sequence. Processing or stability or translated nucleotide sequence. Plant expression regulatory elements refer to nucleotide sequences that can control the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest in plants.
  • Expression control sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment.
  • the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it is derived from plant cells.
  • the promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.
  • operably linked refers to the connection of regulatory elements (for example, but not limited to, promoter sequences, transcription termination sequences, etc.) to nucleic acid sequences (for example, coding sequences or open reading frames) such that the nucleotides The transcription of the sequence is controlled and regulated by the transcription control element.
  • regulatory elements for example, but not limited to, promoter sequences, transcription termination sequences, etc.
  • nucleic acid sequences for example, coding sequences or open reading frames
  • Loss of function of the three copies of wheat MLO gene TaMLO-A1, TaMLO-B1 and TaMLO-D1 genes, such as triple functional knockout mutations, can lead to a phenotype of enhanced powdery mildew resistance, but may also lead to a decrease in yield.
  • the present inventors surprisingly found that under the background of functional deletion of the MLO gene, increasing the expression of TaTMT3B can obtain the same or even increased yield as the wild type while maintaining resistance to powdery mildew.
  • the present invention provides a method of producing an engineered wheat plant, the method comprising:
  • TaTMT3 protein preferably TaTMT3B protein
  • modified wheat plant which has an increased resistance to powdery mildew and a comparable or preferably increased yield relative to the corresponding wild-type wheat plant.
  • the present invention also provides a method of producing an engineered wheat plant, the method comprising:
  • TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes are knocked down and/or knocked out;
  • TaTMT3 protein preferably TaTMT3B protein
  • the modified wheat plant has increased powdery mildew resistance and comparable or preferably increased yield relative to corresponding wild-type wheat plants.
  • the first wheat plant has increased powdery mildew resistance relative to the corresponding wild-type wheat plant.
  • the second plant has increased yield relative to the corresponding wild-type wheat plant.
  • the present invention also provides a method for producing a modified wheat plant, the method comprising increasing the expression of TaTMT3 protein (preferably TaTMT3B protein) in the wheat plant, thereby obtaining the modified wheat plant, which is relative to the corresponding
  • the wild-type wheat plants have increased yields.
  • the wheat plant has increased powdery mildew resistance relative to the corresponding wild-type wheat plant.
  • the present invention also provides the use of TaTMT3 protein (preferably TaTMT3B protein) or its encoding nucleic acid molecule to produce wheat plants with increased yield relative to corresponding wild-type wheat plants.
  • the wheat plant has increased powdery mildew resistance relative to the corresponding wild-type wheat plant.
  • the "corresponding wild-type wheat plant” refers to an unmodified wheat plant from which the modified wheat plant is derived.
  • knockdown refers to the expression and/or activity of a target gene (usually an endogenous target gene) in a wheat plant by manual manipulation (such as genetic manipulation) relative to a wild-type plant that has not received the manipulation. Was downgraded.
  • the expression can be either at the transcription level of the gene or the expression at the translation level. Knockdown of the target gene can cause its function to be weakened. Knockdown of a target gene, for example, also encompasses mutations (such as point mutations) in the encoded product, resulting in a decrease in activity, such as biological activity.
  • knockout refers to the fact that a target gene (usually an endogenous target gene) in a wheat plant is basically not expressed by manual manipulation (such as genetic manipulation) relative to a wild-type plant that has not undergone the manipulation, that is, Substantially no functional expression product is produced, and/or a product that is substantially non-functional is expressed.
  • the expression can be either at the transcription level of the gene or the expression at the translation level.
  • Knockout of the target gene can lead to loss of its function.
  • Knockout of a target gene for example, also encompasses mutations (such as point mutations) in its encoded product, resulting in loss of activity, such as biological activity.
  • knock out the TaMLO-A1, TaMLO-B1 and TaMLO-D1 genes in the wheat plant knock out the TaMLO-A1, TaMLO-B1 and TaMLO-D1 genes in the wheat plant.
  • RNA molecules include, but are not limited to, the introduction of antisense RNA, artificial miRNA (amiRNA), siRNA, shRNA, or gene editing systems that target genes of interest into plants such as meganucleases, zinc finger nucleases ( ZFN), transcription activator-like effector nuclease (TALEN) or CRISPR gene editing system, etc.
  • amiRNA artificial miRNA
  • shRNA shRNA
  • gene editing systems that target genes of interest into plants such as meganucleases, zinc finger nucleases ( ZFN), transcription activator-like effector nuclease (TALEN) or CRISPR gene editing system, etc.
  • the introduction can be achieved by transforming a plant with an expression construct containing a nucleotide sequence encoding the antisense RNA, artificial miRNA (amiRNA), siRNA, shRNA, or a component of the gene editing system.
  • the coding nucleotide sequence is usually operably linked to an expression control element.
  • antisense RNA, amiRNA, siRNA or shRNA targeting the target gene usually results in degradation of the target gene transcription product or translational inhibition.
  • the introduction of a gene editing system that targets the target gene can lead to mutations in the target gene, such as one or more nucleotide substitutions, deletions or additions, or even partial or complete deletion of the gene, thereby rendering the target gene unable to be transcribed or produced Truncated or mutated proteins with reduced or missing functions.
  • the mutation caused by the gene editing system can be located in the regulatory sequence (such as promoter, enhancer, etc.) or coding sequence of the target gene, as long as it can achieve knockdown or knockout of the target gene, preferably in the coding sequence.
  • the mutation in the coding sequence is a frameshift mutation.
  • the mutation in the gene of interest is homozygous in the plant.
  • the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes in wheat plants are knocked out or knocked down by introducing antisense RNA, amiRNA, siRNA, shRNA.
  • the method further includes the step of obtaining a wheat plant that is homozygous for the mutation in the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 gene.
  • the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes in wheat plants are knocked out or knocked down by introducing a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • Transcription activator-like effector nuclease is a restriction enzyme that can be engineered to cut a specific DNA sequence, usually by combining the DNA binding domain of the transcription activator-like effector (TALE) with the DNA cleavage structure Prepared by domain fusion. TALE can be engineered to bind almost any desired DNA sequence.
  • TALE transcription activator-like effector
  • the design and preparation of TALEN suitable for the present invention can be referred to Wang et al. (Nature Biotechnology, Volume 32, Number 9, September, 2014), for example.
  • the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes in wheat plants are knocked out or knocked down by introducing a CRISPR gene editing system.
  • the CRISPR (Clustered regularly interspaced short palindromic repeats, clustered regularly spaced short palindromic repeats) system is an immune system produced during the evolution of bacteria to defend against foreign gene invasion. It has been modified and widely used in eukaryotes. Genome editing of organisms.
  • the CRISPR gene editing system usually contains at least two kinds of CRISPR nuclease and corresponding guide RNA (gRNA).
  • the gRNA includes a targeting part that has homology with the target nucleic acid sequence and a scaffold part responsible for binding to CRISPR nuclease. After CRISPR nuclease forms a complex with gRNA, it can target the target sequence on the genome under the guidance of gRNA, and perform its nucleic acid cleavage or other functions.
  • Nucleases that can be used in the CRISPR gene editing system include but are not limited to Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1 , Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3 or C2c2 protein or their variants or derivatives.
  • the CRISPR gene editing system also covers the so-called base editing system, which is based on the fusion protein of CRISPR nuclease (especially CRISPR nuclease with nickase activity) and cytosine deaminase or adenine deaminase, which can be used in the genome. Achieve precise base substitutions in.
  • “Increase the expression of TaTMT3 protein in the wheat plant” means that the expression and/or activity of the TaTMT3 protein in the wheat plant is up-regulated by manual manipulation (such as genetic manipulation) relative to the corresponding wild-type plant that has not received the manipulation. . "Increase the expression of TaTMT3 protein in the wheat plant” also encompasses the introduction of mutations that increase its activity (such as biological activity) into the TaTMT3 protein, or the introduction of TaTMT3 protein with increased activity (such as biological activity).
  • an expression construct comprising a nucleotide sequence encoding a TaTMT3 protein is introduced into a wheat plant, wherein the nucleotide sequence encoding the TaTMT3 protein is operably linked to an expression control element, thereby increasing TaTMT3 Protein expression.
  • the nucleotide sequence encoding the TaTMT3 protein is operably linked to a strong promoter in a plant.
  • TaTMT3 protein (preferably TaTMT3B protein) can also be increased by modifying the endogenous TaTMT3 gene, for example, by modifying (for example, using the gene editing system described in the present invention) the expression control sequence of the endogenous TaTMT3 gene, such as a promoter. Increase the expression of TaTMT3 protein (preferably TaTMT3B protein).
  • RNA molecules of the present invention such as antisense RNA, artificial miRNA (amiRNA), siRNA, shRNA, etc., gene editing system components, or expression constructs of TaTMT3 protein (preferably TaTMT3B protein) into plants
  • Known ones include, but are not limited to, protoplast electroporation, gene bombardment, PEG-mediated transformation of protoplasts, and Agrobacterium-mediated transformation.
  • the nucleotide sequence encoding the RNA molecule such as antisense RNA, artificial miRNA (amiRNA), siRNA, shRNA, etc., the genome editing component, or the TaTMT3 protein (preferably TaTMT3B protein) is integrated into the In the plant genome, stable and heritable expression can be obtained.
  • the plants are transiently transformed. The instantaneous transformation genome editing system realizes heritable genetic modification, and its components do not need to be integrated into the plant genome.
  • the exemplary coding sequence of the wild-type TaMLO-A1 gene is shown in SEQ ID NO: 1, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 2.
  • the exemplary coding sequence of the wild-type TaMLO-B1 gene is shown in SEQ ID NO: 3, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 4.
  • the exemplary coding sequence of the wild-type TaMLO-D1 gene is shown in SEQ ID NO: 5, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 6.
  • the wild-type MLO gene may be different due to natural genetic polymorphism, so its sequence may be different from the above exemplary sequence, but Perform similar or identical functions in plants.
  • Such MLO sequences are also within the scope of the present invention.
  • the TaTMT3 protein of various aspects of the present invention may be TaTMT3A, TaTMT3B and/or TaTMT3D protein, preferably TaTMT3B protein.
  • the exemplary coding sequence of the TaTMT3B protein of the present invention is shown in SEQ ID NO: 7, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 8.
  • the exemplary coding sequence of the TaTMT3A protein of the present invention is shown in SEQ ID NO: 9, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 10.
  • the exemplary coding sequence of the TaTMT3D protein of the present invention is shown in SEQ ID NO: 11, and the corresponding exemplary amino acid sequence is shown in SEQ ID NO: 12.
  • the present invention also covers SEQ ID NO: 8 having at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, or TaTMT3B with one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared with SEQ ID NO: 8 Protein, which has similar or identical functions to SEQ ID NO: 8.
  • SEQ ID NO: 10 has at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% Or higher sequence identity, or a TaTMT3A protein with one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared with SEQ ID NO: 10, It has similar or same functions as SEQ ID NO: 10.
  • SEQ ID NO: 12 has at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% Or higher sequence identity, or a TaTMT3D protein with one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared with SEQ ID NO: 12, It has similar or same functions as SEQ ID NO: 12.
  • the present invention provides a modified wheat plant or its progeny or part, in which TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes are knocked down and/or knocked out And the expression of TaTMT3 protein (preferably TaTMT3B protein) is increased, and the modified wheat plant has increased powdery mildew resistance and comparable or preferably increased yield relative to wild-type wheat plants.
  • TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes are knocked down and/or knocked out
  • the expression of TaTMT3 protein preferably TaTMT3B protein
  • the modified wheat plant has increased powdery mildew resistance and comparable or preferably increased yield relative to wild-type wheat plants.
  • the engineered wheat plant expression targets antisense RNA, amiRNA, siRNA or shRNA of TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes, for example, comprising The coding nucleotide sequence of the antisense RNA, amiRNA, siRNA or shRNA integrated into its genome and operably linked to the expression control element.
  • the modified wheat plant contains a mutation in the TaMLO-A1, TaMLO-B1, and/or TaMLO-D1 gene, which causes the TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 gene to be Knock down and/or knock out.
  • the modified wheat plant comprises a nucleotide sequence encoding a TaTMT3 protein (preferably a TaTMT3B protein) operably linked to an expression control element.
  • the present invention provides progeny or parts thereof of a modified wheat plant, said modified wheat plant having increased expression of TaTMT3 protein (preferably TaTMT3B protein) and having increased yield relative to wild-type wheat plants.
  • the modified wheat plant comprises a nucleotide sequence encoding TaTMT3 (preferably TaTMT3B) operably linked to an expression control element.
  • the present invention provides an engineered wheat plant or its progeny or part, wherein the engineered wheat plant can be produced by or by the method described in the present invention.
  • the part of the wheat plant is a seed.
  • the present invention also provides the use of the modified wheat plant of the present invention or its progeny or part in plant breeding.
  • the modified wheat plant or its progeny or part can be used to incorporate other wheat traits of interest through conventional breeding or molecular breeding.
  • the "knockdown” or “knockout” of TaMLO-A1, TaMLO-B1 or TaMLO-D1 gene, or increase in TaTMT3 protein expression can be done by detecting the amount of functional transcripts of related genes in plants (such as by quantitative RT-PCR) Or the amount of functional protein (such as by Western blot) to determine. For example, if the presence of TaMLO-A1 transcripts in plants is substantially undetectable by quantitative RT-PCR, it is considered that TaMLO-A1 has been knocked out.
  • the powdery mildew resistance of wheat plants can be determined by methods known in the art. For example, it can be evaluated by inoculating pathogenic bacteria on wheat leaves and observing the survival, growth of the pathogenic bacteria, or the size of the lesions produced.
  • the yield of the wheat plant of the present invention preferably refers to the yield of wheat grains, for example, the yield can be evaluated by the thousand-grain weight of the grains. Yield can also refer to actual yield, such as yield per mu.
  • the yield of the wheat plant of the present invention can also be the biomass of wheat, which can be reflected by the plant height, for example.
  • the modified wheat plant of the present invention has a comparable yield relative to the corresponding wild-type plant, for example, the yield of the modified wheat plant of the present invention and the yield of the corresponding wild-type plant are not statistically significant The difference, especially in the absence of powdery mildew stress.
  • the modified wheat plant of the present invention has increased yield relative to the corresponding wild-type plant, for example, a yield increase of about 5%, about 10%, about 15%, about 20%, about 25%, About 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200% or more.
  • the increase in yield is an increase in the presence of powdery mildew stress.
  • the yield is an increase in the absence of powdery mildew stress.
  • the wheat plants of the present invention include, but are not limited to, common wheat (Triticum aestivum), Ethiopian wheat (T.aethiopicum), Araraticum (T.araraticum), wild wheat (T.boeoticum), Persian wheat (T. .carthlicum), dense ear wheat (T.compactum), wild two-corn wheat (T.dicoccoides), cultivated two-corn wheat (T.dicoccum), durum wheat (T.durum), Ispahan wheat (T.
  • common wheat Triticum aestivum
  • Ethiopian wheat T.aethiopicum
  • Araraticum T.araraticum
  • wild wheat T.boeoticum
  • Persian wheat T. .carthlicum
  • dense ear wheat T.compactum
  • wild two-corn wheat T.dicoccoides
  • cultivated two-corn wheat T.dicoccum
  • durum wheat durum wheat
  • Ispahan wheat T.
  • the wheat plant belongs to common wheat, especially Bobwhite, a cultivar of common wheat.
  • the present invention also provides reagents or kits for the methods of the present invention, which may include antisense RNA, artificial miRNA (amiRNA), siRNA, shRNA, or genes of TaMLO-A1, TaMLO-B1 and/or TaMLO-D1 genes Editing systems such as meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) or CRISPR gene editing systems, or expression constructs encoding these; and/or encoding TaTMT3 protein (preferably TaTMT3B Protein) expression construct.
  • antisense RNA such as meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) or CRISPR gene editing systems, or expression constructs encoding these; and/or encoding TaTMT3 protein (preferably TaTMT3B Protein) expression construct.
  • the TALEN vector is delivered to BW wheat varieties using the gene gun transformation technology.
  • This TALEN vector simultaneously targets TaMLO-A1, TaMLO-B1, and TaMLO-D1 in the wheat genome. Volume 32, Number 9, September, 2014).
  • a new mutant R33 was discovered.
  • the mutant and wild-type wheat were respectively inoculated with powdery mildew, and it was found that compared with the wild type, the mutant showed strong resistance to powdery mildew.
  • the growth phenotype of the mutant and the wild type was observed, and it was found that the growth phenotype of the mutant was no different from the wild type.
  • the thousand-grain weight of the mutant wheat was measured, and it was found that the thousand-grain weight of the mutant plant was significantly increased compared with that of the wild type. (figure 2).
  • mutant R33 is mloaaB r B r dd
  • TaTMT3B was overexpressed in the background of mloaabbdd triticum wheat. It was found that overexpression of TaTMT3B restored the premature aging phenotype caused by the MLO gene mutation ( Figure 7A). At the same time, the plant height and yield also returned to the wild-type level ( Figure 7B and Figure 7C).

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Abstract

本发明属于植物基因工程领域，提供一种获得具有增加的白粉病抗性的小麦的方法，其中该小麦具有与野生型相当的甚至增加的产量。

Description

获得具有增加的白粉病抗性的小麦的方法 技术领域
本发明属于植物基因工程领域。具体而言，本发明涉及一种获得具有增加的白粉病抗性的小麦的方法，其中所述小麦具有与野生型相当的甚至增加的产量。
发明背景
白粉病(Powdery mildew，Pm)是世界范围内最重要的谷物疾病之一。小麦白粉病是由专性寄生真菌小麦白粉菌(Blumeria graminis f.sp tritici)引起的危害日趋严重的世界性真菌性病害。白粉病引起的小麦产量损失一般为13-34％，严重时可达50％。小麦抗病品种的培育是防治白粉病经济、有效和环境友好的策略。抗白粉病的小麦培育主要是将小麦近缘种和野生种的抗性基因通过杂交导入到小麦主栽品种里。由于这些抗性基因大都属于小种特异性抗病基因，其抗性很容易随着新的白粉菌生理小种的出现而打破。目前，大部分抗白粉病小麦品种(系)的抗性已失去或正在丧失。培育具有广谱、持久抗性的小麦品种是当前小麦抗白粉病育种的首要任务和挑战。
将植物中的感病基因(Susceptibility genes,S-genes)进行敲除，获得该感病基因的功能缺失突变体，是使植物获得抗病性的一个有效途径。大麦中的MLO基因(Mildew resistance Locus O,MLO)就是一个典型的感病基因，它对抗白粉病具有负调控作用(Acevedo-Garcia et al.,2014)。MLO基因的功能缺失突变体表现出对几乎所有小种的大麦白粉菌的持久、广谱抗性(Piffanelli et al.,2004)。目前，MLO基因的功能缺失突变体已在番茄、马铃薯、豌豆、辣椒和葡萄等多个物种中发现，这些突变体同样也对相应的白粉菌表现出广谱和持久的抗性(Acevedo-Garcia,et al.,2014；Appiano et al.,2015,Feechan,et al.,2008,Zheng,et al.,2013)。综上所述，植物MLO基因负调控对白粉菌的抗性的功能在进化上是高度保守的。因此，基于MLO基因有望开发出一种通用的创制广谱、持久抗白粉病植物种质的策略。
在六倍体小麦中，与大麦MLO基因同源性高达80％的基因存在三个拷贝，分布在5AL、4BL和4DL上，分别命名为TaMLO-A1、TaMLO-B1和TaMLO-D1。这三个同源基因在核酸水平和氨基酸水平的相似性分别达到98％和99％。小麦TaMLO-B1基因在大麦mlo突变体中表达可以恢复突变体对大麦白粉菌的感病性(Elliott et al.,2002)。此外，利用VIGS(virus-induced gene silencing)沉默小麦的MLO基因可以增强其白粉病抗性(Varallyay et al.,2012)。以上结果表明，小麦的MLO基因与大麦MLO基因功能相似，在进化上具有保守性。由于MLO基因在小麦里的这种多拷贝性以及高度相似性，通过天然突变和传统的生物学手段很难获得三个拷贝同时突变的突变体，这也可能是到目前为止没能获得广谱抗白粉病小麦mlo突变体的一个主要原因。Wang et al.,2014利用 TALEN基因组编辑技术，首次在六倍体小麦KN199中对MLO基因的三个拷贝同时进行了突变，获得的小麦MLO基因纯合突变体tamlo-aabbdd，该突变体对白粉菌表现出几乎完全的抗性。
然而，植物的免疫与生长发育往往是相互拮抗的，这也是作物抗病育种过程中长期存在的挑战。尽管mlo突变体已在大麦、番茄和豌豆等多种植物的抗白粉病育种和农业生产中得到了使用，但mlo突变体在植物抗病育种应用上目前尚存在一定的局限性(Acevedo-Garcia,et al.,2014)。MLO基因突变除产生白粉病抗性外，还导致多种其它表型。对KN199小麦mlo三突植物的后续研究发现，该突变体植物生长到后期出现叶片早衰现象，这种早衰导致产量下降，将影响其在育种和生产中的应用。
发明简述
在一方面，本发明提供一种产生经改造的小麦植物的方法，所述方法包括：
敲低和/或敲除小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因；和
增加小麦植物中TaTMT3蛋白的表达，
由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
在一些实施方案中，所述方法通过向小麦植物导入靶向TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因的反义RNA、amiRNA、siRNA或shRNA敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
在一些实施方案中，所述方法通过导入基因编辑系统，例如大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶(TALEN)或CRISPR基因编辑系统，导致TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变，从而敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
在一些实施方案中，所述方法还包括获得对所述TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变纯合的小麦植物的步骤。
在一些实施方案中，所述方法敲除小麦植物中TaMLO-A1、TaMLO-B1和TaMLO-D1基因。
在一些实施方案中，所述方法通过向小麦植物导入包含编码TaTMT3蛋白的核苷酸序列的表达构建体来增加TaTMT3蛋白的表达，其中所述编码TaTMT3蛋白的核苷酸序列与表达调控元件可操作地连接。
在一方面，本发明提供一种产生经改造的小麦植物的方法，所述方法包括：
提供第一小麦植物，其中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除；
提供第二小麦植物，其中TaTMT3蛋白的表达被增加；和
使第一小麦植物和第二小麦植物杂交，从而获得TaMLO-A1、TaMLO-B1 和/或TaMLO-D1基因被敲低和/或敲除，以及TaTMT3蛋白表达增加的经改造的小麦植物，
其中所述经改造的小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
在一些实施方案中，所述第一小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性。
在一些实施方案中，所述第二植物相对于相应的野生型小麦植物具有增加的产量。
在一方面，本发明一种产生经改造的小麦植物的方法，所述方法包括增加小麦植物中TaTMT3蛋白的表达，由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的产量。
在一些实施方案中，通过向小麦植物导入包含编码TaTMT3蛋白的核苷酸序列的表达构建体来增加TaTMT3蛋白的表达，其中所述编码TaTMT3蛋白的核苷酸序列与表达调控元件可操作地连接。
在一方面，本发明经改造的小麦植物或其后代或部分，其中所述小麦植物可通过或通过本发明的方法产生。
在一方面，本发明经改造的小麦植物或其后代或部分，所述经改造的小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除，并且TaTMT3蛋白表达增加，所述经改造的小麦植物相对于野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
在一方面，本发明经改造的小麦植物其后代或部分，所述经改造的小麦植物具有增加的TaTMT3蛋白表达，且相对于野生型小麦植物具有增加的产量。
在本发明各个方面的一些实施方案中，所述TaMLO-A1基因编码SEQ ID NO:2所示氨基酸序列，所述TaMLO-B1基因编码SEQ ID NO:4所示氨基酸序列，以及所述TaMLO-D1基因编码SEQ ID NO:6所示氨基酸序列。
在本发明各个方面的一些实施方案中，所述TaTMT3蛋白是TaTMT3B蛋白。在本发明各个方面的一些实施方案中，所述TaTMT3B蛋白包含SEQ ID NO:8所示的氨基酸序列。
在本发明各个方面的一些实施方案中，所述小麦植物选自普通小麦(Triticum aestivum)、埃塞俄比亚小麦(T.aethiopicum)、阿拉拉特小麦(T.araraticum)、野生一粒小麦(T.boeoticum)、波斯小麦(T.carthlicum)、密穗小麦(T.compactum)、野生二粒小麦(T.dicoccoides)、栽培二粒小麦(T.dicoccum)、硬粒小麦(T.durum)、依斯帕汗小麦(T.ispahanicum)、科尔希二粒小麦(T.karamyschevii)、马卡小麦(T.macha)、T.militinae、一粒小麦(T.monococcum)、波兰小麦(T.polonicum)、T.repens、斯佩耳特小麦(T.spelta)、印度圆粒小麦(T.sphaerococcum)、提莫非维小麦(T.timopheevii)、东方小麦(T.turanicum)、圆锥小麦(T.turgidum)、乌拉尔图小麦(T.urartu)、瓦维洛夫小麦(T.vavilovii)和茹科夫斯基小麦(T.zhukovskyi)，优选地，所述小麦植物是普通小麦，特别是栽培品种Bobwhite。
附图简述
图1、示出了KN199小麦mlo三突植物的表型。A：与野生型相比，mlo三突植物的生长表型受到影响；B：与野生型相比，mlo三突植物的产量(千粒重)下降。
图2、示出突变体R33的表型。A：与野生型相比，突变体R33对白粉菌具有增强的抗性；B：突变体R33的生长表型与野生型无显著差别；C：与野生型相比，突变体R33的千粒重显著增加。
图3、示出了突变体R33的基因型鉴定。A：琼脂糖凝胶电泳扩增目的条带；B：突变体R33中TaMLO-A1和TaMLO-D1的突变方式。
图4、突变体R33的B基因组上发生缺失突变。A：突变体R33的B基因组缺失304KB的片段；B：缺失片段两段的talen靶序列，红色为talen识别位点，蓝色为mismatch位点，绿色为检测用酶切位点AvaⅡ。
图5、R33突变体删除片段附近基因的表达分析。DUG:deletion upstream gene；DDG:deletion downstream gene。
图6、R33突变体中TaTMT3的表达水平分析。
图7、TaTMT3B过表达恢复了mlo突变体的生长缺陷表型。A：TaTMT3B过表达植物表型；B：TaTMT3B过表达恢复了mlo突变体的株高；C：TaTMT3B过表达恢复了mlo突变体的产量。
具体实施方式
一、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
如本文所使用的，术语“植物”包括整个植物和任何后代、植物的细胞、组织、或部分。术语“植物部分”包括植物的任何部分，包括，例如但不限于：种子(包括成熟种子、没有种皮的未成熟胚、和不成熟的种子)；植物插条(plant cutting)；植物细胞；植物细胞培养物；植物器官(例如，花粉、胚、花、果实、芽、叶、根、茎，和相关外植体)。植物组织或植物器官可以是种子、愈伤组织、或者任何其他被组织成结构或功能 单元的植物细胞群体。植物细胞或组织培养物能够再生出具有该细胞或组织所来源的植物的生理学和形态学特征的植物，并能够再生出与该植物具有基本上相同基因型的植物。与此相反，一些植物细胞不能够再生产生植物。植物细胞或组织培养物中的可再生细胞可以是胚、原生质体、分生细胞、愈伤组织、花粉、叶、花药、根、根尖、丝、花、果仁、穗、穗轴、壳、或茎。
植物“后代”包括植物的任何后续世代。
“经改造的植物”包括在其基因组内包含外源多核苷酸或包含经修饰的基因或表达调控序列的植物。例如外源多核苷酸能够稳定地整合进基因组中，并遗传连续的世代。外源多核苷酸可单独地或作为重组DNA构建体的部分整合进基因组中。修饰的基因或表达调控序列为在植物基因组中所述序列包含单个或多个脱氧核苷酸取代、缺失和添加。
“多核苷酸”、“核酸序列”、“核苷酸序列”或“核酸片段”可互换使用并且是单链或双链RNA或DNA聚合物，任选地可含有合成的、非天然的或改变的核苷酸碱基。
“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列，相应地，其编码核苷酸序列也可以包含起始密码子；反之亦然。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
在肽或蛋白中，合适的保守型氨基酸取代是本领域技术人员已知的，并且一般可以进行而不改变所得分子的生物活性。通常，本领域技术人员认识到多肽的非必需区中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在植物中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的 转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)。本发明的“表达构建体”可包含不同来源的表达调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的表达调控序列和感兴趣的核苷酸序列。
“表达调控序列”和“表达调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。植物表达调控元件指的是能够在植物中控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。
表达调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方案中，启动子是能够控制植物细胞中基因转录的启动子，无论其是否来源于植物细胞。启动子可以是组成型启动子或组织特异性启动子或发育调控启动子或诱导型启动子。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
二、产生经改造的小麦植物的方法
小麦MLO基因的三个拷贝TaMLO-A1、TaMLO-B1和TaMLO-D1基因的功能缺失，例如三重功能性敲除突变，能够导致白粉菌抗性增强的表型，但是也可能导致产量的下降。本发明人令人惊奇地发现，在MLO基因功能性缺失的背景下，增加TaTMT3B的表达，能够在保持白粉菌抗性的同时，获得与野生型相当的甚至增加的产量。
因此，在一方面，本发明提供一种产生经改造的小麦植物的方法，所述方法包括：
敲低(knock down)和/或敲除(knock out)小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因；和
增加小麦植物中TaTMT3蛋白(优选TaTMT3B蛋白)的表达，
由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
在一方面，本发明还提供一种产生经改造的小麦植物的方法，所述方法包括：
提供第一小麦植物，其中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除；
提供第二小麦植物，其中TaTMT3蛋白(优选TaTMT3B蛋白)的表达被增加；和
使第一小麦植物和第二小麦植物杂交，从而获得TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除，以及TaTMT3蛋白(优选TaTMT3B蛋白)表达 增加的经改造的小麦植物。
在一些实施方案中，所述经改造的小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。在一些实施方案中，所述第一小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性。在一些实施方案中，所述第二植物相对于相应的野生型小麦植物具有增加的产量。
在一方面，本发明还提供一种产生经改造的小麦植物的方法，所述方法包括增加小麦植物中TaTMT3蛋白(优选TaTMT3B蛋白)的表达，由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的产量。在一些实施方案中，所述小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性。
在一方面，本发明还提供TaTMT3蛋白(优选TaTMT3B蛋白)或其编码核酸分子在产生相对于相应的野生型小麦植物具有增加的产量的小麦植物中的用途。在一些实施方案中，所述小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性。
“相应的野生型小麦植物”指的是所述经改造的小麦植物所衍生自的未经改造的小麦植物。
如本文所用，“敲低”指的是通过人工操作(例如遗传操作)使得小麦植物中目的基因(通常是内源目的基因)的表达和/或活性相对于未接受所述操作的野生型植物被下调。表达可以是基因的转录水平或是翻译水平上的表达。目的基因的敲低可以导致其功能被减弱。目的基因的敲低例如也涵盖使其编码产物发生突变(例如点突变)而导致活性例如生物学活性降低。
如本文所用，“敲除”指的是通过人工操作(例如遗传操作)使得小麦植物中目的基因(通常是内源目的基因)相对于未接受所述操作的野生型植物基本上不表达，即基本上不产生有功能的表达产物，和/或表达基本上没有功能的产物。表达可以是基因的转录水平或是翻译水平上的表达。目的基因的敲除可以导致其功能丧失。目的基因的敲除例如也涵盖使其编码产物发生突变(例如点突变)而导致活性例如生物学活性丢失。
在一些实施方案中，敲除(knock out)所述小麦植物中TaMLO-A1、TaMLO-B1和TaMLO-D1基因。
本领域已知若干在植物中敲低或敲除目的基因的方法。示例性的方法包括但不限于向植物导入靶向目的基因的反义RNA、人工miRNA(amiRNA)、siRNA、shRNA，或靶向目的基因的基因编辑系统例如大范围核酸酶、锌指核酸酶(ZFN)、转录激活因子样效应物核酸酶(TALEN)或CRISPR基因编辑系统等。
所述导入可以通过用包含编码所述反义RNA、人工miRNA(amiRNA)、siRNA、shRNA、或基因编辑系统组分的核苷酸序列的表达构建体转化植物来实现。在所述表达构建体中，所述编码核苷酸序列通常与表达调控元件可操作地连接。
靶向目的基因的反义RNA、amiRNA、siRNA或shRNA的导入通常导致目的基因转录产物的降解或翻译抑制。
靶向目的基因的基因编辑系统的导入可以导致目的基因中的突变，例如一或多个核 苷酸的取代、缺失或添加，甚至基因的部分或全部缺失，由此导致目的基因无法转录或产生截短的或突变的功能减少或缺失的蛋白质。所述基因编辑系统导致的突变可以位于目的基因的调控序列(如启动子、增强子等)或编码序列，只要其能实现目的基因的敲低或敲除，优选位于编码序列。例如，所述编码序列中的突变是移码突变。优选地，所述目的基因中的突变在植物中是纯合的。
在本发明的一些实施方式中，通过导入反义RNA、amiRNA、siRNA、shRNA敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
在本发明的另一些实施方式中，通过导入基因编辑系统，例如大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶(TALEN)或CRISPR基因编辑系统，导致TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变，从而敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。在一些实施方案中，所述方法还包括获得对所述TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变纯合的小麦植物的步骤。
在本发明的一些具体实施方案中，通过导入转录激活因子样效应物核酸酶(TALEN)敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
“转录激活因子样效应物核酸酶(TALEN)”是可以经工程化而可以切割特定DNA序列的限制性酶，通常通过将转录激活因子样效应物(TALE)的DNA结合结构域与DNA切割结构域融合而制备。TALE经工程化后可以结合几乎任何想要的DNA序列。适用于本发明的TALEN的设计和制备例如可以参考Wang et al.(Nature Biotechnology,Volume 32,Number 9,September,2014)。
在另外一些实施方案中，通过导入CRISPR基因编辑系统敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
CRISPR(Clustered regularly interspaced short palindromic repeats，成簇的规律间隔的短回文重复序列)系统是细菌在进化过程中产生的用于防御外来基因入侵的免疫系统，目前已经被改造并广泛用于真核生物的基因组编辑。
CRISPR基因编辑系统通常至少包含CRISPR核酸酶和相应的向导RNA(gRNA)两种。gRNA包括与靶核酸序列具有同源性的靶向部分以及负责和CRISPR核酸酶结合的支架部分。CRISPR核酸酶与gRNA形成复合物后，能够在gRNA指导下靶向基因组上的靶序列，并执行其核酸切割或其它功能。
CRISPR基因编辑系统可以使用的核酸酶包括但不限于Cas3、Cas8a、Cas5、Cas8b、Cas8c、Cas10d、Cse1、Cse2、Csy1、Csy2、Csy3、GSU0054、Cas10、Csm2、Cmr5、Cas10、Csx11、Csx10、Csf1、Cas9、Csn2、Cas4、Cpf1、C2c1、C2c3或C2c2蛋白或它们的变体或衍生物。
基于CRISPR系统，本领域已经发展出多种基因编辑系统，这些都可以应用于本发明。
例如，CRISPR基因编辑系统还涵盖所谓的碱基编辑系统，其基于CRISPR核酸酶(特别具有切口酶活性的CRISPR核酸酶)和胞嘧啶脱氨酶或腺嘌呤脱氨酶的融合蛋白，能够 在基因组中实现精确的碱基替换。
本领域技术人员有能力选择、设计并获得适于本发明的各种CRISPR基因编辑系统。
“增加所述小麦植物中TaTMT3蛋白的表达”指的是通过人工操作(例如遗传操作)使得小麦植物中TaTMT3蛋白的表达和/或活性相对于未接受所述操作的相应的野生型植物被上调。“增加所述小麦植物中TaTMT3蛋白的表达”也涵盖在TaTMT3蛋白中导入增加其活性(如生物学活性)的突变，或导入具有增加的活性(如生物学活性)的TaTMT3蛋白。
本领域已知若干在植物中增加给定目的蛋白的表达的方法。例如，在一些实施方案中，通过向小麦植物导入包含编码TaTMT3蛋白的核苷酸序列的表达构建体，其中所述编码TaTMT3蛋白的核苷酸序列与表达调控元件可操作地连接，从而增加TaTMT3蛋白的表达。优选地，所述编码TaTMT3蛋白的核苷酸序列与植物中的强启动子可操作地连接。
然而，也可以通过改造内源TaTMT3基因来增加TaTMT3蛋白(优选TaTMT3B蛋白)的表达，例如通过修饰(例如利用本发明所描述基因编辑系统进行修饰)内源TaTMT3基因的表达调控序列例如启动子来增加TaTMT3蛋白(优选TaTMT3B蛋白)的表达。
适合于将本发明所述RNA分子如反义RNA、人工miRNA(amiRNA)、siRNA、shRNA等，基因编辑系统组分、或TaTMT3蛋白(优选TaTMT3B蛋白)的表达构建体导入植物的方法是本领域已知的，包括但不限于原生质体电穿孔、基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化。
在一些实施方案中，编码所述RNA分子如反义RNA、人工miRNA(amiRNA)、siRNA、shRNA等，所述基因组编辑组分，或TaTMT3蛋白(优选TaTMT3B蛋白)的核苷酸序列整合进所述植物基因组中以获得稳定的可遗传的表达。在一些实施方案中，特别是对于基因组编辑系统，被瞬时转化至所述植物。瞬时转化基因组编辑系统即实现可遗传的基因修饰，其组分无需整合进植物基因组。
野生型TaMLO-A1基因的示例性编码序列如SEQ ID NO:1所示，相应的示例性氨基酸序列如SEQ ID NO:2所示。野生型TaMLO-B1基因的示例性编码序列如SEQ ID NO:3所示，相应的示例性氨基酸序列如SEQ ID NO:4所示。野生型TaMLO-D1基因的示例性编码序列如SEQ ID NO:5所示，相应的示例性氨基酸序列如SEQ ID NO:6所示。本领域技术人员可以理解，对于不同的小麦物种、亚种、栽培种或品系，其野生型MLO基因可能由于天然遗传多态性而存在差异，因此其序列可以不同于上述示例性序列，但却在植物中执行类似或相同功能。这样MLO序列也在本发明的范围内。
本发明各个方面的TaTMT3蛋白可以是TaTMT3A、TaTMT3B和/或TaTMT3D蛋白，优选是TaTMT3B蛋白。
本发明的TaTMT3B蛋白的示例性编码序列如SEQ ID NO:7所示，相应的示例性氨基酸序列如SEQ ID NO:8所示。本发明的TaTMT3A蛋白的示例性编码序列如SEQ ID NO:9所示，相应的示例性氨基酸序列如SEQ ID NO:10所示。本发明的TaTMT3D蛋白 的示例性编码序列如SEQ ID NO:11所示，相应的示例性氨基酸序列如SEQ ID NO:12所示。
然而，本领域技术人员可以预期，氨基酸序列中的一些保守型取代或在非关键结构域的取代(特别是由于天然遗传多态性造成的差异)不会实质上影响蛋白质的功能。因此，本发明也涵盖与SEQ ID NO:8具有至少85％、86％、87％、88％、89％、90％、92％、94％、95％、96％、97％、98％或99％或更高序列相同性，或者与SEQ ID NO:8相比具有一个或多个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸取代的TaTMT3B蛋白，其与SEQ ID NO:8具有类似或相同功能。本发明也涵盖与SEQ ID NO:10具有至少85％、86％、87％、88％、89％、90％、92％、94％、95％、96％、97％、98％或99％或更高序列相同性，或者与SEQ ID NO:10相比具有一个或多个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸取代的TaTMT3A蛋白，其与SEQ ID NO:10具有类似或相同功能。本发明也涵盖与SEQ ID NO:12具有至少85％、86％、87％、88％、89％、90％、92％、94％、95％、96％、97％、98％或99％或更高序列相同性，或者与SEQ ID NO:12相比具有一个或多个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸取代的TaTMT3D蛋白，其与SEQ ID NO:12具有类似或相同功能。
在另一方面，本发明提供了经改造的小麦植物或其后代或部分，所述经改造的小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除，并且TaTMT3蛋白(优选TaTMT3B蛋白)表达增加，所述经改造的小麦植物相对于野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。在一些实施方案中，所述经改造的小麦植物表达(优选组成型表达)靶向TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因的反义RNA、amiRNA、siRNA或shRNA，例如，包含整合至其基因组的与表达调控元件可操作连接的所述反义RNA、amiRNA、siRNA或shRNA的编码核苷酸序列。在一些实施方案中，所述经改造的小麦植物在TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因包含突变，所述突变导致TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除。在一些实施方案中，所述经改造的小麦植物包含与表达调控元件可操作连接的TaTMT3蛋白(优选TaTMT3B蛋白)的编码核苷酸序列。
在另一方面，本发明提供了经改造的小麦植物其后代或部分，所述经改造的小麦植物具有增加的TaTMT3蛋白(优选TaTMT3B蛋白)表达，且相对于野生型小麦植物具有增加的产量。在一些实施方案中，所述经改造的小麦植物包含与表达调控元件可操作连接的TaTMT3(优选TaTMT3B)的编码核苷酸序列。
在另一方面，本发明提供了经改造的小麦植物或其后代或部分，其中所述经改造的小麦植物可通过或通过本发明所述的方法产生。
在一些实施方案中，所述小麦植物的部分是种子。
在另一方面，本发明还提供了本发明的经改造的小麦植物或其后代或部分在植物育种中的用途。例如，所述经改造的小麦植物或其后代或部分可以用于通过常规育种或分子育种并入其它感兴趣的小麦性状。
TaMLO-A1、TaMLO-B1或TaMLO-D1基因的“敲低”或“敲除”，或者TaTMT3蛋白表达的增加可以通过检测植物中相关基因功能性转录本的量(如通过定量RT-PCR)或功能性蛋白质的量(如通过蛋白印迹)来确定。例如，如果通过定量RT-PCR基本上检测不到TaMLO-A1的转录物在植物中的存在，则认为TaMLO-A1被敲除。
小麦植物的白粉病抗性可以通过本领域已知的方法确定。例如，可以通过在小麦叶片上接种病原菌，观察病原菌的存活、生长或产生的病斑大小来评估。
本发明所述小麦植物的产量优选是指小麦谷粒的产量，例如可以通过谷粒的千粒重来评估产量。产量也可以指实际产量，例如亩产量。本发明所述小麦植物的产量也可以是小麦的生物量，其例如可以通过株高来反映。
在一些实施方案中，本发明的经改造的小麦植物相对于相应的野生型植物具有相当的产量，例如，本发明的经改造的小麦植物的产量与相应的野生型植物的产量没有统计学显著的区别，特别是在不存在白粉病胁迫下。在一些优选实施方案中，本发明的经改造的小麦植物相对于相应的野生型植物具有增加的产量，例如产量增加约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约50％、约60％、约70％、约80％、约90％、约100％、约150％、约200％或更多。在一些实施方案中，所述产量增加是存在白粉病胁迫下的增加。在一些实施方案中，所述产量是不存在白粉病胁迫下的增加。
本发明所述小麦植物包括但不限于，普通小麦(Triticum aestivum)、埃塞俄比亚小麦(T.aethiopicum)、阿拉拉特小麦(T.araraticum)、野生一粒小麦(T.boeoticum)、波斯小麦(T.carthlicum)、密穗小麦(T.compactum)、野生二粒小麦(T.dicoccoides)、栽培二粒小麦(T.dicoccum)、硬粒小麦(T.durum)、依斯帕汗小麦(T.ispahanicum)、科尔希二粒小麦(T.karamyschevii)、马卡小麦(T.macha)、T.militinae、一粒小麦(T.monococcum)、波兰小麦(T.polonicum)、T.repens、斯佩耳特小麦(T.spelta)、印度圆粒小麦(T.sphaerococcum)、提莫非维小麦(T.timopheevii)、东方小麦(T.turanicum)、圆锥小麦(T.turgidum)、乌拉尔图小麦(T.urartu)、瓦维洛夫小麦(T.vavilovii)和茹科夫斯基小麦(T.zhukovskyi)。在一些优选的实施方案中，所述小麦植物属于普通小麦，特别是普通小麦的栽培品种Bobwhite。
本发明还提供用于本发明的方法的试剂或试剂盒，其可以包含TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因的反义RNA、人工miRNA(amiRNA)、siRNA、shRNA，或基因编辑系统例如大范围核酸酶、锌指核酸酶(ZFN)、转录激活因子样效应物核酸酶(TALEN)或CRISPR基因编辑系统，或编码这些的表达构建体；和/或编码TaTMT3蛋白(优选TaTMT3B蛋白)的表达构建体。
实施例
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
实施例1、KN199小麦mlo三突植物产量下降
生长对本发明人之前获得的KN199小麦mlo三突植物(Wang et al.,Nature Biotechnology,Volume 32,Number 9,September,2014)，并与野生型植物进行比较。结果表明，突变体植物生长到后期出现叶片早衰现象，这种早衰导致产量下降(图1)。
实施例2、突变体R33具有抗病增产的表型
利用基因枪转化技术将TALEN载体递送到BW小麦品种中，此TALEN载体同时靶向小麦基因组中的TaMLO-A1、TaMLO-B1和TaMLO-D1(TALEN载体具体信息参见Wang et al.,Nature Biotechnology,Volume 32,Number 9,September,2014)。在MLO1基因定向编辑突变体的筛选和检测过程中，发现了一个新的突变体R33。对该突变体和野生型小麦分别接种白粉菌，结果发现与野生型相比，突变体表现出对白粉菌很强的抗性。同时，对突变体和野生型的生长表型进行了观察，发现在生长表型上突变体与野生型无异。随后，对突变体小麦的千粒重进行了测定，结果发现，突变体植物的千粒重与野生型相比显著增加。(图2)。
实施例3、突变体R33的基因型鉴定
首先，分别在TaMLO-A1、TaMLO-B1和TaMLO-D1的靶位点两端设计引物，以突变体植物的基因组为模板进行PCR扩增，获得目的片段。琼脂糖凝胶电泳结果显示，A、D基因组上都能扩增到目的条带，经测序分析发现，TaMLO-A1上发生了-2/+52的突变，TaMLO-D1上发生了-32的突变。然而，B基因组上却无法扩增到目的条带(图3)。
为了探究突变体R33中B基因组上发生的改变，进一步开展了一系列的遗传学实验，包括R33与mloaaBBdd、WT以及mloaabbdd之间的杂交(表1)。通过这些遗传学分析，认为B基因组上发生的突变是隐性突变且在TaMLO-B1基因的附近。
表1、突变体R33与其它基因型小麦的杂交实验
Figure PCTCN2021090236-appb-000001
假定突变体R33的基因型为mloaaB rB rdd
实施例4、突变体R33的重测序分析
为明确R33突变体植物的突变方式，对其进行了基于NGS的重测序分析。通过将测序结果与野生型小麦参考基因组目标区域进行比对分析，确定了R33突变体植物中发生的遗传变化：R33突变体B基因组上发生了304.374KB的缺失，从612856038bp到 613160412bp，同时中间插入了344bp的载体序列，具体位置如图4所示。上游缺失位点位于MLO1的一个同源基因，命名为MLOX，切割位点周围序列与TALEN在MLO1上的靶序列高度同源(图4)。
实施例5、突变体R33表型产生的原因
为了明确R33突变体中片段缺失对基因表达的影响，对删除片段附近的基因进行了表达量的检测。结果发现DUG1基因的表达水平与野生型相比显著增加(图5)。
对DUG1进一步的同源性分析发现，其编码一种液泡膜单糖转运蛋白TaTMT3。我们进一步对TaTMT3A、TaTMT3B和TaTMT3D的表达水平进行了检测，结果显示，只有TaTMT3B表达水平显著增加，而TaTMT3A和TaTMT3B较野生型没有变化。设计针对TaTMT3A、TaTMT3B和TaTMT3D的通用引物，对TaTMT3的总体表达水平进行检测，结果表明，TaTMT3总体表达量较野生型显著增加(图6)。有报道显示，该基因在拟南芥中的同源基因AtTMT1的过表达能显著提高产量。
综上，基因编辑造成了R33突变体中TaMLO-A1和TaMLO-D1的功能缺失突变，以及B基因组上TaMLO-B1附近的大片段缺失，最终导致了TaMLO-A1、TaMLO-B1和TaMLO-D1蛋白的功能丧失，以及TaTMT3B表达量的升高，进而使R33突变体获得了抗白粉病同时增产的表型。本发明提供了一种可以平衡小麦白粉病抗性和生长缺陷的方法。
实施例6、过表达TaTMT3B改善mlo-aabbdd三突小麦的表型
为了进一步验证TaTMT3的生物学功能，在mloaabbdd三突小麦背景下对TaTMT3B进行过表达。结果发现，TaTMT3B的过表达恢复了MLO基因突变所造成的早衰表型(图7A)。同时，株高和产量也恢复到野生型水平(图7B和图7C)。
序列表：
SEQ ID NO:1 TaMLO-A1编码核苷酸序列
Figure PCTCN2021090236-appb-000002
Figure PCTCN2021090236-appb-000003
SEQ ID NO:2 TaMLO-A1氨基酸序列
Figure PCTCN2021090236-appb-000004
SEQ ID NO:3 TaMLO-B1编码核苷酸序列
Figure PCTCN2021090236-appb-000005
Figure PCTCN2021090236-appb-000006
SEQ ID NO:4 TaMLO-B1氨基酸序列
Figure PCTCN2021090236-appb-000007
SEQ ID NO:5 TaMLO-D1编码核苷酸序列
Figure PCTCN2021090236-appb-000008
Figure PCTCN2021090236-appb-000009
SEQ ID NO:6 TaMLO-D1氨基酸序列
Figure PCTCN2021090236-appb-000010
SEQ ID NO:7 TMT3B编码核苷酸序列
Figure PCTCN2021090236-appb-000011
Figure PCTCN2021090236-appb-000012
SEQ ID NO:8 TMT3B氨基酸序列
Figure PCTCN2021090236-appb-000013
SEQ ID NO:9 TMT3A编码核苷酸序列
Figure PCTCN2021090236-appb-000014
Figure PCTCN2021090236-appb-000015
SEQ ID NO:10 TMT3A氨基酸序列
Figure PCTCN2021090236-appb-000016
Figure PCTCN2021090236-appb-000017
SEQ ID NO:11 TMT3D编码核苷酸序列
Figure PCTCN2021090236-appb-000018
Figure PCTCN2021090236-appb-000019
SEQ ID NO:12 TMT3D氨基酸序列
Figure PCTCN2021090236-appb-000020

Claims (17)

  1. 一种产生经改造的小麦植物的方法，所述方法包括：
    敲低和/或敲除小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因；和
    增加小麦植物中TaTMT3蛋白的表达，
    由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
  2. 权利要求1的方法，其中通过向小麦植物导入靶向TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因的反义RNA、amiRNA、siRNA或shRNA敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
  3. 权利要求1的方法，其中通过导入基因编辑系统，例如大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶(TALEN)或CRISPR基因编辑系统，导致TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变，从而敲除或敲低小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因。
  4. 权利要求3的方法，还包括获得对所述TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因中的突变纯合的小麦植物的步骤。
  5. 权利要求1-4中任一项的方法，其中敲除小麦植物中TaMLO-A1、TaMLO-B1和TaMLO-D1基因。
  6. 权利要求1-5中任一项的方法，其中通过向小麦植物导入包含编码TaTMT3蛋白的核苷酸序列的表达构建体来增加TaTMT3蛋白的表达，所述编码TaTMT3蛋白的核苷酸序列与表达调控元件可操作地连接；或者通过修饰小麦植物的内源TaTMT3基因的表达调控序列来增加TaTMT3蛋白的表达。
  7. 一种产生经改造的小麦植物的方法，所述方法包括：
    提供第一小麦植物，其中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除；
    提供第二小麦植物，其中TaTMT3蛋白的表达被增加；和
    使第一小麦植物和第二小麦植物杂交，从而获得TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除，以及TaTMT3蛋白表达增加的经改造的小麦植物，
    其中所述经改造的小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
  8. 权利要求7的方法，其中所述第一小麦植物相对于相应的野生型小麦植物具有增加的白粉病抗性。
  9. 权利要求7或8的方法，其中所述第二植物相对于相应的野生型小麦植物具有增加的产量。
  10. 权利要求1-9中任一项的方法，其中TaMLO-A1基因编码SEQ ID NO:2所示氨 基酸序列，所述TaMLO-B1基因编码SEQ ID NO:4所示氨基酸序列，以及所述TaMLO-D1基因编码SEQ ID NO:6所示氨基酸序列。
  11. 一种产生经改造的小麦植物的方法，所述方法包括增加小麦植物中TaTMT3蛋白的表达，由此获得经改造的小麦植物，其相对于相应的野生型小麦植物具有增加的产量。
  12. 权利要求11的方法，其中通过向小麦植物导入包含编码TaTMT3蛋白的核苷酸序列的表达构建体来增加TaTMT3蛋白的表达，所述编码TaTMT3蛋白的核苷酸序列与表达调控元件可操作地连接；或者通过修饰小麦植物的内源TaTMT3基因的表达调控序列来增加TaTMT3蛋白的表达。
  13. 权利要求1-12中任一项的方法，其中所述TaTMT3蛋白是TaTMT3B蛋白，例如包含SEQ ID NO:8所示的氨基酸序列。
  14. 经改造的小麦植物或其后代或部分，其中所述小麦植物可通过或通过权利要求1-13中任一项的方法产生。
  15. 经改造的小麦植物或其后代或部分，所述经改造的小麦植物中TaMLO-A1、TaMLO-B1和/或TaMLO-D1基因被敲低和/或敲除，并且TaTMT3蛋白表达增加，所述经改造的小麦植物相对于野生型小麦植物具有增加的白粉病抗性以及相当的或优选地增加的产量。
  16. 经改造的小麦植物其后代或部分，所述经改造的小麦植物具有增加的TaTMT3蛋白表达，且相对于野生型小麦植物具有增加的产量。
  17. 权利要求1-13中任一项的方法或权利要求14-16中任一项的经改造的小麦植物其后代或部分，其中所述小麦植物选自普通小麦(Triticum aestivum)、埃塞俄比亚小麦(T.aethiopicum)、阿拉拉特小麦(T.araraticum)、野生一粒小麦(T.boeoticum)、波斯小麦(T.carthlicum)、密穗小麦(T.compactum)、野生二粒小麦(T.dicoccoides)、栽培二粒小麦(T.dicoccum)、硬粒小麦(T.durum)、依斯帕汗小麦(T.ispahanicum)、科尔希二粒小麦(T.karamyschevii)、马卡小麦(T.macha)、T.militinae、一粒小麦(T.monococcum)、波兰小麦(T.polonicum)、T.repens、斯佩耳特小麦(T.spelta)、印度圆粒小麦(T.sphaerococcum)、提莫非维小麦(T.timopheevii)、东方小麦(T.turanicum)、圆锥小麦(T.turgidum)、乌拉尔图小麦(T.urartu)、瓦维洛夫小麦(T.vavilovii)和茹科夫斯基小麦(T.zhukovskyi)，优选地，所述小麦植物是普通小麦，特别是栽培品种Bobwhite。
PCT/CN2021/090236 2020-04-27 2021-04-27 获得具有增加的白粉病抗性的小麦的方法 WO2021218961A1 (zh)

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