WO2021249229A1 - 一种增强植物香味的方法 - Google Patents

一种增强植物香味的方法 Download PDF

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WO2021249229A1
WO2021249229A1 PCT/CN2021/097340 CN2021097340W WO2021249229A1 WO 2021249229 A1 WO2021249229 A1 WO 2021249229A1 CN 2021097340 W CN2021097340 W CN 2021097340W WO 2021249229 A1 WO2021249229 A1 WO 2021249229A1
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plant
gene
fragrance
plants
badh2
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PCT/CN2021/097340
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English (en)
French (fr)
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王飞
王彦晓
牛小牧
张金山
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山东舜丰生物科技有限公司
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Priority to CN202180004587.1A priority Critical patent/CN114127299B/zh
Publication of WO2021249229A1 publication Critical patent/WO2021249229A1/zh

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    • 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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • 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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of agronomy, in particular, to a method for enhancing the fragrance of plants.
  • Fragrant rice is favored by consumers for its unique and strong aroma, so aroma has become an important quality indicator of rice.
  • the price of fragrant rice is 2-3 times that of ordinary rice, and the price of BASMATI fragrant rice produced in India and Pakistan is much higher.
  • the volatile aroma of fragrant rice contains more than 100 compounds, among which 2-acetyl-1-pyrroline (2AP) is the most important aroma.
  • the purpose of the present invention is to provide a new method for enhancing the aroma of plants.
  • the first aspect of the present invention provides a method for enhancing plant fragrance or imparting fragrance to plants, including the steps:
  • the BADH2 gene includes the BADH2b gene.
  • the BADH2 gene also includes the BADH2a gene.
  • the BADH2 gene includes BADH2b and BADH2a genes.
  • the method includes administering an inhibitor of the BADH2 gene or its encoded protein to the plant.
  • the enhancing the fragrance of the plant includes increasing the content of 4-aminobutyraldehyde or 2-acetyl-1-pyrroline (2AP) of the plant.
  • the enhancement of the fragrance of the plant is to increase the content of 4-aminobutyraldehyde or 2-acetyl-1-pyrroline (2AP) of the plant.
  • the plants include agricultural crops, forestry plants, vegetables, fruits, flowers, and pastures (including lawn grasses).
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plant is selected from the group consisting of gramineous, leguminous, chenopodiaceae, cruciferous plants, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rape, cabbage, spinach , Sugar beet, strawberry, or a combination thereof.
  • the plant is selected from corn.
  • the expression or activity of the BADH2 gene or its encoded protein in the plant tissue or plant cell is reduced by ⁇ 30%, ⁇ 50%, ⁇ 70%, and more preferably, ⁇ 80%.
  • the "reducing or inhibiting" means that the expression or activity of the BADH2 gene or its encoded protein is reduced to meet the following conditions:
  • the BADH2 gene or its encoded protein in the plant is completely inactivated or partially inactivated, or the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 60%, more preferably ⁇ 40%, and most preferably 0- 30%; where A1 is the expression or activity of the BADH2 gene or its encoded protein in the plant; A0 is the expression or activity of the same BADH2 gene or its encoded protein in wild-type plants of the same type.
  • the reduction or inhibition means that the expression level E1 of the BADH2 gene or its encoded protein in the plant is wild-type compared with the expression level E0 of the BADH2 gene or its encoded protein in the wild-type plant 0-80%, preferably 0-60%, more preferably 0-40%, more preferably 0-30%.
  • the reduction or inhibition of the expression and/or activity of the BADH2 gene or its protein is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, Inhibitors for introducing genes or proteins, or a combination thereof.
  • the gene mutation is obtained by one or more of the following methods: natural mutation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrous acid) , Hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus or bacteria-mediated mutagenesis), gene editing or biosynthesis.
  • natural mutation such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis
  • chemical mutagenesis such as nitrous acid
  • Hydroxylamine EMS, nitrosoguanidine, etc.
  • biological mutagenesis such as virus or bacteria-mediated mutagenesis
  • the mutation region includes exon and/or intron regions.
  • the inhibitor is selected from the group consisting of antisense nucleic acid, antibody, small molecule compound, Crispr reagent, small molecule ligand, or a combination thereof.
  • the gene editing technology is selected from the group consisting of CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.
  • the method includes the steps:
  • the method includes the steps:
  • gRNA targeting the BADH2 gene and the corresponding Cas protein to introduce into the plant or plant cell; in a preferred embodiment, an expression vector containing the gRNA and Cas protein is introduced into the plant or plant cell.
  • the BADH2 gene includes a wild-type BADH2 gene and a mutant BADH2 gene.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein).
  • polypeptide encoded by the mutant BADH2 gene is the same or substantially the same as the polypeptide encoded by the wild-type BADH2 gene.
  • the mutant BADH2 gene includes homology ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98% compared with the wild-type BADH2 gene). % Or 99%) polynucleotides.
  • the mutant BADH2 gene is included in the 5'end and/or 3'end of the wild-type BADH2 gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • the BADH2 gene includes a cDNA sequence, a CDS sequence, a genomic sequence, or a combination thereof.
  • the BADH2 gene is derived from one or more plants selected from the group consisting of gramineous, leguminous, chenopodiaceae, and cruciferous plants.
  • the BADH2 gene is derived from one or more plants selected from the group consisting of Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, Quinoa, lettuce, rape, cabbage, spinach, beets, strawberries.
  • the BADH2 gene is derived from corn.
  • amino acid sequence of the protein encoded by the BADH2a gene is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 1 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and has the same or similar functions (beet Alkaline aldehyde dehydrogenase activity) a polypeptide derived from (i);
  • amino acid sequence and the amino acid sequence shown in SEQ ID NO.:1 have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably More than 98%, such as 99% or 100%) homology, polypeptides with the same or similar functions.
  • amino acid sequence of the protein encoded by the BADH2b gene is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 2 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and has the same or similar functions (beet Alkaline aldehyde dehydrogenase activity) a polypeptide derived from (i);
  • nucleotide sequence of the BADH2a gene is selected from the following group:
  • nucleotide sequence of the BADH2b gene is selected from the following group:
  • the reduction or inhibition of the expression and/or activity of the BADH2b gene or its encoded protein is achieved by mutating the BADH2b gene.
  • the reduction or inhibition of the expression and/or activity of the BADH2b gene and the BADH2a gene or its encoded protein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene.
  • the mutations include insertion mutations, deletion mutations, frameshift mutations, and substitution mutations.
  • the method further includes the step of testing the increased amount of fragrance substance content in the plant.
  • the content of the fragrance substance in the plant is 0.01-20 mg/kg, preferably, 0.05-10 mg/kg, more preferably, 0.1-5 mg/kg, more preferably, 0.1-2 mg /kg, more preferably 0.2-1mg/kg.
  • the fragrance substance includes 2AP.
  • the second aspect of the present invention provides the use of an inhibitor of the BADH2 gene or its encoded protein for enhancing the fragrance of plants or for imparting fragrance to plants; or preparing a composition or preparation for enhancing plant fragrance or for preparation The composition or preparation for imparting fragrance to plants, wherein the BADH2 gene includes the BADH2b gene.
  • the BADH2 gene also includes the BADH2a gene.
  • the BADH2 gene includes BADH2b and BADH2a genes.
  • the composition includes an agricultural composition.
  • the formulation includes an agricultural formulation.
  • said enhancing the fragrance of plants includes increasing the content of fragrance substances in plants.
  • the fragrance substance includes 2AP.
  • the composition comprises (a) an inhibitor of the BADH2 gene or its encoded protein, wherein the BADH2 gene includes the BADH2b gene; and (b) an agronomically acceptable vector.
  • composition further includes BADH2a gene.
  • the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the inhibitor is selected from the group consisting of antisense nucleic acid, antibody, small molecule compound, Crispr reagent, small molecule ligand, or a combination thereof.
  • the antisense nucleic acid is selected from the group consisting of antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.
  • the interfering RNA is selected from the following group: siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.
  • the composition further includes other substances that enhance the aroma of crops.
  • the third aspect of the present invention provides a composition for enhancing plant fragrance or imparting fragrance to plants, comprising:
  • composition further includes BADH2a gene.
  • the composition includes an agricultural composition.
  • the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the composition contains 0.0001-99wt%, preferably 0.1-90wt% of component (a), based on the total weight of the composition.
  • the inhibitor is selected from the group consisting of gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, Crispr reagents, small molecule ligands, or combinations thereof.
  • the antisense nucleic acid is selected from the group consisting of antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.
  • the interfering RNA is selected from the following group: siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.
  • composition further includes other substances that enhance the fragrance of plants.
  • the fourth aspect of the present invention provides a use of the composition according to the third aspect of the present invention for enhancing the fragrance of plants or imparting fragrance to plants.
  • the fifth aspect of the present invention provides a method for preparing genetically engineered plant tissues or plant cells, including the steps:
  • the reduction of the expression and/or activity of the BADH2 gene or its protein is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, and gene introduction Or protein inhibitor, or a combination thereof.
  • the sixth aspect of the present invention provides a method for preparing genetically engineered plants, including the steps:
  • the genetically engineered plant tissue or plant cell prepared by the method of the fifth aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.
  • the seventh aspect of the present invention provides a method for improving plants, the method comprising the steps:
  • step (b) Regenerating the plant cells, plant tissues, and plant parts in step (a) into plants.
  • step (a) gene editing technology is used to transform the plant cell, plant tissue, plant part, so that the BADH2 gene or its encoded protein in the plant cell, plant tissue, plant part The expression or activity is reduced.
  • the BADH2 gene also includes the BADH2a gene.
  • the gene editing technology is selected from the group consisting of CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the method is used to enhance the fragrance of plants.
  • the method is used to increase the content of fragrance substances.
  • the method further includes the step of: testing the plant cell, plant tissue, plant part or plant for an increase in the content of the fragrance substance.
  • the improved plant is to enhance/enhance the fragrance of the plant or impart fragrance to the plant.
  • the eighth aspect of the present invention provides a genetically engineered plant, which is prepared by the method described in the seventh aspect of the present invention.
  • the ninth aspect of the present invention provides a method for screening or identifying fragrance plants to detect the expression level of BADH2 gene and/or its protein in the plant, wherein the BADH2 gene includes the BADH2b gene.
  • the BADH2 gene also includes the BADH2a gene.
  • the detection parts of the detection plant include callus, fruit, seed, flower, stem, leaf, ear, and root of the plant.
  • the tenth aspect of the present invention provides a method for imparting fragrance to plants, including the steps:
  • the BADH2 gene includes BADH2a and BADH2b genes; the plant is corn.
  • the reduction or inhibition of the expression and/or activity of the BADH2 gene or its protein is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, Inhibitors for introducing genes or proteins, or a combination thereof.
  • Figure 1 shows the editing mode of the fragrance gene of the C43 plant under the background of Zheng 58, where the two genes A, ZmBADH2a and ZmBADH2b are edited in the DNA sequence; the two genes B, ZmBADH2a and ZmBADH2b are edited in the cDNA sequence; C , The edited protein sequence of the two proteins ZmBADH2a and ZmBADH2b.
  • the blue mark is the same amino acid sequence as Zheng 58, and the red mark is the amino acid sequence of frameshift mutation.
  • Zheng 58 Maize inbred line Zheng 58
  • C43/Zheng 58 gene-edited plant C43 under the background of Zheng 58.
  • Figure 2 shows the editing method of the fragrance gene of the P64 plant under the background of Zheng 58.
  • the edited form of ZmBADH2a gene in DNA sequence B, the edited form of ZmBADH2a gene in cDNA sequence
  • C the deduced protein sequence of ZmBADH2a after editing.
  • the blue mark is the same amino acid sequence as Zheng 58, and the red mark is the amino acid sequence of frameshift mutation.
  • Zheng 58 Maize inbred line Zheng 58
  • P64/Zheng 58 gene-edited plant P64 under the background of Zheng 58. .
  • Figure 3 shows the content of 2AP in C43 and P64 seeds in the background of Zheng 58; among them, 1 is C43/Zheng 58, 2 is P64/Zheng 58, 3 is unedited Zheng 58, and the gray dotted line in the figure indicates Peak time of 2AP.
  • Figure 4 shows the editing mode of the fragrance gene of the P302 plant under the background of XCW175, where the two genes A, ZmBADH2a and ZmBADH2b are edited in the DNA sequence; the two genes B, ZmBADH2a and ZmBADH2b are edited in the cDNA sequence; C, The edited protein sequence of the two proteins ZmBADH2a and ZmBADH2b.
  • the blue mark is the same amino acid sequence as Zheng 58, and the red mark is the amino acid sequence of frameshift mutation.
  • XCW175 corn inbred line XCW175
  • P302/XCW175 gene-edited plant P302 under the background of XCW175.
  • Figure 5 shows the 2AP content of P302 plants in the XCW175 background.
  • the gray dashed line in the figure indicates the peak time of 2AP, where 1 is P302 and 2XCW175.
  • Figure 6 shows the editing mode of the fragrance gene of Z82 plant under the background of Zheng 58; (A) the edited DNA sequence of Zm00001d050339; (B) the edited cDNA sequence of Zm00001d050339; (C) the deduced protein sequence of Zm00001d050339 after editing.
  • Zheng 58 Maize inbred line Zheng 58; Z82/Zheng 58: gene-edited plant Z82 under the background of Zheng 58.
  • Figure 7 shows the editing mode of the fragrance gene of Z10 plant under the background of Zheng 58; (A) the edited DNA sequence of Zm00001d032257; (B) the edited cDNA sequence of Zm00001d032257; (C) the deduced protein sequence of Zm00001d032257 after editing.
  • Zheng 58 Maize inbred line Zheng 58; Z10/Zheng 58: gene-edited plant Z10 under the background of Zheng 58.
  • Figure 8 shows the editing mode of the fragrance gene of the Z54 plant under the background of Zheng 58;
  • A the editing form of the two genes Zm00001d050339 and Zm00001d032257 in the DNA sequence;
  • B the editing form of the two genes Zm00001d050339 and Zm00001d032257 in the cDNA sequence;
  • C The deduced protein sequence of the two proteins Zm00001d050339 and Zm00001d032257 after editing.
  • Zheng 58 Maize inbred line Zheng 58;
  • Z54/Zheng 58 gene-edited plant Z54 under the background of Zheng 58.
  • Figure 9 shows the content of 2AP in seeds of Z10, Z82 and Z54 under the background of Zheng 58.
  • the gray dashed line in the figure indicates the peak time of 2AP.
  • Figure 10 shows the editing mode of the fragrance gene of the X651 plant under the background of XCW175; (A) the edited DNA sequence of Zm00001d050339; (B) the edited cDNA sequence of Zm00001d050339; (C) the deduced protein sequence of Zm00001d050339 after editing.
  • XCW175 maize inbred line XCW175;
  • X651/XCW175 gene-edited plant X651 under the background of XCW175.
  • Figure 11 shows the editing mode of the fragrance gene of the X610 plant under the background of XCW175; (A) the edited DNA sequence of Zm00001d032257; (B) the edited cDNA sequence of Zm00001d032257; (C) the deduced protein sequence of Zm00001d032257 after editing.
  • XCW175 corn inbred line XCW175;
  • X610/XCW175 gene-edited plant X610 under the background of XCW175.
  • Figure 12 shows the editing mode of the fragrance gene of the X447 plant under the background of XCW175;
  • A the edited form of the DNA sequence of the two genes Zm00001d050339 and Zm00001d032257;
  • B the edited form of the cDNA sequence of the two genes Zm00001d050339 and Zm00001d032257;
  • C The deduced protein sequence of the two proteins Zm00001d050339 and Zm00001d032257 after editing.
  • CW175 corn inbred line XCW175;
  • X447/XCW175 gene-edited plant X447 in the background of XCW175.
  • Figure 13 shows the 2AP content of X610, X651 and X447 plants in the XCW175 background.
  • the gray dashed line in the figure indicates the peak time of 2AP.
  • Fig. 14 is a schematic diagram of the gene editing vector used in this embodiment.
  • the present inventors After extensive and in-depth research, the present inventors, through the research and screening of a large number of plant trait sites, unexpectedly discovered for the first time that when simultaneously inhibiting the expression of BADH2a, BADH2b genes or their encoded proteins, the aroma of plants can be significantly enhanced.
  • the inventor completed the present invention on this basis.
  • enhancing plant fragrance includes imparting fragrance to plants without fragrance, and also includes enhancing the fragrance of fragrance plants.
  • the "Cripsr preparation” refers to a combination of various active ingredients that can achieve gene editing effects, including gRNA (guide RNA) or its coding sequence and Cas protein or its coding sequence, and may further include a vector, and Elements that facilitate homologous recombination or gene expression.
  • the term "homology” or “identity” is used to refer to the sequence matching between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine), then each molecule is the same at that position.
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • the comparison method is a conventional method known to those skilled in the art, such as the BLAST algorithm.
  • genetic engineering refers to the modification and utilization of nucleotides that control biological genetic information through manual intervention, so as to obtain new genetic characteristics, or new varieties, or new products, including all disclosed in the art. Genetic modification techniques, such as genetic mutagenesis, genetic modification, or gene editing. Methods of gene mutagenesis include, but are not limited to, physical mutagenesis (such as ultraviolet mutagenesis), chemical mutagenesis (such as acridine dyes), biological mutagenesis (such as virus, phage mutagenesis), and the like.
  • the genetic engineering of the present invention includes using one or more sgRNA-mediated Cas nucleases to perform gene editing on members of the BADH2 gene family.
  • Badh2 is the coding gene of betaine aldehyde dehydrogenase.
  • Betaine aldehyde dehydrogenase has aldehyde dehydrogenase activity and may catalyze the oxidation of betaine aldehyde, 4-aminobutyraldehyde (AB-ald) and 3-aminopropionaldehyde.
  • 4-aminobutyraldehyde is the synthetic precursor of 2-acetyl-1-pyrroline (2AP), and 2AP is closely related to the fragrance of plants.
  • the BADH2 gene family includes BADH2a and BADH2b.
  • BADH2 gene of the present invention includes the BADH2 gene or variants thereof in monocot or dicot plants, such as maize and rice.
  • nucleotide sequence of the BADH2 gene of the present invention is shown in SEQ ID NO.: 3 or 4.
  • the present invention also includes 50% or more of the preferred gene sequence (SEQ ID NO.: 3 or 4) of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% Above, most preferably 98% or more, such as 99%, or 100%) homology of nucleic acids, which can also effectively enhance the fragrance of plants.
  • “Homology” refers to the level of similarity (ie sequence similarity or identity) between two or more nucleic acids according to the percentage of positional identity.
  • the gene variants can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.
  • the nucleotide sequence in SEQ ID NO.: 3 or 4 can be substituted, deleted or added one or more to generate a derivative sequence of SEQ ID NO.: 3 or 4, due to the degeneracy of codons Even if the homology with SEQ ID NO.: 3 or 4 is low, it can basically encode the amino acid sequence shown in SEQ ID NO.: 1 or 2.
  • the nucleotide sequence in SEQ ID NO.: 3 or 4 has been substituted, deleted, or added at least one nucleotide-derived sequence. A nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO.: 3 or 4 under conditions.
  • variants include (but are not limited to): deletions of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , Insertion and/or substitution, and adding several at the 5'and/or 3'end (usually within 60, preferably within 30, more preferably within 10, most preferably within 5 ) Nucleotides.
  • genes provided in the examples of the present invention are derived from maize, those derived from other similar plants have certain similarities with the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO.: 3 or 4).
  • the gene sequence of BADH2 that is sourced (such as 80% or more, such as 85%, 90%, 95%, or even 98%, 99%, or 100% sequence identity) is also included in the scope of the present invention, as long as the art After reading this application, the skilled person can easily isolate the sequence from other plants based on the information provided in this application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 3 or 4 or a degenerate variant.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, More preferably, hybridization occurs when 95% or more occurs.
  • the BADH2 nucleotide full-length sequence of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. It is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • polypeptide of the present invention and “BADH2 gene-encoded protein” can be used interchangeably, and both refer to BADH2 polypeptides derived from plants (such as corn) and variants thereof.
  • a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.: 1 or 2.
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO. 1 or 2 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, The most preferred is 98% or more, such as 99% or 100%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to having betaine aldehyde dehydrogenase activity.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptide of the present invention may be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plant, insect, and mammalian cells
  • the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes BADH2 protein fragments and analogs having BADH2 protein activity.
  • fragment and analogs having BADH2 protein activity refer to polypeptides that substantially maintain the same biological function or activity as the natural BADH2 protein of the present invention.
  • polypeptide fragments, derivatives or analogs of the present invention may be: (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the base may or may not be encoded by the genetic code; or (ii) a polypeptide with a substitution group in one or more amino acid residues; or (iii) the mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, a polypeptide formed by fusion of polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein).
  • these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • the polypeptide variant is the amino acid sequence shown in SEQ ID NO.: 1 or 2, after several (usually 1-60, preferably 1-30, more preferably 1- 20, preferably 1-10) derived sequences obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually within 20, preferably 10) at the C-terminus and/or N-terminus Within 1, more preferably within 5) amino acids.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or terminal usually does not change the function of the protein.
  • the present invention also includes analogs of the claimed protein.
  • the difference between these analogues and the natural SEQ ID NO.: 1 or 2 may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually without changing the primary structure) forms include: in vivo or in vitro chemically derived forms of proteins such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine).
  • the active substance of the present invention (such as an inhibitor of the BADH2 gene or its encoded protein) can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, gas Sprays, natural and synthetic materials impregnated with active substances, microcapsules in polymers, coatings for seeds.
  • compositions can be produced by known methods, for example, by mixing active substances with extenders, which are liquid or liquefied gaseous or solid diluents or carriers, and optionally surfactants, emulsifiers and/or Dispersant and/or foam former.
  • extenders which are liquid or liquefied gaseous or solid diluents or carriers, and optionally surfactants, emulsifiers and/or Dispersant and/or foam former.
  • organic solvents can also be used as additives.
  • a liquid solvent When a liquid solvent is used as a diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons, such as xylene, toluene or alkyl naphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride Or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffin, such as mineral oil fractions; alcohols, such as ethanol or ethylene glycol and their ethers and lipids; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl Ketone or cyclohexanone; or uncommon polar solvents such as dimethylformamide and dimethylsulfoxide, and water.
  • aromatic hydrocarbons such as xylene, toluene or alkyl naphthalene
  • chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzen
  • the diluent or carrier of liquefied gas refers to a liquid that will become a gas at normal temperature and pressure, such as aerosol propellants, such as halogenated hydrocarbons, butane, propane, nitrogen, and carbon dioxide.
  • the solid carrier can be ground natural minerals, such as kaolin, clay, talc, quartz, activated clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals, such as highly dispersed silicic acid, alumina and silicate. .
  • the solid carrier for the particles is crushed and classified natural zircon, such as calcite, marble, pumice, sepiolite and dolomite, as well as particles synthesized from inorganic and organic coarse powder, and organic materials such as sawdust, coconut shell, Corn on the cob and tobacco stalk particles.
  • Nonionic and anionic emulsifiers can be used as emulsifiers and/or foam formers.
  • polyoxyethylene-fatty acid esters polyoxyethylene-fatty alcohol ethers, such as alkyl aryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and white Protein hydrolysate.
  • Dispersants include, for example, lignin sulfite waste liquor and methyl cellulose.
  • Binders such as carboxymethyl cellulose and natural and synthetic polymers in powder, granule or emulsion form, such as gum arabic, polyvinyl alcohol and polyvinyl acetate can be used in the formulation.
  • Colorants such as inorganic dyes such as iron oxide, diamond oxide and Prussian blue; organic dyes such as organic dyes such as azo dyes or metal phthalocyanine dyes; and trace nutrients such as iron, manganese, boron, and copper , Cobalt, aluminum and zinc salts.
  • the "agricultural preparation” is usually an agricultural plant growth regulator, which contains the BADH2 gene or an inhibitor of its encoded protein as an active ingredient for improving plant properties (e.g., enhancing plant flavor); and agriculturally acceptable Carrier.
  • the "agriculturally acceptable carrier” is an agrochemically acceptable solvent, suspending agent or excipient used to deliver the active substance of the present invention to plants.
  • the carrier can be liquid or solid.
  • the agriculturally acceptable carrier suitable for the present invention is selected from the group consisting of water, buffer, DMSO, surfactants such as Tween-20, or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.
  • the agricultural formulation of the present invention may include an agricultural composition.
  • the agricultural preparation of the present invention can be used in combination with other substances that enhance plant aroma.
  • the other aroma-enhancing substances may be plant growth regulators known to those skilled in the art.
  • the dosage form of the agricultural preparation of the present invention can be various, as long as the dosage form can make the active ingredient reach the plant body effectively.
  • the preferred agricultural preparation is a spray Agent or solution formulation.
  • the agricultural formulation of the present invention usually contains 0.0001-99 wt%, preferably 0.1-90 wt% of the active ingredient of the present invention, which accounts for the total weight of the agricultural formulation.
  • concentration of the active ingredient of the present invention in the commercial preparation or use dosage form can vary within a wide range.
  • concentration of the active ingredient of the present invention in the commercial preparation or dosage form can be from 0.0000001-100% (g/v), preferably between 0.0001 and 50% (g/v).
  • the present invention also provides a method for improving plant traits.
  • the improvement includes: enhancing the fragrance of the plant, including the step of reducing the expression and/or activity of the BADH2 gene or its encoded protein in the plant, or adding the BADH2 gene Or an inhibitor of its encoded protein.
  • the plants or plant seeds can be treated with other substances that can enhance the aroma of plants by conventional methods, so as to improve the properties of the corresponding plants.
  • the present invention found for the first time that by inhibiting the expression or activity of the BADH2a and BADH2b genes or their encoded proteins, the aroma of plants can be significantly enhanced.
  • the present invention found for the first time that by inhibiting the expression or activity of BADH2a and BADH2b genes or their encoded proteins, the content of fragrance substances (such as 2AP) can be increased.
  • the present invention provides a method for quickly improving the aroma of corn, and the improved corn aroma (2AP content) has a substantial increase compared with the wild type.
  • the present invention creates a new corn germplasm resource, which has important theoretical and practical application significance.
  • the protein sequence of the OsBADH2 gene was obtained through the NCBI (https://www.ncbi.nlm.nih.gov) website, and then after Blasting with the Zein database, two homologous genes were found, which are located on chromosome 1 Zm00001d032257( ZmBADH2b) and Zm00001d050339 (ZmBADH2a) located on chromosome 4.
  • Cas9 and sgRNAs targeting ZmBADH2b and ZmBADH2a are used to edit the above two genes in maize.
  • the specific operation method can be carried out in a conventional manner in the art; in this embodiment, a schematic diagram of the constructed gene editing vector As shown in Figure 14; among them, ZmU6 pro is the U6 promoter, Gly-tRNA is the glycine tRNA, ZmU6 Ter is the terminator, UBI pro is the UBI promoter, and NLS is the nuclear localization signal; vector construction can also refer to references (" High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize", Weiwei Qi et al., "BMC Biotechnology", 2016); in this example, Cas9 uses plant codon-optimized Cas9. In other embodiments, Cas9 optimized in other ways can also be used.
  • targetDesign http://skl.scau.edu.cn/targetdesign/ is used to design gRNA, where ZmBADH2-T1(CGCCAGCGATGGTCCCGCTG(SEQ ID NO .:5)), ZmBADH2-T2 (AGTCGCGGCCACGGTTCCTC (SEQ ID NO.: 6)) located in the second exon region and ZmBADH2-T3 (AATTAGGCTAGAGCAAAGAG (SEQ ID NO.) located in the second intron region.
  • ZmBADH2a gene-specific sequences in which there is a 2-base mismatch between the corresponding sequences of ZmBADH2-T1 and ZmBADH2b, and there is a 2-base mismatch between the corresponding sequences of ZmBADH2-T2 and ZmBADH2b, ZmBADH2-T3 There is a 3-base mismatch with the corresponding sequence of ZmBADH2b.
  • ZmBADH2-T1 was combined with ZmBADH2-T2 and ZmBADH2-T3 to be constructed into the U6 promoter-promoted gene editing vector using glycine tRNA spacer, and the vector P0195 was constructed respectively. And P0196.
  • ZmBADH2-T1 and ZmBADH2-T2 were constructed into the vector P0077, and the two gRNAs were separated by glycine tRNA.
  • ZmBADH2-T4 (GGTTGACGACGGGGAGGCGG (SEQ ID NO.: 8)) located in the first exon region and ZmBADH2-T5 (GCGGCGCTCAAGAGGAACCG (SEQ ID NO.: 9)) located in the second exon region are two The consensus sequence of the gene.
  • These two gRNAs were constructed into a gene editing vector initiated by the U6 promoter using a glycine tRNA spacer to construct the vector P0593.
  • ZmBADH2-T6 (CAGCGGTACCATCGCTTGCG) located in the first exon region and ZmBADH2-T7 (GAGGAACCGCGGCCGCGATT) located in the second exon region are both Zm00001d032257 gene-specific sequences, of which the corresponding sequences of ZmBADH2-T6F and Zm00001d050339 exist There is a two-base mismatch between the corresponding sequences of ZmBADH2-T7R and Zm00001d050339. These two gRNAs were constructed into a gene editing vector initiated by the U6 promoter using a glycine tRNA spacer to construct the vector P1801.
  • ZmBADH2-T1F (TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT (SEQ ID NO.: 10))
  • ZmBADH2-T2R (TAGGTCTCTAAACAGTCGCGGCCACGGTTCCTCTGCACCAGCCGGGAATCG (SEQ ID NO.:11)),ZmBADH2-T1F(TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT(SEQ ID NO.:12)) and ZmBADH2-T3R (TAGGTCTCTAAACAATTAGGCTAGAGCAAAGAGTGCACCAGCCGGGAATCG (SEQ ID NO.:13)),ZmBADH2-T4F(TAGGTCTCTTGCAGGTTGACGACGGGGAGGCGGGTTTTAGAGCTAGAAATAGCAAGT (SEQ ID NO.: 14)) and ZmBADH2-T5R (TAGGTCTCTAAACGCGGCGCTCAAGAGGAACCG TGGTGCACCAGCCGGGAATCG (SEQ ID NO.: 15)) and ZmBADH2-T5R (TAGGTCTCTAAACGCGGCGCTCAAG
  • plasmid P0055 As a template for amplification, the fragments of about 200 bp were recovered by tapping respectively as ZmBADH2-T1&2, ZmBADH2-T1&3 and ZmBADH2-T4&5. ZmBADH2-T6&7 was used to digest the recovered fragments with BsaI and recovered with the kit;
  • the plasmid P0055 was digested with BsaI, and the liquid was recovered through the kit, and then ligated with the ZmBADH2-T1&3 that had been digested with BsaI. After the plasmid was amplified, the plasmid was double-cut with EcoRV and PstI, and the gel was tapped The 1020bp fragment was recovered; the P0185 plasmid was double digested with SmaI and PstI to recover the vector fragment; the 1020bp DNA fragment and the P0185 vector were ligated with T4DNA ligase to construct the final vector P0077.
  • the vector in 1 was transferred into Agrobacterium strain EHA105 by heat shock method, and the single clone was picked, cultured in liquid and identified by PCR, and stored in the refrigerator at -80°C for later use.
  • the immature embryos were transferred to a recovery medium, with 30-40 immature embryos per dish, cultured in the dark at 25°C for 10-14 days, and the immature embryos transformed with the P0077 carrier were restored and cultured for 7 days.
  • the immature embryos transferred with P0077 vector are transferred to the screening medium with the corresponding selection pressure of bialaphos. After 14 days of dark culture at 25-28°C, change to the screening medium with a higher selection pressure. Cultivate in the dark at 25 ⁇ 28°C for 14 days.
  • the immature embryos transformed with P0195, P0196, P0593, and P1801 do not need callus selection stage.
  • the resistant callus obtained by the transformation of P0077 vector is put on the differentiation medium for differentiation, clamped into small pieces and then spread flat on the medium, cultivated in the dark at 25-28°C, and the medium is changed once every 14 days.
  • the embryos transfected with P0195, P0196, P0593, and P1801 vectors were directly differentiated on the differentiation medium.
  • primer pairs ZmBADH2a-73F (GAGACGTCCTCGCTTTCCAC (SEQ ID NO.: 34)) and ZmBADH2a-904R (ATGTGCACGCTGCGTTTTAC (SEQ ID NO.: 35)) were used to amplify the corresponding fragments of the ZmBADH2a gene
  • primer pairs BADH2b-jc -F1 (GAAGTCCACTGCCGAGTTGC (SEQ ID NO.: 36)
  • BADH2b-jc-R1 CGACTGAGTTGTCTCACACTGA (SEQ ID NO.: 37)
  • the amplified product was sequenced by the sanger method to confirm the editing form. If the sequencing result showed double peaks, the PCR product was ligated into the T vector, and 5 clones were selected for sequencing to confirm the editing form.
  • the edited plant C43 was obtained by transforming the immature embryo of Zheng 58 with P0077.
  • the editing result was that an A was inserted after +24 nt of ZmBADH2a CDS, which caused a frameshift mutation in the subsequent coding region and formed a new CDS at 265-267 nt.
  • the stop codon TGA will lead to the premature termination of protein translation; deleting a base G at CDS+27nt of ZmBADH2b leads to a frameshift mutation afterwards, and a stop codon TAA is formed at 262-264nt of the new CDS , Leading to the premature termination of protein translation. See Figure 1 (A-C) for specific editing methods.
  • the immature embryos of Zheng 58 were transformed with P0077 to obtain the edited plant P64. Due to the editing of the edited plant P64, a C was inserted after +24 nt of ZmBADH2a CDS, resulting in a frameshift mutation in the subsequent coding region ( Figure 2, AC), and The formation of a stop codon TGA at 265-267nt of the new CDS will result in the premature termination of protein translation; however, the ZmBADH2b gene in the P64 plant has not been edited. Refer to Figure 2 for the specific editing method.
  • the C43 plant has both ZmBADH2a and ZmBADH2b genes edited at the same time, while the P64 plant only has the ZmBADH2a gene edited.
  • the content of 2AP in the seed extracts of C43 and P64 plants was analyzed by mass spectrometry; as shown in Figure 3, the content of 2AP in the seeds of C43 plant was 0.648 mg/kg, which can produce fragrance, while Zheng 58 Wild No 2AP was detected in the seeds of P64 type and P64 plants, and no fragrance was produced (Figure 3).
  • P0593 was used to transform the immature embryos of waxy corn inbred line XCW175 to obtain edited plant P302.
  • the edited plant was edited at two positions of ZmBADH2a and ZmBADH2b respectively.
  • the editing result was that a C and a C were inserted after +77nt of ZmBADH2a CDS. Insert an A after +188nt, the protein deduced from the edited CDS starts to undergo a frameshift mutation at amino acid 27, and a stop codon TGA is formed at 262-264nt of the new CDS, which will cause the premature termination of protein translation. ; Insert AA at +80nt of ZmBADH2b CDS and a deletion of A at +191nt.
  • the protein deduced from CDS after editing begins to undergo a frameshift mutation at amino acid 27, and a stop code is formed at 265-267nt of the new CDS
  • the daughter TAA will cause the premature termination of protein translation. See Figure 4 (A-C) for specific editing methods.
  • the 2AP content of P302 seed extract by mass spectrometry analysis is 0.266mg/kg, and its seeds can produce fragrance.
  • XCW175 wild type does not produce fragrance, and 2AP cannot be detected (Figure 5).
  • P0196 was used to transform the immature embryos of waxy corn inbred line N355 to obtain edited plant 282, which was selfed and three edited plants were identified as 288-4, 288-5 and 288-6 in its progeny plants.
  • the three strains were homozygously edited in both ZmBADH2a and ZmBADH2b, and the editing methods were the same.
  • the editing method is: inserting an A after +24nt of ZmBADH2a CDS, resulting in a frameshift mutation in the subsequent coding region, and forming a stop codon TGA at 265-267nt of the new CDS, which will lead to the premature termination of protein translation.
  • Z10 was edited only in Zm00001d032257 (ZmBADH2b), and its editing resulted in a deletion of 188 bp from +15nt to +203nt in the CDS of Zm00001d032257, resulting in a frameshift mutation in the subsequent coding region ( Figure 7B), and in the 79- of the new CDS.
  • the seed extracts of Z10, Z82, and Z54 were analyzed by mass spectrometry for the 2AP content of aroma substances. It was found that no 2AP was detected in the seeds of wild type, Z10 and Z82, while the 2AP content of Z54 seeds was 0.288mg/kg, and could produce Scent (Figure 9).
  • P0593 was used to transform the immature embryos of waxy corn inbred line XCW175 to obtain edited plants X651 ( Figure 10A), X610 ( Figure 11A) and X447 ( Figure 12A).
  • X651 was edited only in Zm00001d050339, and its editing resulted in the deletion of 2bp in Zm00001d050339 CDS 187-188nt ( Figure 10B), and the formation of a stop codon TGA at 262-264nt of the new CDS, leading to the premature termination of protein translation (Figure 10C);
  • X610 Editing only occurred in Zm00001d032257 which resulted in a deletion of an A at +191nt of CDS in Zm00001d032257, resulting in a frameshift mutation in the subsequent coding region ( Figure 11B), and the formation of a stop codon TAA at 262-264nt of the new CDS.

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Abstract

提供了一种增强植物香味的方法，还提供了BADH2基因或其编码蛋白的抑制剂的用途，用于增强植物的香味；或制备用于增强植物香味的组合物或制剂，其中所述BADH2基因包括BADH2a和BADH2b基因。抑制BADH2基因或其编码蛋白的表达可显著增强植物，尤其是玉米的香味。

Description

一种增强植物香味的方法 技术领域
本发明涉及农学领域，具体地，涉及一种增强植物香味的方法。
背景技术
香稻以其独特浓郁的香味备受消费者的青睐，因此香味成为水稻一个重要的品质性状指标。香米的价格是普通大米的2-3倍，而产自印度和巴基斯坦的BASMATI香米价格更是要高出许多。香稻的挥发性香味物质包含100多种化合物，其中2-乙酰基-1-吡咯啉(2-acetyl-1-pyrroline,2AP)是最主要的香味物质。
我国每年有6亿多亩的玉米种植面积，其中约有两千万亩的鲜食玉米种植面积，玉米种子的市值每年为200亿元左右。在目前的各类报道中，无论是普通籽粒玉米还是鲜食玉米中都没有发现香味玉米的种质。因此，结合现有报道的生物技术，改进玉米香味，提高玉米的附加值，对于丰富玉米种质资源、扩大玉米种植面积、增加玉米适用范围具有重要意义。
因此，本领域迫切需要开发一种新的增强植物香味的方法。
发明内容
本发明的目的在于提供一种新的增强植物香味的方法。
本发明第一方面提供了一种增强植物香味或者赋予植物产生香味的方法，包括步骤：
降低或抑制所述植物中BADH2基因或其编码蛋白的表达量和/或活性，从而增强植物的香味，其中，所述BADH2基因包括BADH2b基因。
在另一优选例中，所述BADH2基因还包括BADH2a基因。
在另一优选例中，所述BADH2基因包括BADH2b和BADH2a基因。
在另一优选例中，所述方法包括给予植物BADH2基因或其编码蛋白的抑制剂。
在另一优选例中，所述增强植物的香味包括提高所述植物的4-氨基丁醛或2-乙酰基-1-吡咯啉(2AP)的含量。
在另一优选例中，所述增强植物的香味为提高所述植物的4-氨基丁醛或2-乙酰基-1-吡咯啉(2AP)的含量。
在另一优选例中，所述植物包括农作物、林业植物、蔬菜、瓜果、花卉、牧草(包括草坪草)。
在另一优选例中，所述的植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物选自下组：禾本科、豆科、藜科、十字花科植物、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、烟草、玉米、高粱、大麦、小麦、小米、大豆、番茄、马铃薯、藜麦、生菜、油菜、白菜、菠菜、甜菜、草莓、或其组合。
在另一优选例中，所述植物选自玉米。
在另一优选例中，所述植物组织或植物细胞中BADH2基因或其编码蛋白的表达量或活性降低了≥30％，≥50％，≥70％，更佳地，≥80％。
在另一优选例中，所述“降低或抑制”是指BADH2基因或其编码蛋白的表达或活性降低满足以下条件：
使所述植物中BADH2基因或其编码蛋白完全失去活性或者失去部分活性，或者，A1/A0的比值≤80％，较佳地≤60％，更佳地≤40％，最佳地为0-30％；其中，A1为所述植物中BADH2基因或其编码蛋白的表达或活性；A0为野生型同种类型植物中相同BADH2基因或其编码蛋白的表达或活性。
在另一优选例中，所述的降低或抑制指与野生型植株中BADH2基因或其编码蛋白的表达水平E0相比，所述植株中BADH2基因或其编码蛋白的表达水平E1为野生型的0-80％，较佳地0-60％，更佳地0-40％，更佳地，0-30％。
在另一优选例中，所述降低或抑制BADH2基因或其蛋白的表达和/或活性通过选自下组的方法实现：基因突变、基因敲除、基因中断、RNA干扰技术、基因编辑技术、导入基因或蛋白的抑制剂、或其组合。
在另一优选例中，所述的基因突变通过以下一种或多种方法获得：自然变异、物理诱变(如紫外线诱变、X射线或Y射线诱变)、化学诱变(如亚硝酸、羟胺、EMS、亚硝基胍等)、生物诱变(如病毒或细菌介导的诱变)、基因编辑或生物合成。
在另一优选例中，所述的突变区域包括外显子和/或内含子区。
在另一优选例中，所述抑制剂选自下组：反义核酸、抗体、小分子化合物、Crispr试剂、小分子配体、或其组合。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR技术、TALEN技术、 ZFN技术、或其组合。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将BADH2基因或其编码蛋白的抑制剂导入所述植物或植物细胞，从而获得改造的植物或植物细胞。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)利用靶向BADH2基因的gRNA以及相应的Cas蛋白导入所述植物或植物细胞；在优选的实施方式中，向所述植物或植物细胞中导入含有所述gRNA和Cas蛋白的表达载体。
在另一优选例中，所述BADH2基因包括野生型BADH2基因和突变型BADH2基因。
在另一优选例中，所述的突变型包括突变后编码蛋白的功能未发生改变的突变形式(即功能与野生型编码蛋白相同或基本相同)。
在另一优选例中，所述的突变型BADH2基因编码的多肽与野生型BADH2基因所编码的多肽相同或基本相同。
在另一优选例中，所述的突变型BADH2基因包括与野生型BADH2基因相比，同源性≥80％(较佳地≥90％，更佳地≥95％，更佳地，≥98％或99％)的多核苷酸。
在另一优选例中，所述的突变型BADH2基因包括在野生型BADH2基因的5＇端和/或3＇端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸。
在另一优选例中，所述的BADH2基因包括cDNA序列、CDS序列、基因组序列、或其组合。
在另一优选例中，所述BADH2基因来源于选自下组的一种或多种植物：禾本科、豆科、藜科、十字花科植物。
在另一优选例中，所述的BADH2基因来源于选自下组的一种或多种植物：拟南芥、水稻、烟草、玉米、高粱、大麦、小麦、小米、大豆、番茄、马铃薯、藜麦、生菜、油菜、白菜、菠菜、甜菜、草莓。
在另一优选例中，所述BADH2基因来源于玉米。
在另一优选例中，所述BADH2a基因的编码蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:1所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:1所示的氨基酸序列经过一个或几个(如1-10个)氨基酸 残基的取代、缺失或添加而形成的，具有所述相同或相近功能(甜菜碱醛脱氢酶活性)由(i)衍生的多肽；
或(iii)氨基酸序列与SEQ ID NO.:1所示氨基酸序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％或100％)的同源性、具有相同或相近功能的多肽。
在另一优选例中，所述BADH2b基因的编码蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:2所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述相同或相近功能(甜菜碱醛脱氢酶活性)由(i)衍生的多肽；
或(iii)氨基酸序列与SEQ ID NO.:2所示氨基酸序列有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％或100％)的同源性、具有相同或相近功能的多肽。
在另一优选例中，所述BADH2a基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:1所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:3所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:3所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
(d)在SEQ ID NO.:3所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述BADH2b基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:2所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:4所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:4所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
(d)在SEQ ID NO.:4所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述降低或抑制所述BADH2b基因或其编码蛋白的表达量和/或活性通过对BADH2b基因进行突变来实现。
在另一优选例中，所述降低或抑制所述BADH2b基因以及BADH2a基因或其编码蛋白的表达量和/或活性通过对BADH2b基因和BADH2a基因同时进行突变来实现。
在另一优选例中，所述突变包括插入突变、缺失突变、移码突变、取代突变。
在另一优选例中，所述方法还包括步骤：测试植物中香味物质含量的增加量。
在另一优选例中，所述植物中的香味物质的含量为0.01-20mg/kg，较佳地，0.05-10mg/kg，更佳地，0.1-5mg/kg，更佳地，0.1-2mg/kg，更佳地，0.2-1mg/kg。
在另一优选例中，所述香味物质包括2AP。
本发明第二方面提供了一种BADH2基因或其编码蛋白的抑制剂的用途，用于增强植物的香味或者用于赋予植物产生香味；或制备用于增强植物香味的组合物或制剂或制备用于赋予植物产生香味的组合物或制剂，其中所述BADH2基因包括BADH2b基因。
在另一优选例中，所述BADH2基因还包括BADH2a基因。
在另一优选例中，所述BADH2基因包括BADH2b和BADH2a基因。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述制剂包括农用制剂。
在另一优选例中，所述增强植物的香味包括提高植物中香味物质的含量。
在另一优选例中，所述香味物质包括2AP。
在另一优选例中，所述组合物包含(a)BADH2基因或其编码蛋白的抑制剂，其中所述BADH2基因包括BADH2b基因；和(b)农学上可接受的载体。
在另一优选例中，所述组合物还包括BADH2a基因。
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述抑制剂选自下组：反义核酸、抗体、小分子化合物、Crispr试剂、小分子配体、或其组合。
在另一优选例中，所述反义核酸选自下组：反义RNA、反义DNA、干扰RNA、核酶、或其组合。
在另一优选例中，所述干扰RNA选自下组：siRNA、shRNA、RNAi、miRNA、dsRNA、hpRNA、ihpRNA、或其组合。
在另一优选例中，所述组合物还包括其他增强农作物的香味的物质。
本发明第三方面提供了一种用于增强植物香味或赋予植物产生香味的组合物，包括：
(a)BADH2基因或其编码蛋白的抑制剂，所述BADH2基因包括BADH2b基因；和
(b)农学上可接受的载体。
在另一优选例中，所述组合物还包括BADH2a基因。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述组合物的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述组合物中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(a)，以所述组合物的总重量计。
在另一优选例中，所述抑制剂选自下组：基因编辑试剂、反义核酸、抗体、小分子化合物、Crispr试剂、小分子配体、或其组合。
在另一优选例中，所述反义核酸选自下组：反义RNA、反义DNA、干扰RNA、核酶、或其组合。
在另一优选例中，所述干扰RNA选自下组：siRNA、shRNA、RNAi、miRNA、dsRNA、hpRNA、ihpRNA、或其组合。
在另一优选例中，所述组合物还包括其他增强植物的香味的物质。
本发明第四方面提供了一种本发明第三方面所述的组合物的用途，用于增强植物的香味或赋予植物产生香味。
本发明第五方面提供了一种制备基因工程的植物组织或植物细胞的方法，包括步骤：
降低或抑制植物组织或植物细胞中的BADH2基因或其编码蛋白的表达和/或活性，从而获得基因工程的植物组织或植物细胞。在另一优选例中，所述降低BADH2基因或其蛋白的表达和/或活性通过选自下组的方法实现：基因突变、基因敲除、基因中断、RNA干扰技术、基因编辑技术、导入基因或蛋白抑制剂、或其组合。
本发明第六方面提供了一种制备基因工程植物的方法，包括步骤：
将本发明第五方面所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得基因工程植物。
本发明第七方面提供了一种改良植物的方法，所述的方法包括步骤：
(a)提供一植物细胞、植物组织、植物部分，向所述植物细胞、植物组织、植物部分中导入BADH2基因或其编码蛋白的抑制剂；或者，降低或抑制植物细胞、植物组织、植物部分中的BADH2基因或其编码蛋白的表达和/或活性；其中所述BADH2基因包括BADH2b基因；
(b)将步骤(a)中的植物细胞、植物组织、植物部分再生成植株。
在另一优选例中，在步骤(a)中，利用基因编辑技术改造所述植物细胞、植物组织、植物部分，从而使所述植物细胞、植物组织、植物部分中的BADH2基因或其编码蛋白的表达或活性降低。
在另一优选例中，所述BADH2基因还包括BADH2a基因。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN。
在另一优选例中，所述方法用于增强植物的香味。
在另一优选例中，所述方法用于提高香味物质的含量。
在另一优选例中，所述方法还包括步骤：对所述植物细胞、植物组织、植物部分或植物，测试香味物质含量的增加量。
在另一优选例中，所述改良植物为增强/提高植物的香味或赋予植物产生香味。
本发明第八方面提供了一种基因工程植物，所述的植物是用本发明第七方面所述的方法制备的。
本发明第九方面提供了一种筛选或鉴定香味植物的方法，检测植物中BADH2基因和/或其蛋白的表达量，其中所述BADH2基因包括BADH2b基因。
在另一优选例中，所述BADH2基因还包括BADH2a基因。
在另一优选例中，所述检测植物的检测部位包括植物的愈伤组织、果实、种子、花、茎、叶、穗、根。
本发明第十方面提供了一种赋予植物产生香味的方法，包括步骤：
降低或抑制所述植物中BADH2基因或其编码蛋白的表达量和/或活性，从而赋予植物产生香味，其中，所述BADH2基因包括BADH2a和BADH2b基因；所述植物为玉米。
在另一优选例中，所述降低或抑制BADH2基因或其蛋白的表达和/或活性通过选自下组的方法实现：基因突变、基因敲除、基因中断、RNA干扰技术、基因 编辑技术、导入基因或蛋白的抑制剂、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了郑58背景下的C43植株的香味基因的编辑方式，其中A,ZmBADH2a和ZmBADH2b两个基因在DNA序列的编辑形式；B,ZmBADH2a和ZmBADH2b两个基因在cDNA序列的编辑形式；C，编辑后的ZmBADH2a和ZmBADH2b两个蛋白的推导的蛋白序列。蓝色标记的为与郑58相同的氨基酸序列，红色标记的为移码突变的氨基酸序列。郑58：玉米自交系郑58；C43/郑58：郑58背景下的基因编辑植株C43。
图2显示了郑58背景下的P64植株的香味基因的编辑方式。ZmBADH2a基因在DNA序列的编辑形式；B,ZmBADH2a基因在cDNA序列的编辑形式；C，编辑后的ZmBADH2a推导蛋白序列。蓝色标记的为与郑58相同的氨基酸序列，红色标记的为移码突变的氨基酸序列。郑58：玉米自交系郑58；P64/郑58：郑58背景下的基因编辑植株P64。。
图3显示了郑58背景下的C43和P64种子中2AP的含量；其中，1为C43/郑58，2为P64/郑58，3为未编辑的郑58，图中灰色虚线所指示的为2AP的出峰时间。
图4显示了XCW175背景下的P302植株的香味基因的编辑方式，其中A,ZmBADH2a和ZmBADH2b两个基因在DNA序列的编辑形式；B,ZmBADH2a和ZmBADH2b两个基因在cDNA序列的编辑形式；C，编辑后的ZmBADH2a和ZmBADH2b两个蛋白的推导的蛋白序列。蓝色标记的为与郑58相同的氨基酸序列，红色标记的为移码突变的氨基酸序列。XCW175：玉米自交系XCW175；P302/XCW175：XCW175背景下的基因编辑植株P302。
图5显示了XCW175背景下的P302植株的2AP的含量。图中灰色虚线所指示的为2AP的出峰时间，其中，1为P302，2XCW175。
图6显示了郑58背景下的Z82植株的香味基因的编辑方式；(A)Zm00001d050339的DNA序列编辑形式；(B)Zm00001d050339的cDNA序列编辑 形式；(C)编辑后的Zm00001d050339的推导蛋白序列。郑58：玉米自交系郑58；Z82/郑58：郑58背景下的基因编辑植株Z82。
图7显示了郑58背景下的Z10植株的香味基因的编辑方式；(A)Zm00001d032257的DNA序列编辑形式；(B)Zm00001d032257的cDNA序列编辑形式；(C)编辑后的Zm00001d032257的推导蛋白序列。郑58：玉米自交系郑58；Z10/郑58：郑58背景下的基因编辑植株Z10。
图8显示了郑58背景下的Z54植株的香味基因的编辑方式；(A)Zm00001d050339和Zm00001d032257两个基因在DNA序列的编辑形式；(B)Zm00001d050339和Zm00001d032257两个基因在cDNA序列的编辑形式；(C)编辑后的Zm00001d050339和Zm00001d032257两个蛋白的推导的蛋白序列。郑58：玉米自交系郑58；Z54/郑58：郑58背景下的基因编辑植株Z54。
图9显示了郑58背景下的Z10、Z82和Z54种子中2AP的含量。图中灰色虚线所指示的为2AP的出峰时间。
图10显示了XCW175背景下的X651植株的香味基因的编辑方式；(A)Zm00001d050339的DNA序列编辑形式；(B)Zm00001d050339的cDNA序列编辑形式；(C)编辑后的Zm00001d050339的推导蛋白序列。XCW175：玉米自交系XCW175；X651/XCW175：XCW175背景下的基因编辑植株X651。
图11显示了XCW175背景下的X610植株的香味基因的编辑方式；(A)Zm00001d032257的DNA序列编辑形式；(B)Zm00001d032257的cDNA序列编辑形式；(C)编辑后的Zm00001d032257的推导蛋白序列。XCW175：玉米自交系XCW175；X610/XCW175：XCW175背景下的基因编辑植株X610。
图12显示了XCW175背景下的X447植株的香味基因的编辑方式；(A)Zm00001d050339和Zm00001d032257两个基因在DNA序列的编辑形式；(B)Zm00001d050339和Zm00001d032257两个基因在cDNA序列的编辑形式；(C)编辑后的Zm00001d050339和Zm00001d032257两个蛋白的推导的蛋白序列。CW175：玉米自交系XCW175；X447/XCW175：XCW175背景下的基因编辑植株X447。
图13显示了XCW175背景下的X610、X651和X447植株的2AP的含量。图中灰色虚线所指示的为2AP的出峰时间。
图14为本实施方式中利用的基因编辑载体示意图。
具体实施方式
经过广泛而深入的研究，本发明人通过对大量的植物性状位点的研究和筛选，首次意外地发现，当同时抑制BADH2a、BADH2b基因或其编码蛋白的表达时，可显著增强植物的香味。本发明人在此基础上完成了本发明。
除非本申请定义，本发明中所使用的科学术语或专业名词具有本领域技术人员所理解的含义，当本领域技术人员理解的含义与本申请所定义的含义出现矛盾时，以本申请所定义的含义为准。
如本文所用，所述“增强植物香味”含义包括赋予没有香味的植物以香味，也包括提高香味植物的香味。
如本文所用，所述“Cripsr制剂”是指可以实现基因编辑效果的各有效成分的组合，包括gRNA(向导RNA)或其编码序列和Cas蛋白或其编码序列，还可以进一步包括载体，以及有利于同源重组或基因表达的元件。
术语“同源性”或“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间。通常，在将两个序列比对以产生最大同一性时进行比较。比对方法为本领域技术人员已知的常规方法，比如BLAST运算法则。
术语“基因工程”是指通过人工干预的方式对控制生物遗传信息的核苷酸进行改造和利用，从而获得新的遗传特性、或新品种、或新产品的技术，包括本领域所公开的所有基因改造技术，如基因诱变、转基因或基因编辑等方法。基因诱变的方法包括但不限于物理诱变(比如紫外线诱变)、化学诱变(比如吖啶类染料)、生物诱变(如病毒、噬菌体诱变)等。在一优选实施方式中，本发明的基因工程改造包括用一个或多个sgRNA介导的Cas核酸酶对BADH2基因家族的成员进行基因编辑。
BADH2基因
Badh2是甜菜碱醛脱氢酶的编码基因，甜菜碱醛脱氢酶具有醛脱氢酶活性，可能催化甜菜碱醛、4-氨基丁醛(AB-ald)和3-氨基丙醛的氧化，而4-氨基丁醛是2-乙酰基-1-吡咯啉(2AP)的合成前体，2AP与植物香味密切相关。在本发明中，BADH2基因家族包括BADH2a、BADH2b。
如本文所用，术语“本发明的BADH2基因”包括单子叶或双子叶植物，如 玉米、水稻中的BADH2基因或其变体。在一优选实施方式中，本发明的BADH2基因的核苷酸序列如SEQ ID NO.:3或4所示。
>Zm00001d050339(ZmBADH2a)基因组DNA序列：
Figure PCTCN2021097340-appb-000001
Figure PCTCN2021097340-appb-000002
>Zm00001d032257(ZmBADH2b)基因组DNA序列：
Figure PCTCN2021097340-appb-000003
Figure PCTCN2021097340-appb-000004
Figure PCTCN2021097340-appb-000005
Figure PCTCN2021097340-appb-000006
本发明还包括与本发明的优选基因序列(SEQ ID NO.:3或4)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％，或100％)同源性的核酸，所述核酸也能有效地增强植物的香味。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。在本文中，所述基因的变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。
在本发明中，SEQ ID NO.:3或4中的核苷酸序列可以经过取代、缺失或添加一个或多个，生成SEQ ID NO.:3或4的衍生序列，由于密码子的简并性，即使与SEQ ID NO.:3或4的同源性较低，也能基本编码出如SEQ ID NO.:1或2所示的氨基酸序列。另外，“在SEQ ID NO.:3或4中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO.:3或4所示的核苷酸序列杂交的核苷酸序列。这些变异形式包括(但并小限于)：若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、插入和/或取代，以及在5’和/或3’端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。
应理解，尽管本发明的实例中提供的基因来源于玉米，但是来源于其它类 似的植物的、与本发明的序列(优选地，序列如SEQ ID NO.:3或4所示)具有一定同源性(如具有80％以上，如85％,90％,95％甚至98％，99％，或100％序列相同性)的BADH2的基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该序列。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:3或4所示的编码区序列相同或者是简并的变异体。
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酞胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的BADH2核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按 正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
BADH2基因编码的多肽
如本文所用，术语“本发明多肽”、“BADH2基因的编码蛋白”、可以互换使用，都是指来源于植物(如玉米)的BADH2的多肽及其变体。在一优选实施方式中，本发明多肽的一种典型的氨基酸序列如SEQ ID NO.:1或2所示。
>Zm00001d050339(ZmBADH2a)蛋白序列：
Figure PCTCN2021097340-appb-000007
>Zm00001d032257((ZmBADH2b)蛋白序列：
Figure PCTCN2021097340-appb-000008
本发明还包括与本发明的SEQ ID NO.:1或2所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％或100％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指具有甜菜碱醛脱氢酶活性。
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有BADH2蛋白活性的BADH2蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然BADH2蛋白相同的生物学功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是：(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的；或(ii)在一个或多个氨基酸残基中具有取代基团的多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明中，所述的多肽变体是如SEQ ID NO.:1或2所示的氨基酸序列，经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表1进行替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO.:1或2差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。
农用制剂
可将本发明的活性物质(如BADH2基因或其编码蛋白的抑制剂)以常规的方法制备成农用制剂，例如溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、用活性物质浸渍的天然的和合成的材料、在多聚物中的微胶囊、用于种子的包衣剂。
这些制剂可用已知的方法生产，例如，将活性物质与扩充剂混合，这些扩充剂就是液体的或液化气的或固体的稀释剂或载体，并可任意选用表面活性剂即乳化剂和/或分散剂和/或泡沫形成剂。例如在用水作扩充剂时，有机溶剂也可用作助剂。
用液体溶剂作稀释剂或载体时，基本上是合适的，如：芳香烃类，例如二甲苯，甲苯或烷基萘；氯化的芳香或氯化的脂肪烃类，例如氯苯，氯乙烯或二氯甲烷；脂肪烃类，例如环己烷或石蜡，例如矿物油馏分；醇类，例如乙醇或乙二醇以及它们的醚和脂类；酮类，例如丙酮，甲乙酮，甲基异丁基酮或环已酮；或不常用的极性溶剂，例如二甲基甲酰胺和二甲基亚砜，以及水。
就液化气的稀释剂或载体说，指的是在常温常压下将成为气体的液体，例如气溶胶推进剂，如卤化的烃类以及丁烷，丙烷，氮气和二氧化碳。
固体载体可用研磨的天然矿物质，例如高岭土，粘土，滑石，石英，活性白土，蒙脱土，或硅藻土，和研磨合成的矿物质，例如高度分散的硅酸，氧化铝和硅酸盐。供颗粒用的固体载体是碾碎的和分级的天然锆石，例如方解石，大理石，浮石，海泡石和白云石，以及无机和有机粗粉合成的颗粒，和有机材料例如锯木屑，椰子壳，玉米棒子和烟草梗的颗粒等。
非离子的和阴离子的乳化列可用作乳化剂和/或泡沫形成剂。例如聚氧乙烯-脂肪酸酯类，聚氧乙烯-脂肪醇醚类，例如烷芳基聚乙二醇醚类，烷基磺酸酯类，烷基硫酸酯类，芳基磺酸酯类以及白蛋白水解产物。分散剂包括，例如木质素亚硫酸盐废液和甲基纤维素。
在制剂中可以用粘合剂，例如羧甲基纤维素和以粉末，颗粒或乳液形式的天然和合成的多聚物，例如阿拉伯胶，聚乙烯基醇和聚乙烯醋酸酯。
可以用着色剂例如无机染料，如氧化铁，氧化钻和普鲁士蓝；有机染料，如有机染料，如偶氮染料或金属钛菁染料；和用痕量营养剂，如铁，猛，硼，铜，钴，铝和锌的盐等。
在本发明中，所述“农用制剂”通常是农用植物生长调节剂，其含有BADH2基因或其编码蛋白的抑制剂作为改良植物性状(如，增强植物香味)的活性成分；以及农业上可接受的载体。
如本文所用，所述“农业上可接受的载体”是用于将本发明的活性物质传送给植物的农药学上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。适用于本发明的农业上可接受的载体选自下组：水、缓冲液、DMSO、表面活性剂如Tween-20、或其组合。任何本领域技术人员已知的农业上可接受的载体均可用于本发明中。
本发明的农用制剂可包括农用组合物。
本发明的农用制剂可与其他增强植物香味的物质联用。所述其他增强香味的物质可以是本领域技术人员已知的植物生长调节剂。
本发明所述的农用制剂的剂型可以是多种多样的，只要能够使活性成分有效地到达植物体内的剂型都是可以的，从易于制备和施用的立场看，优选的农用制剂是一种喷雾剂或溶液制剂。
本发明所述的农用制剂通常含有占所述农用制剂总重量的0.0001-99wt％，较佳地0.1-90wt％的本发明的活性成分。商品制剂或使用剂型中的本发明的活性成分的浓度可在广阔的范围内变动。商品制剂或使用剂型中的本发明的活性成分的浓度可从0.0000001-100％(g/v)，最好在0.0001与50％(g/v)之间。
植物性状的改良
本发明还提供了一种改良植物性状的方法，所述的改良包括：增强植物的香味，包括步骤：降低所述植物中BADH2基因或其编码蛋白的表达量和/或活性，或添加BADH2基因或其编码蛋白的抑制剂。
在本发明中，还可进一步用常规方法将其他可以增强植物香味的物质处理植物或植物种子，从而改良对应植物的性状。
本发明的主要优点包括：
(1)本发明首次发现，通过抑制BADH2a和BADH2b基因或其编码蛋白的表达或活性，可显著增强植物香味。
(2)本发明首次发现，通过抑制BADH2a和BADH2b基因或其编码蛋白的表达或活性，可提高香味物质(如2AP)的含量。
(3)本发明提供了一种快速改良玉米香味的方法，改进的玉米香味(2AP含量)与野生型相比，有大幅度的增加。
(4)本发明创造了一个新的玉米种质资源，具有重要的理论和实际应用意义。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非有特别说明，否则实施例中所用的材料和试剂均为市售产品。
实施例
1、靶点设计及载体构建
通过NCBI(https://www.ncbi.nlm.nih.gov)网站获得OsBADH2基因的蛋白序列，然后与玉米蛋白数据库进行Blast后发现了两个同源基因，分别是位于1号染色体的Zm00001d032257(ZmBADH2b)和位于4号染色体的Zm00001d050339(ZmBADH2a)。
本实施例中利用Cas9和靶向ZmBADH2b和ZmBADH2a的sgRNA在玉米中针对上述两个基因进行编辑，具体操作方法可按照本领域常规的方式进行；本实施方式中，所构建的基因编辑载体的示意图如图14所示；其中，ZmU6 pro为U6启动子，Gly-tRNA为甘氨酸tRNA，ZmU6 Ter为终止子，UBI pro为UBI启动子，NLS为核定位信号；载体构建亦可借鉴参考文献(“High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize”，Weiwei Qi等，《BMC Biotechnology》，2016)；本实施例中，Cas9采用植物密码子优化的Cas9，在其他的实施方式中，也可以采用其他方式优化的Cas9。
具体而言，本实施方式中，利用target Design(http://skl.scau.edu.cn/targetdesign/)设计gRNA,其中位于第一个外显子区的ZmBADH2-T1(CGCCAGCGATGGTCCCGCTG(SEQ ID NO.:5))，位于第二个外显子区的ZmBADH2-T2(AGTCGCGGCCACGGTTCCTC(SEQ ID NO.:6))和位于与第二个内含子区的ZmBADH2-T3(AATTAGGCTAGAGCAAAGAG(SEQ ID NO.:7)都是ZmBADH2a基因特异性的序列，其中ZmBADH2-T1与ZmBADH2b的对应序列存在2个碱基的错配，ZmBADH2-T2与ZmBADH2b的对应序列存在2个碱基的错配， ZmBADH2-T3与ZmBADH2b的对应序列存在3个碱基的错配。将ZmBADH2-T1分别与ZmBADH2-T2和ZmBADH2-T3组合构建入U6启动子启动的利用甘氨酸tRNA间隔的基因编辑载体中,分别构建成载体P0195和P0196。另外将ZmBADH2-T1和ZmBADH2-T2构建入载体P0077中，两个gRNA用甘氨酸tRNA间隔开。
位于第一个外显子区的ZmBADH2-T4(GGTTGACGACGGGGAGGCGG(SEQ ID NO.:8))和位于第二个外显子区的ZmBADH2-T5(GCGGCGCTCAAGAGGAACCG(SEQ ID NO.:9))为两个基因的共有序列。将这两个gRNA构建入U6启动子启动的利用甘氨酸tRNA间隔的基因编辑载体中，构建成载体P0593。
位于第一个外显子区的ZmBADH2-T6(CAGCGGTACCATCGCTTGCG)和位于第二个外显子区的ZmBADH2-T7(GAGGAACCGCGGCCGCGATT)都是Zm00001d032257基因特异性的序列，其中ZmBADH2-T6F与Zm00001d050339的对应序列存在2个碱基的错配，ZmBADH2-T7R与Zm00001d050339的对应序列存在2个碱基的错配。将这两个gRNA构建入U6启动子启动的利用甘氨酸tRNA间隔的基因编辑载体中，构建成载体P1801。
具体构建方法如下：
1)、分别利用引物对
ZmBADH2-T1F(TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT(SEQ ID NO.:10))和
ZmBADH2-T2R(TAGGTCTCTAAACAGTCGCGGCCACGGTTCCTCTGCACCAGCCGGGAATCG(SEQ ID NO.:11))，ZmBADH2-T1F(TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT(SEQ ID NO.:12))和ZmBADH2-T3R(TAGGTCTCTAAACAATTAGGCTAGAGCAAAGAGTGCACCAGCCGGGAATCG(SEQ ID NO.:13))，ZmBADH2-T4F(TAGGTCTCTTGCAGGTTGACGACGGGGAGGCGGGTTTTAGAGCTAGAAATAGCAAGT(SEQ ID NO.:14))和ZmBADH2-T5R(TAGGTCTCTAAACGCGGCGCTCAAGAGGAACCG TGGTGCACCAGCCGGGAATCG(SEQ ID NO.:15))，ZmBADH2-T6F(TAGGTCTCTTGCACAGCGGTACCATCGCTTGCGGTTTTAGAGCTAGAAA TAGCAAGT)和ZmBADH2-T7R(TAGGTCTCTAAACAATCGCGGCCGCGGTTCCTCTGCACCAGCCGGGAATCG)；
以质粒P0055为模板扩增，分别割胶回收约200bp的片段为ZmBADH2-T1&2,ZmBADH2-T1&3和ZmBADH2-T4&5，ZmBADH2-T6&7对回收片段用BsaI酶切，并利用试剂盒回收；
2)、分别对骨架载体P0174和P0522进行BsaI酶切，分别回收23Kb片段；
3)、分别将ZmBADH2-T1&2，ZmBADH2-T1&3与P0174用T4连接酶进行连接，构建成最终载体P0195和P0196；将ZmBADH2-T4&5，ZmBADH2-T6&7与P0522进行连接，构建成最终载体P0593和P1801；
4)、将质粒P0055用BsaI酶切，并通过试剂盒进行液体回收，并与BsaI酶切好的ZmBADH2-T1&3进行连接，经质粒扩增后，对质粒用EcoRV和PstI进行双酶切，割胶回收1020bp的片段；将P0185质粒利用SmaI和PstI进行双酶切，回收载体片段；将1020bp的DNA片段与P0185的载体用T4DNA连接酶进行连接，构建成最终载体P0077。
5)、将上述3)和4)中的连接产物分别转化大肠杆菌感受态Trans-T1，涂布于Kan平板培养，挑8个菌落液体培养2h，PCR菌液检测，选择正确的单克隆2个，进行菌液送测。
6)、选择测序正确的单克隆进行扩繁、保菌、提质粒，转化农杆菌EHA105，挑取5个单菌落培养，PCR检测正确后保菌备用。
2、遗传转化
2.1转化农杆菌
将1中的载体利用热击法转入农杆菌菌株EHA105中，挑取单克隆经液体培养以及PCR鉴定后保存于-80℃冰箱中备用。
2.2菌种活化
从-80冰箱取出菌，在YEP固体培养基上划线，28℃暗培养，通常1～2天可以长出菌落，取菌落重新涂布在新的YEP平板上于28℃暗培养，12～24小时平板菌可长好。
2.3准备农杆菌侵染液
从新活化的菌板上刮取新鲜菌体，重悬到预先加入乙酰丁香酮(AS)的侵染液中，用1ml移液器将菌体打散至看不到颗粒状菌体，调整OD550至0.3～ 0.4，在摇床上室温低速振荡培养2～4h。
2.4取玉米幼胚
取授粉后10天左右的玉米果穗，拨去苞叶和花丝，浸没在75％酒精中消毒5～10min，期间晃动2～3次，挑取1.5～2mm的幼胚，放入加有AS的侵染培养基(不含农杆菌)中，每管放50～70个幼胚。
2.5侵染
将待转化幼胚用侵染液洗3遍，至侵染液澄清，将侵染液倒干净加入1ml菌液，温和颠倒10下，静置5～10min。取干净培养皿，放3张灭菌滤纸，侵染完毕后颠倒几下后迅速将菌液倒在滤纸上，手持培养皿，变换方向使携带幼胚的菌液在滤纸上均匀分布。
2.6共培养
待最上一层滤纸看不到菌液时用镊子将上层滤纸夹起，沾有幼胚的一面贴在共培养培养基上，用镊子驱赶滤纸和培养基之间的气泡后，用镊子夹住滤纸一角快速揭下，留在滤纸上的幼胚用剥胚刀转移到培养基上，幼胚盾面朝上，22℃暗培养3天。
2.7恢复培养
共培养3天后，将幼胚转移到恢复培养基，每皿30～40个幼胚，25℃暗培养10～14天，单转P0077载体的幼胚恢复培养7天。
2.8筛选
恢复结束后，将转P0077载体的幼胚转移到带双丙铵磷的相应筛选压的筛选一培养基上，25～28℃暗培养14天后，换较高筛选压的筛选二培养基上，25～28℃暗培养14天。
转P0195,P0196,P0593,和P1801这4个载体的幼胚无需愈伤筛选阶段。
2.9分化
将转P0077载体得到的抗性愈伤放到分化培养基上进行分化，夹成小块后平铺在培养基上，25～28℃暗培养，14天换一次培养基。
转P0195,P0196,P0593,和P1801载体额幼胚直接于分化培养基上分化。
2.10生根
将分化产生的小苗转接到生根培养基，每瓶3～4个小苗，25～28℃光照培养直至长成完整植株，生根培养7天后将有白色小根长出，可取样检测。
3、阳性苗的筛选
对于再生苗，取少量叶片以CTAB法提取基因组DNA。
对于转P0077载体的植株以引物对ZmCas9-jc-F2(TCACCGACGAGTACAAGGTC(SEQ ID NO.:16))和ZmCas9-jc-R1(GTCTGGACGAGCTGGATGAA(SEQ ID NO.:17))，ZmCas9-jc-F5(TTCAAGGAGGACATCCAGAAG(SEQ ID NO.:18))和ZmCas9-jc-R5(TCTCGTCGTACTTGGTGTTCAT(SEQ ID NO.:19))，35S-F2(TCATTTGGAGAGGACACGCT(SEQ ID NO.:20))和sp110(GGAGAAACTCGAGTCAAATCTCG(SEQ ID NO.:21))等3对引物分别检测，任何一对引物可以扩增到目的产物，则该再生苗即为转基因阳性苗。
对于转P0195和P0196的植株分别用引物对Cas9-jc-F1(AAGAAGCGGAAGGTCGGTAT(SEQ ID NO.:22))和Cas9-jc-R1(CTCAGGTGGTAGATGGTGGG(SEQ ID NO.:23))，Cas9-jc-F3(CAGAAAGAGCGAGGAAACCA(SEQ ID NO.:24))和Cas9-jc-R3(CCTCAAACAGTGTCAGGGTCA(SEQ ID NO.:25))，SIE1-jc-F1(CCCGAGGATAATGAGCAGAA(SEQ ID NO.:26))和NOS-jc-R1(CCGATCTAGTAACATAGATGACA(SEQ ID NO.:27))等3对引物分别检测，任何一对引物可以扩增到目的产物，则该再生苗即为转基因阳性苗。
对于转P0593和P1801的植株分别用引物对Cas9-jc-F1(AAGAAGCGGAAGGTCGGTAT(SEQ ID NO.:28))和Cas9-jc-R1(CTCAGGTGGTAGATGGTGGG(SEQ ID NO.:29))，Cas9-jc-F3(CAGAAAGAGCGAGGAAACCA(SEQ ID NO.:30))和Cas9-jc-R3(CCTCAAACAGTGTCAGGGTCA(SEQ ID NO.:31))，35S-F2(TCATTTGGAGAGGACACGCT(SEQ ID NO.:32))和sp110(GGAGAAACTCGAGTCAAATCTCG(SEQ ID NO.:33))等3对引物分别检测，任何一对引物可以扩增到目的产物，则该再生苗即为转基因阳性苗。
4、基因检测
对于转基因阳性苗以引物对ZmBADH2a-73F(GAGACGTCCTCGCTTTCCAC(SEQ ID NO.:34))和ZmBADH2a-904R(ATGTGCACGCTGCGTTTTAC(SEQ ID NO.:35))扩增ZmBADH2a基因的相应片段，以引物对BADH2b-jc-F1(GAAGTCCACTGCCGAGTTGC(SEQ ID NO.:36))和BADH2b-jc-R1(CGACTGAGTTGTCTCACACTGA(SEQ ID NO.:37))扩增ZmBADH2b基因的相应片段。扩增产物经sanger法进行测序，确认编辑形式，若为测序结果显示双峰，则将PCR产物连接入T载体，选取5 个克隆进行测序，确认编辑形式。
5、实验结果
利用P0077转化郑58的幼胚获得了编辑植株C43,其编辑结果是在ZmBADH2a CDS的+24nt后插入一个A,导致其后的编码区发生移码突变，并在新CDS的265-267nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止；在ZmBADH2b的CDS+27nt处删除一个碱基G，导致其后发生移码突变，并且在新的CDS的262-264nt形成一个终止密码子TAA,导致将会导致蛋白翻译的提前终止。具体编辑方式参见图1(A-C)。
利用P0077转化郑58的幼胚获得了编辑植株P64,编辑植株P64由于其编辑导致在ZmBADH2a CDS的+24nt后插入一个C,导致其后的编码区发生移码突变(图2，A-C)，并在新CDS的265-267nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止；而在P64植株中的ZmBADH2b基因没有发生编辑。具体编辑方式参见图2。
综上，C43植株在ZmBADH2a和ZmBADH2b两个基因同时发生编辑，而P64植株仅在ZmBADH2a基因发生编辑。对C43和P64两个植株的种子提取物通过质谱分析其香味物质2AP的含量；如图3所示，C43植株的种子中2AP的含量为0.648mg/kg、其可以产生香味，而郑58野生型以及P64植株的种子中检测不到2AP、也不会产生香味(图3)。
利用P0593转化糯玉米自交系XCW175的幼胚获得了编辑植株P302，该编辑植物分别在ZmBADH2a、ZmBADH2b的各两个位置发生了编辑，其编辑结果是在ZmBADH2a CDS的+77nt后插入一个C以及+188nt后插入一个A,由编辑后CDS的推导出的蛋白质在第27位氨基酸开始发生移码突变，并在新CDS的262-264nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止；在ZmBADH2b的CDS+80nt处插入AA以及+191nt处缺失一个A，由编辑后CDS的推导出的蛋白质在第27位氨基酸开始发生移码突变，并在新CDS的265-267nt形成一个终止密码子TAA，将会导致蛋白翻译的提前终止。具体编辑方式参见图4(A-C)。
P302种子提取物通过质谱分析其香味物质2AP含量为0.266mg/kg，并且其种子可以产生香味。而XCW175野生型不产生香味，也检测不到2AP(图5)。
另外，利用P0196转化糯玉米自交系N355的幼胚获得了编辑植株282,将 其自交并在其后代植株中鉴定到3株编辑植株为288-4、288-5和288-6，这三株在ZmBADH2a和ZmBADH2b都发生纯合编辑，并且编辑方式相同。其编辑方式为：在ZmBADH2a CDS的+24nt后插入一个A,导致其后的编码区发生移码突变，并在新CDS的265-267nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止；在ZmBADH2b的CDS+27nt处删除一个碱基G，导致其后发生移码突变，并且在新的CDS的262-264nt形成一个终止密码子TAA,导致将会导致蛋白翻译的提前终止。将三株植株自交，并取授粉后27天处于糯玉米鲜食期未成熟籽粒，通过质谱分析其香味物质2AP含量，三株2AP的含量分别为0.774mg/kg、0.826mg/kg、0.503mg/kg。而对照的N355自交系相同时期的籽粒中检测不到2AP。
利用P0195、P1801和P0593分别转化郑58的幼胚获得了编辑植株Z82(图6A)、Z10(图7A)和Z54(图8A)，其中Z82只在Zm00001d050339(ZmBADH2a)上发生编辑，其编辑导致Zm00001d050339 CDS的+24nt处缺失一个G,导致其后的编码区发生移码突变(图6B)，并在新CDS的259-261nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止(图6C)；Z10只在Zm00001d032257(ZmBADH2b)发生编辑，其编辑导致Zm00001d032257 CDS的+15nt到+203nt处缺失188bp,导致其后的编码区发生移码突变(图7B)，并在新CDS的79-81nt形成一个终止密码子TAG，将会导致蛋白翻译的提前终止(图7C)；Z54在Zm00001d050339和Zm00001d032257都发生编辑，其编辑导致Zm00001d050339 CDS 188-189nt缺失2bp(图8B)，并在新CDS的262-264nt形成一个终止密码子TGA，导致蛋白翻译的提前终止(图8C)，Zm00001d032257 CDS+191nt后插入一个A(图8B)，并在新CDS的268-270nt形成一个终止密码子TGA，导致蛋白翻译的提前终止(图8C)。
Z10、Z82和Z54的种子提取物通过质谱分析香味物质2AP含量，结果发现在野生型、Z10和Z82的种子中检测不到2AP，而Z54种子中的2AP含量为0.288mg/kg、并且可以产生香味(图9)。
利用P0593转化糯玉米自交系XCW175的幼胚获得了编辑植株X651(图10A)、X610(图11A)以及X447(图12A)。其中X651只在Zm00001d050339发生编辑，其编辑导致Zm00001d050339 CDS 187-188nt缺失2bp(图10B)，并在新CDS的262-264nt形成一个终止密码子TGA，导致蛋白翻译的提前终止(图10C)；X610 只在Zm00001d032257发生编辑，其编辑导致Zm00001d032257 CDS的+191nt处缺失一个A,导致其后的编码区发生移码突变(图11B)，并在新CDS的262-264nt形成一个终止密码子TAA，将会导致蛋白翻译的提前终止(图11C)；X447植株的Zm00001d050339和Zm00001d032257基因同时发生编辑，编辑分别导致在Zm00001d050339 CDS的+188nt处缺失了一个A(图12B),并在新CDS的199-201nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止(图12C)；在Zm00001d032257的CDS+83nt到+193nt缺失110bp(图12B)，并在新CDS的157-159nt形成一个终止密码子TGA，将会导致蛋白翻译的提前终止(图12C)。
对X610、X651和X447的种子提取物通过质谱分析香味物质2AP含量，结果发现在野生型、X610和X651的种子中检测不到2AP，而X447种子中的2AP含量为为0.126mg/kg、并且可以产生香味(图13)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种增强植物香味的方法，其特征在于，包括步骤：
    降低或抑制所述植物中BADH2基因或其编码蛋白的表达量和/或活性，从而增强植物的香味，其中，所述BADH2基因包括BADH2a和BADH2b基因；所述植物为玉米。
  2. 一种赋予植物产生香味的方法，其特征在于，包括步骤：
    降低或抑制所述植物中BADH2基因或其编码蛋白的表达量和/或活性，从而赋予植物产生香味，其中，所述BADH2基因包括BADH2a和BADH2b基因；所述植物为玉米。
  3. 根据权利要求1或2所述的方法，其特征在于，所述降低或抑制BADH2基因或其蛋白的表达和/或活性通过选自下组的方法实现：基因突变、基因敲除、基因中断、RNA干扰技术、基因编辑技术、导入基因或蛋白的抑制剂、或其组合。
  4. 一种BADH2基因或其编码蛋白的抑制剂的用途，其特征在于，用于增强植物的香味或赋予植物产生香味；或者，制备用于增强植物香味或赋予植物产生香味的组合物或制剂，其中所述BADH2基因包括BADH2a和BADH2b基因；所述植物为玉米。
  5. 一种用于增强植物香味或赋予植物产生香味的组合物，其特征在于，包括：
    (a)BADH2基因或其编码蛋白的抑制剂，所述BADH2基因包括BADH2a和BADH2b基因；和
    (b)农学上可接受的载体；
    优选的，所述抑制剂选自下组：基因编辑试剂、反义核酸、抗体、小分子化合物、Crispr试剂、小分子配体、或其组合。
  6. 一种权利要求5所述的组合物的用途，其特征在于，用于增强植物的香味或赋予植物产生香味，所述植物为玉米。
  7. 一种制备基因工程的植物组织或植物细胞的方法，其特征在于，包括步骤：
    降低或抑制植物组织或植物细胞中的BADH2基因或其编码蛋白的表达和/或活性，从而获得基因工程的植物组织或植物细胞，所述BADH2基因包括BADH2a和BADH2b基因，所述植物为玉米。
  8. 一种制备基因工程植物的方法，其特征在于，包括步骤：
    将权利要求7所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得基因工程植物。
  9. 一种改良植物的方法，其特征在于，所述的方法包括步骤：
    (a)提供一植物细胞、植物组织、植物部分，向所述植物细胞、植物组织、植物部分中导入BADH2基因或其编码蛋白的抑制剂；或者，降低或抑制植物细胞、植物组织、植物部分中的BADH2基因或其编码蛋白的表达和/或活性；所述BADH2基因包括BADH2a和BADH2b基因；
    (b)将步骤(a)中的植物细胞、植物组织、植物部分再生成植株；
    所述植物为玉米；
    优选的，所述改良植物为增强/提高植物的香味或赋予植物产生香味。
  10. 一种筛选或鉴定香味植物的方法，其特征在于，检测植物中BADH2基因和/或其蛋白的表达量，所述BADH2基因包括BADH2a和BADH2b基因；优选的，所述植物为玉米。
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