WO2020156087A1 - 一种bxl基因或其编码蛋白的应用 - Google Patents

一种bxl基因或其编码蛋白的应用 Download PDF

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WO2020156087A1
WO2020156087A1 PCT/CN2020/071199 CN2020071199W WO2020156087A1 WO 2020156087 A1 WO2020156087 A1 WO 2020156087A1 CN 2020071199 W CN2020071199 W CN 2020071199W WO 2020156087 A1 WO2020156087 A1 WO 2020156087A1
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plant
gene
bxl
plants
composition
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PCT/CN2020/071199
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French (fr)
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张惠明
贺丹霞
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中国科学院分子植物科学卓越创新中心
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Publication of WO2020156087A1 publication Critical patent/WO2020156087A1/zh
Priority to US17/427,647 priority Critical patent/US20220177904A1/en

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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • 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
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    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • 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 present invention relates to the field of agronomy, in particular to the application of a BXL gene or its encoded protein.
  • Plants experience various biotic or abiotic stresses during their growth. Drought and pathogenic bacteria, as the two main factors that harm plant growth, have been widely focused by people.
  • the purpose of the present invention is to provide a new gene that can help plants resist external stress, especially drought stress and pathogenic bacteria.
  • the first aspect of the present invention provides the use of an inhibitor of the BXL gene or its encoded protein for improving plant properties; or preparing a composition or preparation for improving plant properties.
  • the improved plant traits include:
  • the stress resistance is selected from the group consisting of drought resistance, salt resistance, osmotic pressure resistance, heat resistance, or a combination thereof.
  • the pathogen is selected from the group consisting of Pseudomonas syringae, cauliflower, or a combination thereof.
  • the pathogen is selected from the group consisting of: Pseudomonas syringae tomato pathogenic strain DC3000, Pseudomonas syringae tomato pathogenic strain T1, Pseudomonas syringae tomato pathogenic strain 3435, Cauliflower 4326. Cauliflower fungus 4981, or a combination thereof.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the composition includes an agricultural composition.
  • the formulation includes an agricultural formulation.
  • the composition comprises (a) an inhibitor of the BXL gene or its encoded protein; and (b) an agronomically acceptable carrier.
  • 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 acids, antibodies, small molecule compounds, Crispr reagents, and bacterial volatiles (including acetylene, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
  • the inhibitor is selected from the group consisting of abscisic acid (ABA), salt (NaCl), salicylic acid (SA), mannitol (Mannitol), volatiles of Bacillus amyloliquefaciens GB03 (including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol), or a combination thereof.
  • ABA abscisic acid
  • NaCl salt
  • SA salicylic acid
  • mannitol Malannitol
  • volatiles of Bacillus amyloliquefaciens GB03 including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol
  • the composition further includes other substances that improve the palatability of feed.
  • the other substances that improve the palatability of feed are selected from the following group: cellulose hydrolase enzyme, pectin synthase, pectin additive, galactase, ethylene molecule, abscisic acid, or a combination thereof.
  • the composition also includes other substances that enhance plant stress resistance.
  • the other substances that enhance plant resistance to stress are selected from the group consisting of abscisic acid, analogs of abscisic acid, callose, proline, or a combination thereof.
  • the other anti-pathogenic bacteria substance is selected from the following group: alkaloids, flavonoids, tannins, phenylpropanoids, or combinations thereof.
  • the composition further includes other substances that reduce the content of lignin and increase the content of fiber and pectin.
  • the other substances that reduce the content of lignin and increase the content of fiber and pectin are selected from the following group: glycosyl hydrolase, arabinosidase, xylosidase, or a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, Cruciferous plants, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, Capricaceae, Rutaceae, Malvaceae, Rosaceae, Hibiscus, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, leguminous plants, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, alfalfa, apple, camelina, soybean, rice, rape, radish, pepper, cherry, date, cabbage, cleome, citrus, durian , Or a combination thereof.
  • the BXL gene is selected from the following group: BXL1, BXL2, BXL3, BXL4, BXL7, AT5G10560, AT3G19620, AT5G09700, or a combination thereof.
  • the BXL gene includes wild-type BXL gene and mutant BXL 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 BXL gene is the same or substantially the same as the polypeptide encoded by the wild-type BXL gene.
  • the mutant BXL gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98%) compared with the wild-type BXL gene. % Or 99%) polynucleotides.
  • mutant BXL gene is included in the 5'end and/or 3'end of the wild-type BXL gene truncated or 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • the BXL gene includes a cDNA sequence, a genomic sequence, or a combination thereof.
  • the BXL gene is derived from a plant, preferably from a dicotyledonous plant, more preferably, from one or more plants selected from the group consisting of Arabidopsis, alfalfa, apple, camelina , Soybean, rape, radish, pepper, cherry, date, cabbage, cleome, citrus, durian.
  • the BXL gene is selected from the group consisting of the BXL1 gene of Arabidopsis thaliana (gene accession number: AT5G49360), the xylosidase 1 gene of Camelina sativa (L.) Crantz ⁇ - D-xylosidase 1, gene accession number: LOC104723790), xylosidase gene of alfalfa ( ⁇ -D-xylosidase of Medicago Sativa Linn, gene accession number: Medtr2G034720.1), xylosidase of Brassica napus Enzyme gene (Brassica napus L.
  • ⁇ -D-xylosidase gene accession number: LOC106365857
  • rose xylosidase 2 gene Rosa sp. ⁇ -D-xylosidase 2 gene, gene accession number: LOC112179881
  • amino acid sequence of the protein encoded by the BXL gene is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 1 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and has the function of regulating agronomic traits,
  • a polypeptide derived from (i); or (iii) the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.:1 is ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98%), with improvement Polypeptides for plant agronomic traits.
  • nucleotide sequence of the BXL gene is selected from the following group:
  • the second aspect of the present invention provides a composition comprising:
  • the composition includes an agricultural composition.
  • the agricultural composition is selected from the group consisting of a feed composition, an organic fertilizer composition, a pesticide composition, or a combination thereof.
  • the feed composition includes a solid feed composition or a liquid feed composition.
  • the feed composition is a plant breeding additive.
  • 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 content (wt%) of the inhibitor of the BXL gene or its encoded protein in the composition is 0.05%-10%, preferably, 0.1%-8%, more preferably , 0.5%-6%.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), antisense nucleic acid, Crispr reagent, siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles, or combinations thereof.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of abscisic acid (ABA), salt (NaCl), salicylic acid (SA), mannitol (Mannitol), amylolytic spores Volatile matter of Bacillus coli GB03 (including acetone, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol), or a combination thereof.
  • ABA abscisic acid
  • NaCl salt
  • SA salicylic acid
  • mannitol Malannitol
  • amylolytic spores Volatile matter of Bacillus coli GB03 including acetone, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol, or a combination thereof.
  • the composition further includes other substances that improve the palatability of feed.
  • the other substances that improve the palatability of feed are selected from the following group: cellulose hydrolase enzyme, pectin synthase, pectin additive, galactase, ethylene molecule, abscisic acid, or a combination thereof.
  • the composition also includes other substances that enhance plant stress resistance.
  • the other substances that enhance plant resistance to stress are selected from the group consisting of abscisic acid, analogs of abscisic acid, callose, proline, or a combination thereof.
  • the other anti-pathogenic bacteria substance is selected from the following group: alkaloids, flavonoids, tannins, phenylpropanoids, or combinations thereof.
  • the composition further includes other substances that reduce the content of lignin and increase the content of fiber and pectin.
  • the other substances that reduce the content of lignin and increase the content of fiber and pectin are selected from the following group: glycosyl hydrolase, arabinosidase, xylosidase, or a combination thereof.
  • the third aspect of the present invention provides a use of the composition of the second aspect of the present invention for improving agronomic traits of plants.
  • the fourth aspect of the present invention provides a method for improving the palatability of feed, including the steps:
  • the method includes administering an inhibitor of the plant BXL gene or its encoded protein.
  • the method includes the steps:
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), antisense nucleic acid, Crispr reagent, siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), or a combination thereof.
  • the inhibitor is selected from the group consisting of abscisic acid (ABA), salt (NaCl), salicylic acid (SA), mannitol (Mannitol), volatiles of Bacillus amyloliquefaciens GB03 (including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol), or a combination thereof.
  • ABA abscisic acid
  • NaCl salt
  • SA salicylic acid
  • mannitol Malannitol
  • volatiles of Bacillus amyloliquefaciens GB03 including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol
  • the "reduction" means that the expression or activity of the BXL gene or its encoded protein is reduced to meet the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 50%, more preferably ⁇ 20%, and most preferably 0-10%; wherein, A1 is the expression or activity of the BXL gene or its encoded protein; A0 is wild The expression or activity of the same BXL gene or its encoded protein in plant plants of the same type.
  • the reduction means that the expression level E1 of the BXL gene or its encoded protein in the plant is 0-80% of that of the wild-type BXL gene or its encoded protein E0. , Preferably 0-60%, more preferably 0-40%.
  • the reduction of the expression or activity of the BXL gene or its encoded protein in the plant is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology, ZFN (Zinc finger endonuclease technology), TALEN (transcription activator-like effector nuclease), or a combination thereof.
  • the fifth aspect of the present invention provides a method for improving plant traits, including the steps:
  • the improved plant traits include:
  • the improved plant traits further include:
  • the reduction of lignin content refers to a reduction of lignin content by ⁇ 50% compared with wild-type plants, preferably, ⁇ 70%, more preferably, ⁇ 80%, more preferably, ⁇ 90%.
  • the increase in fiber and pectin content means that compared with wild-type plants, the fiber and pectin content has increased by ⁇ 30%, preferably, ⁇ 40%, more preferably, ⁇ 50%, More preferably, ⁇ 60%.
  • the reduction of stomata opening means that compared with wild-type plants, the stomata opening is reduced by ⁇ 50%, preferably, ⁇ 50%, more preferably, ⁇ 60%, more preferably Ground, ⁇ 70%.
  • the trait improvement of the plant includes:
  • the "reduction" means that the expression or activity of the BXL gene or its encoded protein is reduced to meet the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 50%, more preferably ⁇ 20%, and most preferably 0-10%; wherein, A1 is the expression or activity of the BXL gene or its encoded protein; A0 is wild The expression or activity of the same BXL gene or its encoded protein in plant plants of the same type.
  • the reduction means that the expression level E1 of the BXL gene or its encoded protein in the plant is 0-80% of that of the wild-type BXL gene or its encoded protein E0. , Preferably 0-60%, more preferably 0-40%.
  • the reduction of the expression or activity of the BXL gene or its encoded protein in the plant is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology, ZFN (Zinc finger endonuclease technology), TALEN (transcription activator-like effector nuclease), or a combination thereof.
  • the method includes administering an inhibitor of the plant BXL gene or its encoded protein.
  • the method includes the steps:
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), antisense nucleic acid, Crispr reagent, siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), or a combination thereof.
  • the inhibitor is selected from the group consisting of abscisic acid (ABA), salt (NaCl), salicylic acid (SA), mannitol (Mannitol), volatiles of Bacillus amyloliquefaciens GB03 (including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol), or a combination thereof.
  • ABA abscisic acid
  • NaCl salt
  • SA salicylic acid
  • mannitol Malannitol
  • volatiles of Bacillus amyloliquefaciens GB03 including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol
  • the sixth aspect of the present invention provides a method for preparing genetically engineered plant tissues or plant cells, including the steps:
  • the method further includes introducing the BXL gene or an inhibitor of its encoded protein into the plant tissue or plant cell.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), antisense nucleic acid, Crispr reagent, siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
  • the inhibitor of the BXL gene or its encoded protein is selected from the group consisting of small molecule compounds, antibodies, bacterial volatiles (including acetophenone, di, tri-butanediol, di, tri- Diacetyl, isoamyl alcohol, n-butanol), or a combination thereof.
  • the inhibitor is selected from the group consisting of abscisic acid (ABA), salt (NaCl), salicylic acid (SA), mannitol (Mannitol), volatiles of Bacillus amyloliquefaciens GB03 (including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol), or a combination thereof.
  • ABA abscisic acid
  • NaCl salt
  • SA salicylic acid
  • mannitol Malannitol
  • volatiles of Bacillus amyloliquefaciens GB03 including Acetylene, di, tri-butanediol, di, tri-butanedione, isoamyl alcohol, n-butanol
  • the seventh 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 sixth aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.
  • the method includes using RNA interference technology, Crispr technology, ZFN (zinc finger endonuclease technology), TALEN (transcription activator-like effector nuclease) to reduce BXL in plant tissues or plant cells.
  • RNA interference technology Crispr technology
  • ZFN zinc finger endonuclease technology
  • TALEN transcription activator-like effector nuclease
  • the eighth aspect of the present invention provides a genetically engineered plant, which is prepared by the method of the seventh aspect of the present invention.
  • Figure 1 shows the tissue location of the BXL1 gene in Arabidopsis. The figure clearly shows that BXL1 can be expressed in vascular tissues. In leaves, the present invention discovered for the first time that BXL1 can be specifically expressed in stomata.
  • Figure 2 shows the gene expression of BXL1 in wild-type, mutant and overexpressing plants. Quantitative PCR detects the expression level of the BXL1 gene at the transcript level of the BXL1 mutant or overexpression strain. The results showed that the expression of the BXL1 gene in the overexpressed plants was significantly increased compared to the wild-type plants. However, the expression of the mutant BXL1 gene was significantly suppressed compared to the wild-type plant.
  • FIG. 3 shows the basic phenotype of BXL1 gene resistance to pathogenic bacteria and drought.
  • A The figure shows that the BXL1 mutant plants have better resistance to pathogenic bacteria, and the degree of infection is reduced compared to the wild type.
  • B The figure shows that the mutant plants can better resist the drought environment. Under the same drought conditions, compared to the wild-type plants, the mutants can maintain a higher survival rate.
  • Figure 4(A)(B) shows the effect of BXL1 gene on the normal growth of plants.
  • the graph (A) shows that the BXL1 mutant plant can have a slow seed germination rate, and the graph (B) shows that the mutant plant shows a slow growth rate.
  • the BXL1 mutant showed a smaller plant morphology, and the flowering time was slower.
  • the length of the fruit pod was shorter than that of the wild type.
  • Figure 5 (A) and (B) show that the BXL1 gene has no effect on the final biomass of a single plant and the seed weight of a single plant seed.
  • Figure (A) shows that when the BXL1 mutant plant reaches the flowering stage, there is no significant change in the dry weight of a single plant when compared to the wild type when it reaches flowering.
  • Figure (B) shows the final harvested seeds of the mutant plant. Compared with the seeds harvested from a wild-type single plant, the thousand-seed weight of seeds did not show a difference in dry weight.
  • FIG. 6 shows that the BXL1 gene regulates the opening of plant stomata.
  • the stomata opening in the BXL1 mutant was significantly smaller than that in the wild type.
  • FIG. 7 shows that the BXL1 gene is regulated by various stress factors. Salicylic acid, salt, abscisic acid, mannitol, these stress factors can significantly down-regulate the expression of BXL1 gene in a specific time.
  • the mock in the figure represents water treatment, as a control group.
  • the inventors discovered a BXL gene or its encoded protein for the first time by studying and screening a large number of plant trait sites.
  • the protein encoded by it is a glycosyl hydrolase.
  • the expression of the BXL gene or its encoded protein can significantly improve plant traits, including (i) enhancing plant resistance; and/or (ii) resistance to pathogenic bacteria; and/or (iii) reducing lignin content, increasing fiber and Pectin content.
  • suppressing the expression of the BXL gene or its encoded protein also (a) delays seed germination; (b) delays flowering time; (c) reduces stomata opening. The inventor completed the present invention on this basis.
  • BXL1 The gene encoding glycosyl hydrolase is named ⁇ -D-xylosidase 1 (BXL1), which has the dual functions of encoding ⁇ -D-xylosidase and ⁇ alpha ⁇ -L-arabinofuranosidase.
  • BXL1 exists in areas outside the cytoplasm, and is specifically expressed in tissues with secondary cell wall thickening, such as vascular tissue and secondary cell walls.
  • BXL1 is one of the three members of glycosyl hydrolase family, which controls the germination of seeds and hydrolyzes arabinan and xylan. When a normal seed germinates, a complete viscous fluid is released around the seed to help soften the seed coat and promote seedling morphology.
  • BXL1 plays different roles in different tissues. It mainly plays the role of xylosidase in the stem tissues, and mainly plays the role of arabinofuranosidase during the germination of seeds.
  • BXL1 In Arabidopsis, there are 7 homologous genes in BXL1, namely BXL2, BXL3, BXL4, BXL7, AT5G10560, AT3G19620, AT5G09700.
  • the homology analysis of the similarity of protein domains showed that the homology degree of the protein encoding 7 homologous genes (BXL2, BXL3, BXL4, BXL7, AT5G10560, AT3G19620, AT5G09700) and BXL1 protein was 99 respectively. %, 98%, 98%, 97%, 97%, 96%, 97%.
  • BXL1 expression tissues found that the BXL1 gene exists in stems, fruit pods, and vascular tissues.
  • the present invention conducts a more in-depth study on BXL1, and through tissue staining, it is found that the BXL1 gene can be specifically expressed in the guard cells of the stoma.
  • Physiological and biochemical research results show that the BXL1 gene plays an important role in helping plants resist external stress, especially drought stress and pathogenic bacteria. This discovery not only opens a new idea for studying the genes related to the secondary cell wall in plant drought and disease resistance, but also adds new content to the function of the BXL1 gene.
  • BXL gene of the present invention and “BXL gene” are used interchangeably, and both refer to BXL genes or variants thereof derived from monocotyledonous or dicotyledonous plants (such as Arabidopsis, alfalfa, apple, etc.) .
  • the nucleotide sequence of the BXL gene of the present invention is shown in SEQ ID NO.: 2.
  • the present invention also includes 50% or more of the preferred gene sequence (SEQ ID NO.: 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 nucleic acid, which can also effectively regulate the agronomic traits of plants (such as Arabidopsis, alfalfa, etc.).
  • “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.
  • variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, etc.
  • the nucleotide sequence in SEQ ID NO.: 2 can be substituted, deleted, or added one or more to generate a derivative sequence of SEQ ID NO.: 2. Due to the codon degeneracy, even if it is SEQ ID NO.: 2 has low homology, and can basically encode the amino acid sequence shown in SEQ ID NO.:1.
  • the nucleotide sequence in SEQ ID NO.: 2 has been substituted, deleted or added at least one nucleotide-derived sequence means that it can be used under moderately stringent conditions, and more preferably under highly stringent conditions.
  • variants include (but are not limited to): deletion 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 Arabidopsis thaliana and alfalfa, the sequences of the present invention derived from other similar plants (preferably, the sequence is shown in SEQ ID NO.: 2 Arabidopsis thaliana))
  • the gene sequence of BXL with certain homology (conservation) is also included in the scope of the present invention, as long as those skilled in the art can easily obtain information from other sources after reading this application. The sequence was isolated from plants.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include: 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.: 2 or a degenerate variant.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide.
  • 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 aforementioned 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. It 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 BXL gene of the present invention is preferably derived from Arabidopsis thaliana, it is highly homologous to the Arabidopsis BXL gene from other plants (such as having more than 80%, such as 85%, 90%, 95% or even 98%, 99%). %, or 100% sequence identity) other genes are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.
  • the full-length BXL nucleotide sequence of the present invention or its fragments can usually be obtained by PCR amplification method, recombinant 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 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 connecting them 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 “protein encoded by the BXL gene” can be used interchangeably, and both refer to BXL polypeptides derived from plants (such as Arabidopsis) and variants thereof.
  • a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.: 1 (Arabidopsis).
  • the present invention relates to a BXL polypeptide for controlling traits and variants thereof.
  • the amino acid sequence of the polypeptide is shown in SEQ ID NO.:1.
  • the polypeptide of the present invention can effectively regulate the traits of plants (such as monocots or dicots).
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO. 1 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 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: "regulate the agronomic traits of plants (such as monocots or dicots)".
  • 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 prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insect and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or exclude the initial methionine residue.
  • the present invention also includes BXL protein fragments and analogs having BXL protein activity.
  • fragment and “analog” refer to polypeptides that substantially retain the same biological function or activity as the natural BXL protein of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be: (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been 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 fusion of 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, after several (usually 1-60, preferably 1-30, and more preferably 1-20) , Preferably 1-10) substitution, deletion or addition of at least one amino acid derived sequence, and adding one or several (usually within 20, preferably within 10) at the C-terminal and/or N-terminal , 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 invention also includes analogs of the claimed protein.
  • the difference between these analogs and the natural SEQ ID NO.: 1 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 with residues different from natural L-amino acids (such as D-amino acids), and analogs with 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 unchanged primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, 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, phosphothreonine).
  • the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant mutein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the invention also provides a recombinant vector including the gene of the invention.
  • the downstream of the promoter of the recombinant vector contains a multiple cloning site or at least one restriction site.
  • the target gene of the present invention needs to be expressed, the target gene is ligated into a suitable multiple cloning site or restriction site, so that the target gene and the promoter are operably linked.
  • the recombinant vector includes (from 5'to 3'direction): a promoter, a target gene, and a terminator.
  • the recombinant vector may also include elements selected from the following group: 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance and green fluorescent protein, etc.); enhancer; or operator.
  • the polynucleotide sequence encoding the protein of the present invention can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. As long as it can replicate and stabilize in the host, any plasmid and vector can be used.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide.
  • the vector including the gene of the present invention and the expression cassette can be used to transform an appropriate host cell to allow the host to express protein.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc.
  • conventional methods can be used to regenerate plants to obtain transgenic plants.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as rice cells).
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the replication initiation point, polyoma enhancers on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis,
  • the active substance of the present invention (such as the inhibitor of BXL gene or its encoded protein) can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, gas Aerosols, 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 polyethylene glycol ethers, alkyl sulfonate esters, alkyl sulfate esters, aryl sulfonate esters 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 an inhibitor of the BXL gene or its encoded protein as an improved plant trait (e.g., enhances plant resistance (such as drought resistance, salt tolerance) , Osmotic pressure, heat resistance, etc.); and/or resistance to pathogenic bacteria; and/or reduce lignin content, increase fiber and pectin content; and/or delay seed germination; and/or delay flowering time; and/or reduce Small stomata opening; and/or active ingredients to improve the palatability of feed); and agriculturally acceptable carriers.
  • an agricultural plant growth regulator which contains an inhibitor of the BXL gene or its encoded protein as an improved plant trait (e.g., enhances plant resistance (such as drought resistance, salt tolerance) , Osmotic pressure, heat resistance, etc.); and/or resistance to pathogenic bacteria; and/or reduce lignin content, increase fiber and pectin content; and/or delay seed germination; and/or delay flowering time; and/or reduce
  • 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, surfactant 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 preparation of the present invention may include a feed composition, an organic fertilizer composition, or a pesticide composition.
  • the agricultural preparation of the present invention includes a solid feed composition or a liquid feed composition.
  • the feed composition of the present invention is a plant breeding additive.
  • the agricultural preparation of the present invention can be combined with other substances that improve the palatability of feed (such as cellulose hydrolase enzyme, pectin synthase, pectin additive, galactase, ethylene molecule, abscisic acid, etc.).
  • other substances that improve the palatability of feed such as cellulose hydrolase enzyme, pectin synthase, pectin additive, galactase, ethylene molecule, abscisic acid, etc.
  • the agricultural preparation of the present invention can be made into a mixture with other anti-stress agents (such as drought-resistant agents, salt tolerants, osmotic pressure agents, heat-resistant agents, etc.) to be present in their commercial preparations or use dosage forms prepared from these preparations Among them, these other drought-resistant agents include (but are not limited to): drought-resistant seed coating agents, drought-resistant water-retaining agents, or drought-resistant sprays; these other salt-tolerant agents include (but are not limited to): salt-tolerant microbial agents, Salt thickener; these other osmotic pressure agents include (but are not limited to): sprays, biological protein protectors, trehalose; these other heat-resistant agents include (but are not limited to): heat-resistant film, biological Wax preparations, hydration film, trehalose.
  • these other drought-resistant agents include (but are not limited to): drought-resistant seed coating agents, drought-resistant water-retaining agents, or drought-resistant sprays; these other salt-tolerant agents include (but are not limited
  • the agricultural preparations of the present invention are prepared as a mixture with other anti-pathogenic agents and present in their commercial preparations or in dosage forms prepared from these preparations.
  • These other anti-pathogenic agents include (not limited to): saponin , Phenolics, disease-fighting compounds, organic sulfides, unsaturated fatty acids.
  • the agricultural preparations of the present invention can also be prepared as a mixture with synergists and present in their commercial preparations or in dosage forms prepared from these preparations.
  • synergists are compounds that enhance the action of the active compound. It is active by itself, and no synergist is necessary.
  • 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, based on 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 a 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 the traits of plants (such as monocots or dicots), the improvement comprising: (i) enhancing plant resistance; and/or (ii) resistance to pathogenic bacteria; and/or (iii) reducing the content of lignin, increasing the content of fiber and pectin, including the steps of reducing the expression and/or activity of the BXL gene or its encoded protein in the plant, or adding an inhibitor of the BXL gene or its encoded protein.
  • conventional methods can be used to further combine other plants that can (i) enhance plant resistance; and/or (ii) resist pathogenic bacteria; and/or (iii) reduce lignin content and increase fiber and pectin content. Substances treat plants or plant seeds to improve the traits of corresponding plants.
  • the present invention screens for the first time a BXL gene, which encodes a glycosyl hydrolase, which can improve plant traits by inhibiting the expression or activity of the BXL gene or its encoded protein.
  • the present invention found for the first time that reducing the expression of the BXL gene or its encoded protein can significantly (i) enhance plant stress resistance; and/or (ii) resist pathogenic bacteria; and/or (iii) reduce lignin content and increase fiber And pectin content.
  • the present invention found for the first time that reducing the expression of the BXL gene or its encoded protein can improve the palatability of feed.
  • reducing the expression of the BXL gene or its encoded protein can also (a) delay seed germination; (b) delay flowering time; (c) reduce stomata opening; (d) enhance plant resistance (E) Enhance the resistance of plants to disease bacteria; (f) Improve the palatability of plants as feed.
  • Arabidopsis models involved in the following examples are Col-0, SALK_012090C (bxl1-1), and SALK_086578C (bxl1-2) from the Arabidopsis Biological Resource Center (ABRC).
  • Figure (A) shows that when the BXL1 mutant plant reaches the flowering stage, there is no significant change in the dry weight of a single plant when compared to the wild type when it reaches flowering.
  • Figure (B) shows the final harvested seeds of the mutant plant. Compared with the wild-type seeds harvested from a single plant, there is no difference in the thousand-seed weight of a single seed.
  • Pro BXL1 :GUS and 35S:BXL1:flag materials were constructed. Take the 2.8k upstream of the start codon of BXL1 as its promoter analysis (not including the 5'UTR region), and fuse it into pGWB533 by the Getway method. The full-length BXL1CDs were fused into pGWB511 by Getway method. Transcription level detection of the expression of BXL1 in different materials, as shown in Figure 2, quantitative PCR detected the expression level of the BXL1 gene at the transcript level of the BXL1 mutant or overexpression strain. The results showed that overexpressed plants, the expression of BXL1 gene was significantly increased, compared with wild-type plants. However, the expression of the mutant BXL1 gene was significantly suppressed.
  • the primers used for detection are: F 5'-AGTTGATGTTGATGCTTGCA-3' (SEQ ID NO.: 3), R 5'-AAGTTGCGGTTGGACCAAAA-3' (SEQ ID NO.: 4).
  • GUS staining by histochemical method found that BXL1 can be specifically expressed in the stomata.
  • the BXL1 gene can be specifically expressed in the stomata.
  • the number of stomata of the mutant and overexpression materials did not change compared with the wild-type material, but the stomata opening was smaller in the mutant material.
  • the data is shown in Figure 6, which shows that the BXL1 gene regulates plant stomata The opening.
  • the stomata opening in the BXL1 mutant was significantly smaller than that in the wild type.
  • Stomatal opening experiments for detecting different genotypes For each genotype, fifty stomata openings (stoma opening widths) were calculated as technical repetitions. The biological independent experiments were repeated three times and the results were consistent.
  • the mock in the figure serves as a control group, which means water treatment, because all the compound solvents used in the experiment are water.
  • This result shows that, in addition to being resistant to drought and pathogenic bacteria, BXL1 can also resist high salt stress and osmotic stress.
  • the specific operation is as follows: cultivate 14-day-old wild Arabidopsis seedlings in 1% sucrose (weight to volume ratio) 1/2MS medium, and spray 2 ⁇ M SA, 300mM NaCl, 400mM Mannitol, 100 ⁇ M ABA water solution on the surface of the leaves evenly , Water (containing the same volume of absolute ethanol added to other treatments) as a control, put it back to the original growth environment, collect samples within a specific time, and perform downstream gene expression testing.
  • the specific method of drought treatment is as follows: Nutrient soil and vermiculite are mixed evenly according to the volume ratio of 1:2, and the same volume of mixed soil is evenly filled into each bowl, and then watered in the tray, so that each contains the same The soil bowl is evenly filled with water. Pick out 7-day-old seedlings of the same size and plant them in the prepared soil, put them under long-day conditions (16 hours of light, 8 hours of darkness, and light intensity of 220 ⁇ m.s -1 ), and water them normally for 10 days, then Stop watering and continuously record the drought phenotype. After the drought phenotype appears, you can rewater and record the phenotype. The results are shown in Figure 3-(B). Figure (B) shows that the mutant plants can better resist the drought environment.
  • the mutant Can maintain a higher survival rate.
  • BXL1 has homologous genes in many dicotyledonous plants, such as alfalfa, apple, flax, rape, radish, pepper, cherry, jujube, brasenia and alfalfa.
  • alfalfa a gene that is highly homologous to BXL1 is named Medtr2g034720.1.
  • the invention realizes the expression regulation of the Medtr2g034720.1 gene in alfalfa through gene editing technology, so as to change the cell wall composition of alfalfa, regulate the palatability of alfalfa as feed, and at the same time enhance the alfalfa's ability to resist biological and abiotic stress.
  • CRISPR short palindrome repetitions with regular aggregation intervals
  • the experimental design of the present invention selects the psgR-Cas9-At carrier, and designs the sg-RNA online at www.atum.bio/eCommerce/cas9/input.
  • the thermal cycle is 37 degrees for thirty minutes, 95 degrees for five minutes and then down to 25 degrees at five degrees per minute. Finally, the linearized vector backbone and the processed oligonucleotide are connected with T4 ligase. Connect for ten minutes at room temperature, the reaction system is as follows:
  • the ligated product is transformed into E. coli.
  • a medium containing 50 micrograms per milliliter of ampicillin antibiotics was used to screen positive single clones. Extract plasmids from positive clones and send them for sequencing. Ensure that the target gene has been connected to the vector.
  • Alfalfa plants with the Medtr2g034720.1 gene knocked out by CRISPR technology are expected to produce changes in growth phenotype, such as delayed germination and prolonged flowering time. However, the overall biomass will not decrease.
  • alfalfa plants lacking the Medtr2g034720.1 gene will have a different cell wall composition than wild-type plants. The main manifestation is that the content of lignin may be less, and the content of fiber and pectin will increase, thereby improving its use as feed. The palatability.
  • the alfalfa lacking the Medtr2g034720.1 gene can produce better resistance to drought and pathogenic bacteria, thereby improving the growth ability of alfalfa and expanding the growth range of alfalfa. This provides a powerful way for China to develop alfalfa as feed industry. stand by.

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Abstract

一种BXL基因或其编码蛋白的应用，具体地，当抑制BXL基因或其编码蛋白的表达时，可显著改善植物的性状，包括：(i)增强植物抗逆性；和/或(ii)抗病原菌；和/或(iii)减少木质素含量、增加纤维和果胶含量。此外， BXL基因或其编码蛋白的抑制剂还可用于饲料组合物，用于改善饲料适口性。

Description

一种BXL基因或其编码蛋白的应用 技术领域
本发明涉及农学领域，具体地，涉及一种BXL基因或其编码蛋白的应用。
背景技术
植物的生长过程中会经历各种各样生物或者非生物胁迫。干旱和致病菌作为危害植物生长的两大主要因子，受到人们的广泛专注。
然而目前的改良干旱和致病菌的主要手段为常规的浇灌技术和喷洒农药。常规的浇灌技术有：挖沟渠，造水库，拦河坝，引水，或者现在大棚常用的节水浇灌技术---滴灌。然而，这些常规的改良干旱的手段存在很多缺点，比如，实施难度较大，人力物力耗费较多，不经济，工程更改性差，改善干旱的范围有限，适用范围有限等。人们通过喷洒农药的方法来实现改良致病菌的措施，虽然直接，也存在很多不足，比如：农药的残留问题，危害人体健康，造成环境污染，污染水资源，破坏大气清洁，违背人类倡导的绿色理念。
因此，本领域迫切需有开发可帮助植物抵抗外界胁迫，尤其是抵抗干旱胁迫和病原菌的侵害的新的基因。
发明内容
本发明的目的在于提供一种可帮助植物抵抗外界胁迫，尤其是抵抗干旱胁迫和病原菌的侵害的新的基因。
本发明第一方面提供了一种BXL基因或其编码蛋白的抑制剂的用途，用于改良植物性状；或制备用于改良植物性状的组合物或制剂。
在另一优选例中，所述改良植物性状包括：
(i)增强植物抗逆性；和/或
(ii)抗病原菌；和/或
(iii)减少木质素含量、增加纤维和果胶含量。
在另一优选例中，所述抗逆性选自下组：抗旱性、耐盐性、耐渗透压、耐热性、或其组合。
在另一优选例中，所述病原菌选自下组：丁香假单胞杆菌、花菜菌、或其 组合。
在另一优选例中，所述病原菌选自下组：丁香假单胞菌番茄致病菌株DC3000、丁香假单胞菌番茄致病菌株T1、丁香假单胞菌番茄致病菌株3435、花菜菌4326、花菜菌4981、或其组合。
在另一优选例中，组合物或制剂还用于选自下组的一种或多种用途：
(a)推迟种子萌发；
(b)延迟开花时间；
(c)减小气孔开度；
(d)增强抗逆能力；
(e)增强抗病原菌能力；
(f)改善作为饲料的适口性。
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，所述组合物包含(a)BXL基因或其编码蛋白的抑制剂；和(b)农学上可接受的载体。
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、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、siRNA、shRNA、miRNA、小分子配体、或其组合。
在另一优选例中，所述抑制剂选自下组：脱落酸(ABA)、盐(NaCl)、水杨酸(SA)、甘露醇(Mannitol)、解淀粉芽孢杆菌GB03的挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述组合物还包括其他改善饲料适口性的物质。
在另一优选例中，所述其他改善饲料适口性的物质选自下组：纤维素水解酶酶、果胶合成酶、果胶添加剂、半乳糖酶、乙烯分子、脱落酸、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抗逆性的物质。
在另一优选例中，所述其他增强植物抗逆性的物质选自下组：脱落酸、脱落酸的类似物、胼胝质、脯氨酸、或其组合。
在另一优选例中，所述其他抗病原菌的物质选自下组：生物碱、黄酮类化合物、单宁、苯丙素、或其组合。
在另一优选例中，所述组合物还包括其他减少木质素含量、增加纤维和果胶含量的物质。
在另一优选例中，所述其他减少木质素含量、增加纤维和果胶含量的物质选自下组：糖基水解酶、阿拉伯糖苷酶、木糖苷酶、或其组合。
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，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、十字花科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、山柑科、芸香科、锦葵科、蔷薇科、木槿科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、苜蓿、苹果、亚麻荠、大豆、水稻、油菜、萝卜、辣椒、樱桃、枣、甘蓝、醉蝶花、柑橘、榴莲、或其组合。
在另一优选例中，BXL基因选自下组：BXL1、BXL2、BXL3、BXL4、BXL7、AT5G10560、AT3G19620、AT5G09700、或其组合。
在另一优选例中，所述BXL基因包括野生型BXL基因和突变型BXL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BXL基因编码的多肽与野生型BXL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BXL基因包括与野生型BXL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BXL基因包括在野生型BXL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BXL基因包括cDNA序列、基因组序列、或其组合。
在另一优选例中，所述BXL基因来自植物，较佳地，来自双子叶植物，更佳地，来自选自下组的一种或多种植物：拟南芥、苜蓿、苹果、亚麻荠、大豆、油菜、萝卜、辣椒、樱桃、枣、甘蓝、醉蝶花、柑橘、榴莲。
在另一优选例中，所述BXL基因选自下组：拟南芥的BXL1基因(基因登录号为：AT5G49360)、亚麻荠的木糖苷酶1基因(Camelina sativa(L.)Crantz的β-D- 木糖苷酶1，基因登录号为：LOC104723790)、苜蓿的木糖苷酶基因(Medicago Sativa Linn的β-D-木糖苷酶，基因登录号为：Medtr2G034720.1)、甘蓝型油菜的木糖苷酶基因(Brassica napus L.的β-D-木糖苷酶，基因登录号为：LOC106365857)、蔷薇木糖苷酶2基因(Rosa sp.的β-D-木糖苷酶2基因，基因登录号为：LOC112179881)、或其组合。
在另一优选例中，所述BXL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，具有所述调控农艺性状功能的、由(i)衍生的多肽；或(iii)氨基酸序列与SEQ ID NO.:1所示氨基酸序列的同源性≥90％(较佳地≥95％，更佳地≥98％)，具有改良植物农艺性状功能的多肽。
在另一优选例中，所述BXL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.:2所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:2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.:2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。
本发明第二方面提供了一种组合物，包括：
(a)BXL基因或其编码蛋白的抑制剂；和
(b)农学上可接受的载体。
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，所述农用组合物选自下组：饲料组合物、有机肥组合物、农药组合物、或其组合。
在另一优选例中，所述饲料组合物包括固体饲料组合物或液体饲料组合物。
在另一优选例中，所述饲料组合物为植物养殖添加剂。
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，所述组合物中，所述BXL基因或其编码蛋白的抑制剂的含量(wt％)为0.05％-10％，较佳地，0.1％－8％，更佳地，0.5％－6％。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、反义核酸、Crispr试剂、siRNA、shRNA、miRNA、小分子配体、或其组合。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物、或其组合。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：脱落酸(ABA)、盐(NaCl)、水杨酸(SA)、甘露醇(Mannitol)、解淀粉芽孢杆菌GB03的挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述组合物还包括其他改善饲料适口性的物质。
在另一优选例中，所述其他改善饲料适口性的物质选自下组：纤维素水解酶酶、果胶合成酶、果胶添加剂、半乳糖酶、乙烯分子、脱落酸、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抗逆性的物质。
在另一优选例中，所述其他增强植物抗逆性的物质选自下组：脱落酸、脱落酸的类似物、胼胝质、脯氨酸、或其组合。在另一优选例中，所述其他抗病原菌的物质选自下组：生物碱、黄酮类化合物、单宁、苯丙素、或其组合。
在另一优选例中，所述组合物还包括其他减少木质素含量、增加纤维和果胶含量的物质。
在另一优选例中，所述其他减少木质素含量、增加纤维和果胶含量的物质选自下组：糖基水解酶、阿拉伯糖苷酶、木糖苷酶、或其组合。
本发明第三方面提供了一种本发明第二方面所述的组合物的用途，用于改良植物农艺性状。
本发明第四方面提供了一种改良饲料适口性的方法，包括步骤：
降低所述植物中BXL基因或其编码蛋白的表达量和/或活性，从而改良饲料适口性。
在另一优选例中，所述方法包括给予植物BXL基因或其编码蛋白的抑制剂。
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BXL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BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、反义核酸、Crispr试剂、siRNA、shRNA、miRNA、小分子配体、或其组合。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述抑制剂选自下组：脱落酸(ABA)、盐(NaCl)、水杨酸(SA)、甘露醇(Mannitol)、解淀粉芽孢杆菌GB03的挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述“降低”是指BXL基因或其编码蛋白的表达或活性降低满足以下条件：
A1/A0的比值≤80％，较佳地≤50％，更佳地≤20％，最佳地为0-10％；其中，A1为BXL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植株中相同BXL基因或其编码蛋白的表达或活性。
在另一优选例中，所述的降低指与野生型BXL基因或其编码蛋白的表达水平E0相比，所述植株中BXL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％。
在另一优选例中，所述的降低植株中BXL基因或其编码蛋白的表达或活性通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、ZFN(锌指核酸内切酶技术)、TALEN(类转录激活因子效应物核酸酶)、或其组合。
本发明第五方面提供了一种改良植物性状的方法，包括步骤：
降低所述植物中BXL基因或其编码蛋白的表达量和/或活性，从而改良植物的性状。
在另一优选例中，所述改良植物性状包括：
(i)增强植物抗逆性；和/或
(ii)抗病原菌；和/或
(iii)减少木质素含量、增加纤维和果胶含量。
在另一优选例中，所述改良植物性状还包括：
(a)推迟种子萌发；
(b)延迟开花时间；
(c)减小气孔开度；
(d)增强抗逆能力；
(e)增强抗病原菌能力；
(f)改善作为饲料的适口性。
在另一优选例中，所述减少木质素含量指与野生型植物相比，木质素含量减少了≥50％，较佳地，≥70％，更佳地，≥80％，更佳地，≥90％。在另一优选例中，所述增加纤维和果胶含量指与野生型植物相比，纤维和果胶含量增加了≥30％，较佳地，≥40％，更佳地，≥50％，更佳地，≥60％。
在另一优选例中，所述减小气孔开度指与野生型植物相比，气孔开度减小了≥50％，较佳地，≥50％，更佳地，≥60％，更佳地，≥70％。
在另一优选例中，当所述植物中BXL的活性E1与所述植物中的野生型BXL本底活性E0之比≤1/2，较佳地≤1/5，更佳地≤1/10时，所述植物的性状改良包括：
(i)增强植物抗逆性；和/或
(ii)抗病原菌；和/或
(iii)减少木质素含量、增加纤维和果胶含量；和/或
(iv)推迟种子萌发；和/或
(v)延迟开花时间；和/或
(vi)减小气孔开度。
在另一优选例中，所述“降低”是指BXL基因或其编码蛋白的表达或活性降低满足以下条件：
A1/A0的比值≤80％，较佳地≤50％，更佳地≤20％，最佳地为0-10％；其中，A1为BXL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植株中相同BXL基因或其编码蛋白的表达或活性。
在另一优选例中，所述的降低指与野生型BXL基因或其编码蛋白的表达水平E0相比，所述植株中BXL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％。
在另一优选例中，所述的降低植株中BXL基因或其编码蛋白的表达或活性通 过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、ZFN(锌指核酸内切酶技术)、TALEN(类转录激活因子效应物核酸酶)、或其组合。
在另一优选例中，所述方法包括给予植物BXL基因或其编码蛋白的抑制剂。
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BXL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(如转基因)的植物或植物细胞。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、反义核酸、Crispr试剂、siRNA、shRNA、miRNA、小分子配体、或其组合。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述抑制剂选自下组：脱落酸(ABA)、盐(NaCl)、水杨酸(SA)、甘露醇(Mannitol)、解淀粉芽孢杆菌GB03的挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
本发明第六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：
降低植物组织或植物细胞中的BXL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，所述方法还包括向植物组织或植物细胞中导入BXL基因或其编码蛋白的抑制剂。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、反义核酸、Crispr试剂、siRNA、shRNA、miRNA、小分子配体、或其组合。
在另一优选例中，所述BXL基因或其编码蛋白的抑制剂选自下组：小分子化合物、抗体、细菌挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
在另一优选例中，所述抑制剂选自下组：脱落酸(ABA)、盐(NaCl)、水杨酸(SA)、甘露醇(Mannitol)、解淀粉芽孢杆菌GB03的挥发物(包括乙偶烟、二,三-丁二醇、二,三-丁二酮、异戊醇、正丁醇)、或其组合。
本发明第七方面提供了一种制备基因工程植物的方法，包括步骤：
将本发明第六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。
在另一优选例中，所述方法包括用RNA干扰技术、Crispr技术、ZFN(锌指核酸内切酶技术)、TALEN(类转录激活因子效应物核酸酶)降低植物组织或植物细胞中的BXL基因或其编码蛋白的表达和/或活性。
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。
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BXL1基因在拟南芥中的组织定位。图中可以明确表明BXL1可以在维管组织中表达。在叶片中，本发明首次发现了BXL1可以特异的在气孔中表达。
图2为BXL1在野生型，突变体和过表达植物里的基因表达情况。定量PCR检测出BXL1突变体或者过表达株系的转录本水平的BXL1基因的表达水平。结果显示，过表达的植株，相比较于野生型植株，BXL1基因的表达被明显的提高。然而突变体的BXL1基因的表达相比较于野生型植株被明显的抑制。
图3(A)(B)两个图的结果显示了BXL1基因抗致病菌和干旱的基本表型。(A)图表示BXL1突变体植株可以有更好的抵抗病原菌的能力，被感染的程度相比较于野生型有所减轻。(B)图表示了突变体植株可以更好的抵抗干旱环境，在同样的干旱条件下，相比较于野生型植株，突变体可以保持更高的存活率。
图4(A)(B)两个图的结果显示了BXL1基因对植物正常生长的影响。(A)图表示BXL1突变体植株可以有缓慢的种子萌发的速度，(B)图表示了突变体植株呈现出缓慢的生长速度。在同一生长时期，相比较于野生型，BXL1突变体表 现出了更小的植物形态，开花时间也会缓慢一些，开始结果时，果荚的长度相比较与野生型也短些。
图5(A)(B)两个图的结果显示了BXL1基因对植物最终的单株植株的生物量以及单株植物种子的种子千粒重量并没有影响。(A)图表示BXL1突变体植株在达到开花时期时，相比较于野生型在达到开花时的单颗植株的干重并没有明显改变，(B)图表示了突变体植株最终收获的种子，相比较于野生型单株收获的种子，种子千粒重并没有呈现出干重差异。
图6显示了BXL1基因调控了植物气孔的开度。BXL1突变体中的气孔开度相比较于野生型，明显变小。
图7显示，BXL1基因受到多种胁迫因子的调控。水杨酸，盐，脱落酸，甘露醇，这些胁迫因子都能够显著下调BXL1基因在特定的时间里的表达。图中mock表示的是水处理，作为对照组。
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，首次意外地发现了一种BXL基因或其编码蛋白，其所编码的蛋白为糖基水解酶，当抑制BXL基因或其编码蛋白的表达时，可显著改善植物的性状，包括(i)增强植物抗逆性；和/或(ii)抗病原菌；和/或(iii)减少木质素含量、增加纤维和果胶含量。此外，本发明人还发现，抑制BXL基因或其编码蛋白的表达时还会(a)推迟种子萌发；(b)延迟开花时间；(c)减小气孔开度等。本发明人在此基础上完成了本发明。
BXL基因
编码糖基水解酶的基因被命名为β-D-木糖苷酶1(BXL1)，具有编码β-D-木糖苷酶和{alpha}-L-阿拉伯呋喃糖苷酶的双重功能。BXL1存在细胞质外的区域，在细胞壁二次增厚的组织中特异性表达，如维管组织和次生细胞壁。BXL1属于糖基水解酶家族三中的一员，控制种子的萌芽，水解阿拉伯聚糖和木聚糖。正常种子在萌发时，在种子周围会释放完整的粘性液，以此来帮助种皮软化，促发幼苗形态建成。
BXL1在不同的组织中起到不同的作用，在茎干组织中主要起到木糖苷酶的作用，而在种子的萌发过程中，则主要起到阿拉伯呋喃糖苷酶的作用。
拟南芥中，BXL1存在7个同源的基因，分别是BXL2、BXL3、BXL4、BXL7、AT5G10560、AT3G19620、AT5G09700。
对蛋白结构域的相似性进行了同源度分析，结果表明，编码7个同源基因(BXL2、BXL3、BXL4、BXL7、AT5G10560、AT3G19620、AT5G09700)的蛋白与BXL1蛋白的同源度分别为99％，98％，98％，97％，97％，96％，97％。
对BXL1表达组织的研究发现，BXL1基因存在于茎干，果荚，以及维管组织中。
本发明对BXL1进行了更为深入研究，通过组织染色，发现BXL1基因可以特异地在气孔的保卫细胞中表达。生理生化的研究结果表明，BXL1基因在帮助植物抵抗外界胁迫方面起着重要作用，尤其是抵抗干旱胁迫和病原菌的侵害。这一发现不仅为研究次生细胞壁相关的基因在植物抗旱抗病方面打开一个新思路，也为BXL1基因的功能增添了新的内容。
如本文所用，术语“本发明的BXL基因”、“BXL基因”可互换使用，均指来源于单子叶或双子叶植物(如拟南芥、苜蓿、苹果等)的BXL基因或其变体。在一优选实施方式中，本发明的BXL基因的核苷酸序列如SEQ ID NO.:2所示。
代表性的其他物种的BXL(如BXL1)同源基因包括(但并不限于)：拟南芥的BXL基因(BXL1)、苜蓿的BXL同源基因(Medtr2G0347201和拟南芥相似性80％)、亚麻荠的BXL同源基因(β-D-木糖苷酶1(LOC104723790)和拟南芥相似性100％)、甘蓝型油菜的BXL同源基因(β-D-木糖苷酶1(LOC106365857)和拟南芥相似性99％)。
本发明还包括与本发明的优选基因序列(SEQ ID NO.:2)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调控植物(如拟南芥、苜蓿等)的农艺性状。“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.:2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.:2的衍生序列，由于密码子的简并性，即使与SEQ ID NO.:2的同源性较低，也能基本编码出如SEQ ID NO.:1所示的氨基酸序列。另外，“在SEQ ID NO.:2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.:2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拟南芥和苜蓿，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.:2所示(拟南芥))具有一定同源性(保守性)的BXL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。
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.:2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。
应理解，虽然本发明的BXL基因优选来自拟南芥，但是来自其它植物的与拟南芥BXL基因高度同源(如具有80％以上，如85％,90％,95％甚至98％，99％，或100％序列相同性)的其它基因也在本发明考虑的范围之内。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BXL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。
BXL基因编码的多肽
如本文所用，术语“本发明多肽”、“BXL基因的编码蛋白”、可以互换使用，都是指来源于植物(如拟南芥)的BXL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所示(拟南芥)。
本发明涉及一种调控性状的BXL多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO.:1所示。本发明的多肽能够有效调控植物(如单子叶植物或双子叶植物)的性状。
本发明还包括与本发明的SEQ ID NO.:1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：“调控植物(如单子叶植物或双子叶植物)的农艺性状”。
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BXL蛋白活性的BXL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BXL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
Figure PCTCN2020071199-appb-000001
Figure PCTCN2020071199-appb-000002
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。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明还提供了一种包括本发明的基因的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达本发明目的基因时，将目的基因连接入适合的多克隆位点或酶切位点内，从而将目的基因与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5’到3’方向)：启动子，目的基因，和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3’多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。
在本发明中，编码本发明蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的基因的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。使用本发明的基因构建重组表达载体时，可在其转录起始核苷酸前加上任何一种增强型、组成型、组织特异型或诱导型启动子。
包括本发明基因、表达盒的载体可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如水稻细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
农用制剂
可将本发明的活性物质(如BXL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BXL基因或其编码蛋白的抑制剂作为改良植物性状(如，增强植物抗逆性(如抗旱性、耐盐性、耐渗透压、耐热性等)；和/或抗病原菌；和/或减少木质素含量、增加纤维和果胶含量；和/或推迟种子萌发；和/或延迟开花时间；和/或减小气孔开度；和/或改善作为饲料的适口性)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饲料组合物、有机肥组和物、或农药组合物。
在一优选实施方式中，本发明的农用制剂包括包括固体饲料组合物或液体饲料组合物。
在一优选实施方式中，本发明的饲料组合物为植物养殖添加剂。
本发明的农用制剂可与其他改善饲料适口性的物质(如纤维素水解酶酶、果胶合成酶、果胶添加剂、半乳糖酶、乙烯分子、脱落酸等)联用。
本发明的农用制剂可与其他抗逆剂(如抗旱剂、耐盐剂、耐渗透压剂、耐热剂等)制成一种混合物存在于它们的商品制剂中或从这些制剂制备的使用剂型中，这些其他的抗旱剂包括(并不限于)：抗旱种衣剂、抗旱保水剂、或抗旱喷洒剂等；这些其他的耐盐剂包括(但并不限于)：耐盐微生物菌剂、耐盐增稠剂；这些其他的耐渗透压剂包括(但并不限于)：喷雾剂、生物蛋白质保护 剂、海藻糖；这些其他的耐热剂包括(但并不限于)：耐热薄膜、生物蜡制剂、水化膜、海藻糖。
本发明的农用制剂抗与其他的抗病原菌剂制成一种混合物存在于它们的商品制剂中或从这些制剂制备的使用剂型中，这些其他的抗病原菌剂包括(并不限于)：皂角苷、酚类物质、抗病化合物、有机硫化物、不饱和脂肪酸。
此外，本发明的农用制剂也可与增效剂制成一种混合物存在于它们的商品制剂中或从这些制剂制备的使用剂型中，这些增效剂是提高活性化合物作用的化合物，由于活性化合物本身有活性，也可不必加增效剂。
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(如单子叶植物或双子叶植物)性状的改良
本发明还提供了一种改良植物(如单子叶植物或双子叶植物)性状的方法，所述的改良包括：(i)增强植物抗逆性；和/或(ii)抗病原菌；和/或(iii)减少木质素含量、增加纤维和果胶含量，包括步骤：降低所述植物中BXL基因或其编码蛋白的表达量和/或活性，或添加BXL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(i)增强植物抗逆性；和/或(ii)抗病原菌；和/或(iii)减少木质素含量、增加纤维和果胶含量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)本发明首次筛选到一种BXL基因，该基因编码糖基水解酶，通过抑制BXL基因或其编码蛋白的表达或活性，可改良植物性状。
(2)本发明首次发现，降低BXL基因或其编码蛋白的表达可显著(i)增强植物抗逆性；和/或(ii)抗病原菌；和/或(iii)减少木质素含量、增加纤维和果胶含量。
(3)本发明首次发现，降低BXL基因或其编码蛋白的表达可改善饲料的适口性。
(4)本发明首次发现，降低BXL基因或其编码蛋白的表达还可(a)推迟种子萌发；(b)延迟开花时间；(c)减小气孔开度；(d)增强植物的抗逆性；(e)增强植物的抗病菌能力；(f)改善植物作为饲料的适口性。
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
以下实例涉及到的拟南芥型号为Col-0,SALK_012090C(bxl1-1),SALK_086578C(bxl1-2)来自拟南芥生物资源中心(ABRC)。
以拟南芥为例。
1，突变体获得
从解淀粉芽孢杆菌菌株GB03(一种商品化的模式促进植物生长的有益植物根际促生菌，购自ATCC，全球生物资源中心)诱导的全基因组转录水平测序的数据中，发现了一个显著下调的基因，beta-xylosidase 1(BXL1)。在Tair网站查到该基因的T-DNA插入突变体的种子编号，在拟南芥生物资源中心(ABRC)订购了SALK_0120909C和SALK_86578C两个T-DNA插入突变体。通过基因型鉴定，确定无误后，观察发现，BXL1基因功能缺失突变体在生长过程中，表现为推迟萌芽，稍慢的营养生长和延迟的开花时间，结果见图4，(A)图表示BXL1突变体植株可以有缓慢的种子萌发的速度，(B)图表示了突变体植株呈现出缓慢的生长速度。在同一生长时期，相比较于野生型，BXL1突变体表现出了更小的植物形态，开花时间也会缓慢一些，开始结果时，果荚的长度相比较与野生型也短些。
与野生型相比，同一生长时期，BXL1功能缺失型植株表型出较小的叶片面积，然而当突变体达到开花阶段时，植株的叶面积与同一时期的野生型相比并没有减少；抽薹开花之后，突变体呈现较矮的茎干，较细小的果荚，然而成 熟之后，植株的生物量和单株的种子总重量与野生型相比并没有呈现出显著差异，如图5所示，图5，(A)(B)两个图的结果显示了BXL1基因对植物最终的单株植株的生物量以及单株植物种子的种子千粒重并没有影响。(A)图表示BXL1突变体植株在达到开花时期时，相比较于野生型在达到开花时的单颗植株的干重并没有明显改变，(B)图表示了突变体植株最终收获的种子，相比较于野生型单株收获的种子，并没有呈现出单株种子千粒重差异。
2，转基因获得过表达材料
利用转基因技术，构建了Pro BXL1:GUS和35S:BXL1:flag材料。取BXL1起始密码子上游的2.8k作为其启动子分析(不包括5’UTR区域)，通过Getway方法融合入pGWB533。全长的BXL1CDs通过Getway方法融合入pGWB511。转录水平检测不同材料BXL1的表达，结果如图2所示，定量PCR检测出BXL1突变体或者过表达株系的转录本水平的BXL1基因的表达水平。结果显示，过表达的植株，BXL1基因的表达被明显的提高了，相比较于野生型植株。然而突变体的BXL1基因的表达被明显的抑制了。
检测所用引物为：F 5’-AGTTGATGTTGATGCTTGCA-3’(SEQ ID NO.:3)，R 5’-AAGTTGCGGTTGGACCAAAA-3’(SEQ ID NO.:4)。通过组织化学法进行GUS染色，发现BXL1可以特异的在气孔中表达，如图1所示，BXL1基因可以特异的在气孔中表达。在显微镜观测下，突变体和过表达材料的气孔数目相较于野生型材料没有变化，但是气孔开度在突变体材料里更小，数据见图6，图6显示了BXL1基因调控了植物气孔的开度。BXL1突变体中的气孔开度相比较于野生型，明显变小。检测不同基因型的气孔开度实验，每个基因型每次计算五十个气孔的开度(气孔的张开宽度)作为技术重复，生物学独立实验重复了三次，结果均一致。
3，干旱和病原菌的胁迫处理
鉴于BXL1的组织表达特异性和生长表型，检测了生物胁迫和非生物胁迫因子(水杨酸SA,盐NaCl,甘露醇Mannitol,脱落酸ABA)对BXL1基因转录水平的影响，发现这些因子都可以使BXL1显著下调，结果见图7，BXL1基因受到多种胁迫因子的调控。水杨酸，盐，脱落酸，甘露醇，这些胁迫因子都能够显著下调BXL1基因的表达在特定的时间里。图中mock作为对照组，表示的是水处理，因为所有用来实验的化合物溶剂是水。此结果表明，BXL1除了可以抗干旱和抗病原菌侵害之外，还可以抵抗高盐胁迫，以及渗透胁迫。具体操 作为：还有1％的蔗糖(重量体积比)1/2MS培养基培养14天大的野生拟南芥苗，2μM SA，300mM NaCl,400mM Mannitol,100μM ABA水溶液均匀的喷洒在叶子的表面，水(含有加入其它处理中的同样体积的无水乙醇)作为对照，放回原来的生长环境，在特定的时间内收集样品，进行下游的基因表达检测。基于此，进行了干旱和病原菌的实验。发现功能缺失型突变体bxl1可以更好的抵抗干旱，同时也可以增强对丁香假单胞菌假单胞菌Pst DC3000(一种商品化的模式致病菌，购自ATCC,全球生物资源中心)的抗性，结果如图3所示，图3，(A)(B)两个图的结果显示了BXL1基因抗致病菌和干旱的基本表型。(A)图表示BXL1突变体植株可以有更好的抵抗病原菌的能力，被感染的程度相比较于野生型有所缓减。干旱处理的具体方法为：营养土和蛭石按照1:2的体积比例混合均匀，均匀的装入相同体积的混合土壤到每一个钵里，然后在托盘里浇水，让每一个装有相同土壤的钵子均匀的吸满水。挑出相同大小的7天大的苗子种植在准备好的土壤里，放到长日照条件下(16小时光照，8小时黑暗，光强为220μm.s -1)，正常浇水10天，然后停止浇水，连续记录干旱表型。出现干旱表型后，可进行复水，记录表型，结果见图3-(B)，(B)图表示了突变体植株可以更好的抵抗干旱环境，在同样的干旱条件下，突变体可以保持更高的存活率。抗病实验的细节为：野生型，突变体和过表达的转基因植株种植在短日照的条件下(14小时光照，10小时黑暗，110μm.s -1)至4周大小，Pst DC3000预先在28度培养箱里活化好，用无菌的10mM MgCl 2悬浮DC3000至终浓度为5×10 7cfu/mL(约OD＝0.1)，加入终浓度为0.02％(V/V)的表面活性剂Silwet L-77。菌液均匀的喷洒在植物叶子上，然后附上保鲜膜保持6个小时，3天之后随机取样进行定量分析，5天之后，记录病原菌的侵染表型，结果见图3-(B)干旱实验独立重复了4次，结果呈现一致表型。图3-(A)抗病实验独立重复两次，结果显示一致。
实施例2
BXL1在很多双子叶植物中均存在同源基因，比如苜蓿，苹果，亚麻，油菜，萝卜，辣椒，樱桃，枣，莼菜以及苜蓿。在苜蓿中，与BXL1高度同源的基因被命名为Medtr2g034720.1。本发明通过基因编辑技术实现苜蓿中Medtr2g034720.1基因的表达调控，来达到改变苜蓿细胞壁成分，调节苜蓿作为饲料的适口性，同时增强苜蓿的抵抗生物和非生物胁迫能力。
CRISPR(定期聚集间隔短回文重复)是一项很强大的基因编辑技术，已经在动、植物中能够广泛应用。本发明的实验设计选择psgR-Cas9-At载体，在线设计sg-RNA，网址为www.atum.bio/eCommerce/cas9/input。为了有效的将本发明的目的片段构建到psgR-Cas9-At载体上，我们合成两种形式的寡核苷酸接头，分别为：5’–GATTGNNNNNNNNNNNNNNNNNNN–3’(SEQ ID NO.:5)和5’–TGGCGNNNNNNNNNNNNNNNNNNN–3’(SEQ ID NO.:6)。反向都是3’–CNNNNNNNNNNNNNNNNNNNCAAA–5(SEQ ID NO.:7)。寡核苷酸退火和克隆到骨架载体的流程为：
先用BbsI f对psgR-Cas9-At/Os载体在37度下酶切三十分钟:
Figure PCTCN2020071199-appb-000003
然后对酶切产物进行纯化回收。然后对每对寡核苷酸磷酸化并退火。反应体系为：1ul
Figure PCTCN2020071199-appb-000004
热循坏程序为37度三十分钟95度五分钟然后以每分钟五度降至二十五度。最后将线性化的载体骨架和处理好的寡核苷酸用T4连接酶连接起来。室温链接十分钟，反应体系如下：
XμL来自步骤2的BbsI消化的psgR-Cas9-At/Os(50ng)(X ul BbsI digested psgR-Cas9-At/Os from step 2(50ng))
来自步骤3的1ul磷酸化和退火的寡核苷酸双链体(1：200稀释)(1 ul phosphorylated and annealed oligo duplex from step 3(1:200dilution))
5ul 2X快速连接缓冲液(NEB)(5ul 2X Quickligation Buffer(NEB))
Figure PCTCN2020071199-appb-000005
最后将连接好的产物转化进入大肠杆菌。并用含有五十微克每毫升氨苄抗生素的培养基进行阳性单克隆的筛选。对阳性克隆摇菌提质粒，送测序。确保目的基因已经连入载体。
通过CRISPR技术敲除Medtr2g034720.1基因的苜蓿植株，预计会产生生长表型上的变化，比如萌芽推迟，开花时间延长等。然而总体的生物量并不会降低。作为饲料的主要材料，缺失Medtr2g034720.1基因的苜蓿植株较比野生型植株细胞壁成分会有变化，主要表现为，木质素含量可能较少，纤维含量和果胶含量会增多，从而改善其作为饲料的适口性。同时，缺失Medtr2g034720.1基因的苜蓿能产生更好的抵抗干旱和病原菌的能力，从而提高了苜蓿的生长能力，扩大了苜蓿的生长范围，这为我国发展以苜蓿为饲料的产业提供了有力的支持。
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 (23)

  1. 一种BXL基因或其编码蛋白的抑制剂的用途，其特征在于，用于改良植物性状；或制备用于改良植物性状的组合物或制剂。
  2. 如权利要求1所述的用途，其特征在于，所述改良植物性状包括：
    (i)增强植物抗逆性；和/或
    (ii)抗病原菌；和/或
    (iii)减少木质素含量、增加纤维和果胶含量。
  3. 如权利要求1所述的用途，其特征在于，所述组合物或制剂还用于选自下组的一种或多种用途：
    (a)推迟种子萌发；
    (b)延迟开花时间；
    (c)减小气孔开度；
    (d)增强抗逆能力；
    (e)增强抗病原菌能力；
    (f)改善作为饲料的适口性。
  4. 如权利要求1所述的用途，其特征在于，所述植物包括草本植物和木本植物。
  5. 如权利要求4所述的用途，其特征在于，所述草本植物选自下组：茄科、禾本科植物、豆科植物、十字花科植物、或其组合。
  6. 如权利要求4所述的用途，其特征在于，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、山柑科、芸香科、锦葵科、蔷薇科、木槿科、或其组合。
  7. 如权利要求1所述的用途，其特征在于，所述的植物选自下组：拟南芥、苜蓿、苹果、亚麻荠、大豆、水稻、油菜、萝卜、辣椒、樱桃、枣、甘蓝、醉蝶花、柑橘、榴莲、或其组合。
  8. 如权利要求1所述的用途，其特征在于，所述BXL基因选自下组：BXL1、BXL2、BXL3、BXL4、BXL7、AT5G10560、AT3G19620、AT5G09700、或其组合。
  9. 如权利要求1所述的用途，其特征在于，所述BXL基因来自植物，较佳地，来自双子叶植物，更佳地，来自选自下组的一种或多种植物：拟南芥、苜蓿、苹果、亚麻荠、大豆、油菜、萝卜、辣椒、樱桃、枣、甘蓝、醉蝶花、柑橘、榴莲。
  10. 如权利要求1所述的用途，其特征在于，所述BXL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，具有所述调控农艺性状功能的、由(i)衍生的多肽；或(iii)氨基酸序列与SEQ ID NO.:1所示氨基酸序列的同源性≥90％(较佳地≥95％，更佳地≥98％)，具有改良植物农艺性状功能的多肽。
  11. 如权利要求1所述的用途，其特征在于，所述BXL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.:2所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.:2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.:2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。
  12. 一种组合物，其特征在于，包括：
    (a)BXL基因或其编码蛋白的抑制剂；和
    (b)农学上可接受的载体。
  13. 如权利要求12所述的组合物，其特征在于，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。
  14. 如权利要求12所述的组合物，其特征在于，所述组合物还包括其他改善饲料适口性的物质。
  15. 如权利要求12所述的组合物，其特征在于，所述组合物还包括其他增强植物抗逆性的物质。
  16. 如权利要求12所述的组合物，其特征在于，所述组合物还包括其他减少木质素含量、增加纤维和果胶含量的物质。
  17. 一种权利要求12所述的组合物的用途，其特征在于，用于改良植物农艺性状。
  18. 一种改良饲料适口性的方法，其特征在于，包括步骤：
    降低所述植物中BXL基因或其编码蛋白的表达量和/或活性，从而改良饲 料适口性。
  19. 如权利要求18所述的方法，其特征在于，所述的降低植株中BXL基因或其编码蛋白的表达或活性通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、ZFN、TALEN、或其组合。
  20. 一种改良植物性状的方法，其特征在于，包括步骤：
    降低所述植物中BXL基因或其编码蛋白的表达量和/或活性，从而改良植物的性状。
  21. 如权利要求20所述的方法，其特征在于，所述改良植物性状包括：
    (i)增强植物抗逆性；和/或
    (ii)抗病原菌；和/或
    (iii)减少木质素含量、增加纤维和果胶含量。
  22. 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：
    降低植物组织或植物细胞中的BXL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。
  23. 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22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。
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