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WO2025086073A1 - Protéine 2-odd, gène, vecteur, cellule, composition et utilisation associée - Google Patents

Protéine 2-odd, gène, vecteur, cellule, composition et utilisation associée Download PDF

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Publication number
WO2025086073A1
WO2025086073A1 PCT/CN2023/126049 CN2023126049W WO2025086073A1 WO 2025086073 A1 WO2025086073 A1 WO 2025086073A1 CN 2023126049 W CN2023126049 W CN 2023126049W WO 2025086073 A1 WO2025086073 A1 WO 2025086073A1
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Prior art keywords
amino acid
acid sequence
seq
mutated
phenylalanine
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Chinese (zh)
Inventor
杨光富
林红艳
董进
叶宝琴
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Central China Normal University
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Central China Normal University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to a 2ODD protein, a gene, a vector, a cell, a composition and applications thereof.
  • the gene HIS1 in rice namely 4-hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No.1
  • multiple homologous genes of HIS1 were found in rice, named HSL (HIS1-Like).
  • HSL genes were also found in other plants, such as barley, wheat, sorghum, corn, and Arabidopsis.
  • HIS1 and HSL found that the protein encoded by HIS1 and its homologous gene HSL can catalyze triketone HPPD inhibitors, and the inhibitory activity of the catalytic product on HPPD is greatly reduced. Since HPPD is an important target of herbicides, the important function of HIS1 and HSL in detoxifying HPPD inhibitors can be used to develop crops resistant to HPPD herbicides.
  • HPPD inhibitor herbicides There are 13 types of HPPD inhibitor herbicides currently on the market. Among the more than 20 types of herbicides in the world, HPPD inhibitor herbicides have grown rapidly in recent years and have attracted much attention from the industry. Their low resistance risk is the cornerstone for this type of product to achieve leading growth. Based on the good product characteristics of HPPD inhibitor herbicides, their future market still has growth potential.
  • HPPD inhibitor herbicides are mostly used in corn and wheat fields, and are not widely used in cash crop planting areas such as peanuts, soybeans, rice, and sorghum due to crop safety issues.
  • HPPD inhibitors in the research and development stage have not been further developed due to the lack of certain crop safety.
  • Herbicide-resistant crops have brought huge benefits to farmers and the environment in the past 20 years, such as glyphosate-resistant corn and soybeans.
  • the purpose of the present invention is to overcome the defect that the inhibitory activity and crop safety of the existing HPPD inhibitor herbicides cannot be taken into account at the same time, and to provide a class of Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase proteins resistant to HPPD inhibitors, their encoding genes and their applications.
  • the first aspect of the present invention provides an Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein, which has an amino acid sequence selected from at least one of the following:
  • (6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted or added to the third amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the third amino acid sequence;
  • the second aspect of the present invention provides a gene encoding an Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect.
  • the third aspect of the present invention provides a recombinant vector, which contains the gene described in the second aspect.
  • the fourth aspect of the present invention provides a transgenic cell, which contains the gene described in the second aspect.
  • the fifth aspect of the present invention provides a composition comprising the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect.
  • the sixth aspect of the present invention provides the use of at least one of the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.
  • the seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to the herbicide.
  • the eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in a mutant crop containing the recombinant vector described in the third aspect into a target plant through hybridization, breeding or backcrossing, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to the herbicide.
  • the ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase gene of a target plant by a CRISPR/Cas gene editing method, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect above to obtain resistance to the herbicide.
  • the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein resistant to HPPD inhibitors and its encoding gene provided by the present invention have at least the following advantages:
  • the target plant By transferring the gene encoding the enzyme provided by the present invention into the target plant, the target plant can acquire resistance to HPPD inhibitors (herbicides), but the catalytic activity of the enzyme itself is basically unaffected. Therefore, the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein and its encoding gene provided by the present invention can be used to cultivate plants with herbicide resistance.
  • HPPD inhibitors herebicides
  • the first aspect of the present invention provides an Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein having an amino acid sequence selected from at least one of the following:
  • (6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted or added to the third amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the third amino acid sequence;
  • unchanged enzyme activity means that under the same assay conditions, the percentage (relative activity) between the enzyme activity of the proteins derived from the first amino acid sequence, the second amino acid sequence, the third amino acid sequence, the fourth amino acid sequence, the fifth amino acid sequence, the sixth amino acid sequence, the seventh amino acid sequence, and the eighth amino acid sequence and the enzyme activity of the wild type is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).
  • the first amino acid sequence is an amino acid sequence selected from at least one of the following:
  • the first amino acid sequence is the amino acid sequence shown by H141F or H141F/F205L.
  • the amino acid sequence derived from the mutation of histidine at position 141 in the amino acid sequence shown in SEQ ID NO:1 to phenylalanine is called H141F.
  • the second amino acid sequence is the following amino acid sequence:
  • (2c) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to tryptophan.
  • the second amino acid sequence is at least one of the amino acid sequences shown by F140H/L204F/F298L/I335F, L204F/F298L/I335A, and L204F/F298L/I335W.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:2, in which the phenylalanine at position 140 is mutated to histidine, the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to phenylalanine, is called F140H/L204F/F298L/I335F.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:2, in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to alanine is called L204F/F298L/I335A.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:2, in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to tryptophan, is called L204F/F298L/I335W.
  • the third amino acid sequence is the following amino acid sequence:
  • the third amino acid sequence is at least one of the amino acid sequences shown by T206F, T206F/F301L, and T206F/F301L/Y339F.
  • the amino acid sequence derived from the mutation of threonine at position 206 in the amino acid sequence shown in SEQ ID NO:3 to phenylalanine is called T206F.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:3 in which the threonine at position 206 is mutated to phenylalanine and the phenylalanine at position 301 is mutated to leucine is called T206F/F301L.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:3, in which the threonine at position 206 is mutated to phenylalanine, the phenylalanine at position 301 is mutated to leucine, and the tyrosine at position 339 is mutated to phenylalanine is called T206F/F301L/Y339F.
  • the fourth amino acid sequence is the following amino acid sequence:
  • the fourth amino acid sequence is at least one of the amino acid sequences shown by Y142H, Y142H/I207F, and Y142H/I207F/F301L.
  • the amino acid sequence derived from the mutation of tyrosine at position 142 in the amino acid sequence shown in SEQ ID NO:4 to histidine is called Y142H.
  • the amino acid sequence derived from the mutation of tyrosine at position 142 to histidine and the mutation of isoleucine at position 207 to phenylalanine in the amino acid sequence shown in SEQ ID NO:4 is called Y142H/I207F.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:4, in which the tyrosine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, and the phenylalanine at position 301 is mutated to leucine is called Y142H/I207F/F301L.
  • the fifth amino acid sequence is the following amino acid sequence:
  • the fifth amino acid sequence is at least one of the amino acid sequences shown by F285L and F285L/H322F.
  • the amino acid sequence derived from the mutation of phenylalanine at position 285 in the amino acid sequence shown in SEQ ID NO:5 to leucine is called F285L.
  • the amino acid sequence derived from the mutation of phenylalanine at position 285 to leucine and the mutation of histidine at position 322 to phenylalanine in the amino acid sequence shown in SEQ ID NO:5 is called F285L/H322F.
  • the sixth amino acid sequence is the following amino acid sequence:
  • the sixth amino acid sequence is at least one of the amino acid sequences shown by Q142H, Q142H/I207F, Q142H/I207F/F301L, Q142H/I207F/F301L/R338F, and Q142H/R338F.
  • amino acid sequence derived from the mutation of glutamine at position 142 in the amino acid sequence shown in SEQ ID NO:6 to histidine is called Q142H.
  • the amino acid sequence derived from the mutation of glutamine at position 142 to histidine and the mutation of isoleucine at position 207 to phenylalanine in the amino acid sequence shown in SEQ ID NO:6 is called Q142H/I207F.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:6, in which the glutamine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, and the phenylalanine at position 301 is mutated to leucine, is called Q142H/I207F/F301L.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:6, in which the glutamine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, the phenylalanine at position 301 is mutated to leucine, and the arginine at position 338 is mutated to phenylalanine, is called Q142H/I207F/F301L/R338F.
  • the glutamine at position 142 in the amino acid sequence shown in SEQ ID NO: 6 is mutated to histidine
  • the amino acid sequence derived from the mutation of arginine at position 338 to phenylalanine is called Q142H/R338F.
  • the seventh amino acid sequence is the following amino acid sequence:
  • the seventh amino acid sequence is at least one of the amino acid sequences shown by Q140H/Y205F and Q140H/Y205F/K336F.
  • amino acid sequence derived from the mutation of glutamine at position 140 to histidine and the mutation of tyrosine at position 205 to phenylalanine in the amino acid sequence shown in SEQ ID NO:7 is called Q140H/Y205F sequence.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:7, in which the glutamine at position 140 is mutated to histidine, the tyrosine at position 205 is mutated to phenylalanine, and the lysine at position 336 is mutated to phenylalanine is called Q140H/Y205F/K336F sequence.
  • the eighth amino acid sequence is the following amino acid sequence:
  • the eighth amino acid sequence is at least one of the amino acid sequences shown by Q140H/Y205F and Q140H/Y205F/K336F.
  • amino acid sequence derived from the mutation of glutamine at position 140 to histidine and the mutation of tyrosine at position 205 to phenylalanine in the amino acid sequence shown in SEQ ID NO:8 is called Q140H/Y205F.
  • the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:8, in which the glutamine at position 140 is mutated to histidine, the tyrosine at position 205 is mutated to phenylalanine, and the lysine at position 336 is mutated to phenylalanine is called Q140H/Y205F/K336F.
  • the method for obtaining the above-mentioned protein is well known to those skilled in the art.
  • the amino acid sequence of the protein when known, it can be directly obtained by chemical synthesis, or by first obtaining a gene encoding the protein and then obtaining the protein by biological expression.
  • Modifications include: chemical derivatization forms of proteins in vivo or in vitro such as acetylation or hydroxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the protein or in further processing steps, which can be completed by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).
  • the protein in order to facilitate purification, can also be modified by adding tags commonly used in the art.
  • tags commonly used in the art.
  • it can be obtained by connecting the common purification tags shown in Table 1 (such as at least one of GST, Poly-His, FLAG, His-SUMO and c-myc) to the amino terminal and/or carboxyl terminal of the protein.
  • the tag will not affect the activity of the protein provided by the present invention. In actual application, it can be selected whether to add a tag according to needs.
  • the second aspect of the present invention provides a gene encoding an Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect above.
  • the nucleotide sequence of the gene is at least one nucleotide sequence selected from the following:
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 11;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 21;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:31;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:41;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:51;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:61;
  • nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:71;
  • a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:81.
  • nucleotide sequences of the genes encoding the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and the specific mutation patterns of the proteins provided by the present invention, and obtain the nucleotide sequences by biological methods (such as PCR methods, mutation methods) or chemical synthesis methods. Therefore, this part of the nucleotide sequences should be included in the scope of the present invention.
  • nucleotide sequences SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81 and the specific mutation mode of the protein provided by the present invention it can also be carried out by methods well known in the art, such as the method of Sambrook et al.
  • site-directed mutagenesis can be performed on the basis of SEQ ID NO: 11, and the site-directed mutagenesis method includes but is not limited to ZFN site-directed mutagenesis method, TALEN site-directed mutagenesis method and/or genome site-directed mutagenesis method such as CRISPR-Cas9.
  • the 5' end and/or 3' end of the nucleotide sequence provided by the present invention may also be connected to the coding sequence of the common purification tag shown in Table 1.
  • the nucleotide sequence provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis.
  • PCR polymerase chain reaction
  • those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and amplify the nucleotide sequence using PCR.
  • the amino acid sequence can be obtained in large quantities by recombination.
  • the obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the nucleotide sequence is separated from the host cell after the proliferation by conventional methods.
  • the nucleotide sequence can also be synthesized by artificial chemical synthesis methods known in the art.
  • the third aspect of the present invention provides a recombinant vector, which contains the gene described in the second aspect.
  • the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, etc., which can be selected according to specific circumstances, and can be exemplified by pGWC, pB2GW7.0 or pET-28a, etc.
  • the recombinant vector construction can be obtained by enzyme digestion with various nucleases that have a cleavage site in the vector multiple cloning site to obtain a linear plasmid, and connected with a gene fragment cut with the same nuclease to obtain a recombinant plasmid.
  • the fourth aspect of the present invention provides a transgenic cell, which contains the gene described in the second aspect.
  • the transgenic cell is a prokaryotic cell.
  • the recombinant vector can be transformed, transduced or transfected into a host cell by methods known in the art, such as chemical transformation by calcium chloride method, high voltage electroporation transformation, preferably electroporation transformation.
  • the host cell can be a prokaryotic cell or a eukaryotic cell, which can be selected according to actual conditions.
  • the cell can be a DH5 ⁇ strain, an Agrobacterium strain GV3101, etc.
  • the fifth aspect of the present invention provides a composition, which contains the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect.
  • composition provided by the present invention contains the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect of the present invention as an active ingredient, and the content of the protein is 50-90% by weight based on the total weight of the composition.
  • the composition may also contain solvents known to those skilled in the art (such as glycerol, sugars, protein protectants such as protease inhibitors, etc.).
  • the sixth aspect of the present invention provides the use of at least one of the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.
  • the herbicide is a HPPD inhibitor.
  • the herbicide is at least one of triketone compounds, pyrazole compounds, isoxazole compounds, diketonitrile compounds and heterocyclic amide compounds.
  • the herbicide is at least one of mesotrione, cypermethrin, furansulfuron, bicyclonazole, quinclorac (Y13161), methylquinclorac (Y13287), Y18024, Y16550 and benzylpyrazone, and its molecular structure is as follows:
  • the seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to the herbicide.
  • the eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in a mutant crop containing the recombinant vector described in the third aspect into a target plant through hybridization, breeding or backcrossing, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to herbicides.
  • the ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HIS1/HSL gene of the target plant by the CRISPR/Cas gene editing method, so that the target plant expresses the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein described in the first aspect above to obtain resistance to the herbicide.
  • the transfer of the recombinant vector into the target plant specifically refers to: the nucleotide sequence of the protein with herbicide resistance provided by the present invention is transferred into the target plant by a transgenic method, so that the target plant acquires resistance to HPPD inhibitor herbicides; the nucleotide sequence of the protein provided by the present invention can also be transferred into the target plant by hybridization, breeding, backcrossing and other methods, so that the target plant acquires resistance to HPPD inhibitor herbicides; in addition, the HIS1/HSL gene of the target plant can be directly modified by gene editing technology such as CRISPR/Cas, so that the target plant acquires resistance to HPPD inhibitor herbicides. More specifically, the protein can be used as a parent material, hybridized with other excellent plant varieties and further backcrossed, and the herbicide resistance trait can be further transferred to other target plant varieties.
  • the transgenic method used in the present invention is well known to those skilled in the art, and includes direct or indirect transformation methods.
  • Direct transformation methods include chemical induction, liposome method, gene gun method, electroporation method and microinjection method.
  • plants has the broadest meaning.
  • plants include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones and plant parts used for asexual reproduction (e.g., cuttings, layering, shoots, rhizomes, underground stems, clumps, root collars, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.).
  • plant further encompasses whole plants, plant parents and offspring, and plant parts, including seeds, branches, stems, leaves, roots (including tubers), flowers, florets, fruits, fleshy stems, peduncles, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues and cells of plants, and tissues and organs.
  • plant also encompasses plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores. Among them, all of the above-mentioned include the target genes provided by the present invention. Gene/nucleic acid.
  • the plant is a crop plant.
  • crop plants include rice, wheat, corn, sorghum, soybean, sunflower, rape, alfalfa, cotton, tomato, potato or tobacco.
  • the crop plant is a cereal, such as rice, wheat, corn, sorghum, barley, millet, rye or oats.
  • the beneficial effects achieved by the present invention are obtaining proteins, genes, recombinant vectors, transgenic cells, compositions, and plants that can replace the prior art and make crops resistant to herbicides, and obtaining applications and methods of crops with herbicide resistance, and being able to obtain crop varieties with herbicide resistance through transgenic or non-transgenic methods, and being able to more efficiently control weeds in cultivated paddy fields and cultivated dry fields, thereby being able to increase crop yields and yield stability.
  • room temperature refers to 25 ⁇ 2°C.
  • Agarose gel recovery kit was purchased from TIANGEN Company;
  • the ligation kit was purchased from New England BioLabs.
  • the coding gene of the protein shown in Table 2 and the nucleotide sequence shown in SEQ ID NO: 11 were synthesized.
  • the coding gene of the protein shown in Table 3 and the nucleotide sequence shown in SEQ ID NO: 21 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 31 the coding gene of the protein shown in Table 4 and the nucleotide sequence shown in SEQ ID NO: 31 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 41 According to the nucleotide sequence shown in SEQ ID NO: 41, the coding gene of the protein shown in Table 5 and the nucleotide sequence shown in SEQ ID NO: 41 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 51 the coding gene of the protein shown in Table 6 and the nucleotide sequence shown in SEQ ID NO: 51 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 61 According to the nucleotide sequence shown in SEQ ID NO: 61, the coding gene of the protein shown in Table 7 and the nucleotide sequence shown in SEQ ID NO: 61 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 71 the coding gene of the protein shown in Table 8 and the nucleotide sequence shown in SEQ ID NO: 71 were synthesized.
  • nucleotide sequence shown in SEQ ID NO: 81 the coding gene of the protein shown in Table 9 and the nucleotide sequence shown in SEQ ID NO: 81 were synthesized.
  • the extension time at 72°C depends on the specific fragment length and the efficiency of the enzyme used. Steps 2 to 4 in the above procedure are repeated for 25 cycles.
  • the gel recovery kit was purchased from TIANGEN (Cat. No. DP209); the amount of gel recovery was 300 ⁇ L of sol solution PN (provided by the kit) for 100mg of gel, and incubated in a 50°C water bath, constantly turning it upside down to mix well to help dissolve the gel;
  • the vector and PCR product are double-digested (the digestion solution is purchased together with the restriction endonuclease).
  • the digestion system is shown in Table 12:
  • the recovered gene fragments were ligated according to the following system.
  • the ligation reagent Solution 1 (including T4 DNA ligase and matching buffer) was purchased from New England BioLabs (Cat. No. M0202S), and then incubated at 16°C for more than 5 hours.
  • the ligation system is shown in Table 13:
  • the ligation product is transformed into Escherichia coli JM109 competent cells (purchased from Promega, product number ST1105) and cultured.
  • the specific steps are as follows:
  • Protein expression is carried out in Escherichia coli BL21 (DE3) cells (purchased from Kangti Life Science Technology Co., Ltd., product number KTSM109), and the process is as follows:
  • step b Expand the vial of bacteria in step a into a large bottle of LB medium (containing antibiotics) at a volume ratio of 1:100, and culture at 37°C and 220rpm for 3h; when the OD600 value reaches 0.7h, lower the temperature to 20°C and add 0.2mM isopropyl- ⁇ -D-thiogalactoside (IPTG) for induction, and the induction time is 14h;
  • LB medium containing antibiotics
  • a Resuspend the collected E. coli with cell lysis solution, with the volume ratio of the cell lysis solution to the LB culture medium of the collected E. coli being 30 mL:1 L; stir on a magnetic stirrer for 20 min to mix the cells, during which 1 mM of serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), 40 g/mL of lysozyme for lysing bacterial cell walls, 1 ⁇ g/mL of deoxyribonuclease I for decomposing nucleic acids, and 10 mM of cofactor MgCl 2 are added; then ultrasonically disrupt the cells;
  • PMSF serine protease inhibitor phenylmethylsulfonyl fluoride
  • the protein after affinity chromatography was diluted and loaded onto an ion exchange column. After loading, a linear gradient (NaCl from 0 to 500 mM) elution was performed with a NaCl buffer solution (the buffer solution was Tris, pH 7.0). This step was completed with the aid of a protein purifier. SDS-PAGE was run to analyze the results, and proteins with better properties and purity were combined and concentrated for the next step of molecular sieve chromatography.
  • HPLC high performance liquid chromatography
  • HEPES buffer for activity measurement Prepare a 1 M stock solution, adjust its pH to 7.0 with sodium hydroxide, dilute to 20 mM before use, and filter with a 0.22 ⁇ m filter membrane;
  • Preparation of substrate i.e. HPPD inhibitor: first prepare a stock solution with a concentration of 10 mM using DMSO, and dilute it with activity detection buffer before use;
  • Preparation of sodium ascorbate Prepare to a concentration of 20 mM with deionized water. Store at -80°C;
  • the sample is detected by HPLC, the sample volume is 60 ⁇ L, the residual substrate absorption peak area at 286 nm is detected, and the amount of substrate consumed in different reactions is calculated based on the standard curve of the substrate (i.e., control), so as to evaluate the catalytic activity of each protein. Each sample is repeated three times.
  • Control refers to adding inhibitors to the reaction system but not adding enzymes, which is regarded as the remaining amount of substrate when the inhibitor is not catalyzed.
  • Sample Add the same volume and concentration (50 ⁇ M) of inhibitor to the enzyme reaction system and observe the catalytic efficiency of the enzyme on the inhibitor.
  • Catalytic efficiency (1-(peak area of substrate in sample)/(peak area of substrate in control)) ⁇ 100%. The larger the value, the better the catalytic effect.
  • Triketones mesotrione, cypermethrin, furansulfuron, bicyclonazole technical, methyl quinazoline, compound A;
  • pyraclostrobin technical and fenpyraclostrobin (Baowei), the catalytic results of rice HIS1 protein and HSL proteins of rice, wheat, barley, sorghum and corn on the above HPPD inhibitors were tested respectively; the specific results are shown in Tables 14 to 21.
  • Table 14 Catalytic results of rice HIS1 mutants to different HPPD inhibitors
  • H141F refers to a mutant in which the 141st position of rice HIS1 mutated from histidine to phenylalanine
  • H141F/F205L refers to a mutant in which the 141st position of rice HIS1 mutated from histidine to phenylalanine and the 205th position of rice HIS1 mutated from phenylalanine to leucine.
  • Table 15 Catalytic results of rice HSL1 mutants to different HPPD inhibitors
  • L204F/F298L/I335A refers to a mutant of rice HSL1 in which the 204th position of leucine mutated to phenylalanine, the 298th position of phenylalanine mutated to leucine, and the 335th position of isoleucine mutated to alanine
  • L204F/F298L/I335W refers to a mutant of rice HSL1 in which the 204th position of leucine mutated to phenylalanine, the 298th position of phenylalanine mutated to leucine, and the 335th position of isoleucine mutated to tryptophan
  • F140H/L204F/F298L/I335F refers to a mutant of rice HSL1 in which the 140th position of phenylalanine mutated to histidine, the 204th position of leucine mutated to phenylalanine, the 298th position of pheny
  • Table 16 Catalytic results of rice HSL2 mutants to different HPPD inhibitors
  • T206F refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine
  • T206F/F301L refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine and the phenylalanine at position 301 mutated to leucine
  • T206F/F301L/Y339F refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine, the phenylalanine at position 301 mutated to leucine and the tyrosine at position 339 mutated to phenylalanine.
  • Table 17 Catalytic results of rice HSL4 mutants to different HPPD inhibitors
  • Y142H refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine
  • Y142H/I207F refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine and the isoleucine at position 207 mutated to phenylalanine
  • Y142H/I207F/F301L refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine, the isoleucine at position 207 mutated to phenylalanine, and the phenylalanine at position 301 mutated to leucine.
  • Table 18 Catalytic results of wheat HSL2 mutants to different HPPD inhibitors
  • F285L refers to a mutant in which the phenylalanine at position 285 of wheat HSL2 mutated to leucine
  • F285L/H322F refers to a mutant in which the phenylalanine at position 285 of wheat HSL2 mutated to leucine and the histidine at position 322 mutated to phenylalanine.
  • Q142H/R338F refers to a mutant in which the glutamine at position 142 of corn HSL1A mutated to histidine and the arginine at position 338 mutated to phenylalanine
  • Q142H/I207F/F301L/R338F refers to a mutant in which the glutamine at position 142 of corn HSL1A mutated to histidine, the isoleucine at position 207 mutated to phenylalanine, the phenylalanine at position 301 mutated to leucine, and the arginine at position 338 mutated to phenylalanine.
  • Q140H/Y205F refers to a mutant in which the glutamine at position 140 of corn HSL1B mutated to histidine and the tyrosine at position 205 mutated to phenylalanine
  • Q140H/Y205F/K336F refers to a mutant in which the glutamine at position 140 of corn HSL1B mutated to histidine, the tyrosine at position 205 mutated to phenylalanine, and the lysine at position 336 mutated to phenylalanine.
  • Table 21 Catalytic results of sorghum HSL1 mutants to different HPPD inhibitors
  • Q140H/Y205F refers to a mutant in which the glutamine at position 140 of sorghum HSL1 mutated to histidine and the tyrosine at position 205 mutated to phenylalanine
  • Q140H/Y205F/K336F refers to an amino acid sequence in which the glutamine at position 140 of sorghum HSL1 mutated to histidine, the tyrosine at position 205 mutated to phenylalanine, and the lysine at position 336 mutated to phenylalanine.
  • the single point mutation H141F of rice HIS1 has a catalytic efficiency of 31% for bacterium and 37% for Y16550, which are 9% and 26% higher than those of the wild type, respectively;
  • the double point mutation H141F/F205L of rice HIS1 has a catalytic efficiency of 27% for bacterium and 26% for Y16550, which are 5% and 15% higher than those of the wild type, respectively.
  • the triple-point mutation L204F/F298L/I335A of rice HSL1 has a catalytic efficiency of 87% for BBC-OH, 72% for Y13287, 21% for Y18024, and 69% for Y16550, which are 38%, 72%, 21%, and 52% higher than those of the wild type, respectively.
  • the triple-point mutation L204F/F298L/I335W of rice HSL1 has a catalytic efficiency of 76% for mesotrione, 100% for BBC-OH, 64% for Y13287, 48% for Y18024, and 42% for Y16550, which are 36%, 51%, 64%, 48%, and 35% higher than those of the wild type, respectively.
  • the four-point mutation F140H/L204F/F298L/I335F of rice HSL1 has a catalytic efficiency of 85% for mesotrione and 25% for BBC-OH.
  • the catalytic efficiency of Y13287 and Y18024 was 92% and 96%, respectively, which were increased by 45%, 51%, 92% and 96% compared with the wild type.
  • the single point mutation T206F of rice HSL2 has a catalytic efficiency of 20% for mesotrione, 26% for BBC-OH, 8% for furansulfuron, 9% for Y13287, 16% for Y18024, and 11% for Y16550, which are 11%, 26%, 8%, 9%, 16%, and 6% higher than those of the wild type, respectively.
  • the double point mutation T206F/F301L of rice HSL2 has a catalytic efficiency of 47% for mesotrione, 100% for BBC-OH, 25% for furansulfuron, 83% for Y13287, 30% for Y18024, and 17% for Y16550, which are 38%, 100%, 25%, 83%, 30%, and 12% higher than those of the wild type, respectively.
  • the triple-point mutations T206F/F301L/Y339F of rice HSL2 have a catalytic efficiency of 44% for mesotrione, 100% for BBC-OH, 21% for furansulfuron, 86% for Y13287, 27% for Y18024, 12% for bauhinia, and 16% for Y16550, which are catalytic efficiencies increased by 35%, 100%, 21%, 86%, 27%, 12% and 11% respectively compared with the wild type.
  • the single point mutation Y142H of rice HSL4 has a catalytic efficiency of 30% for furansuline, 75% for Y13287, and 8% for bauer, which are 13%, 2%, and 6% higher than those of the wild type, respectively.
  • the double point mutation Y142H/I207F of rice HSL4 has a catalytic efficiency of 27% for furansuline, 76% for Y13287, and 5% for bacterium, which are 10%, 3%, and 3% higher than those of the wild type, respectively.
  • the triple-point mutation Y142H/I207F/F301L of rice HSL4 has a catalytic efficiency of 22% for mesotrione and 4% for bacteriochlorant, which are 1% and 2% higher than those of the wild type, respectively.
  • the single point mutation F285L of wheat HSL2 had a catalytic efficiency of 10% for BBC-OH, 32% for Y13287, and 34% for Y18024, which were 4%, 32%, and 34% higher than those of the wild type, respectively;
  • the double point mutation 285L/H322F of wheat HSL2 has a catalytic efficiency of 17% for BBC-OH, 12% for Y13287, and 9% for Y18024, which are 11%, 12%, and 9% higher than those of the wild type, respectively.
  • the double point mutation Q142H/R338F of maize HSL1A has a catalytic efficiency of 21% for mesotrione and 77% for furansulfuron, which are 8% and 28% higher than those of the wild type, respectively.
  • the catalytic efficiency of the four-point mutation Q142H/I207F/F301L/R338F of corn HSL1A for mesotrione is 15%, which is 2% higher than that of the wild type.
  • the double point mutation Q140H/Y205F of maize HSL1B has a catalytic efficiency of 18% for mesotrione, 100% for BBC-OH, and 9% for Y16550, which are 10%, 5%, and 3% higher than those of the wild type, respectively.
  • the catalytic efficiency of the triple-point mutation Q140H/Y205F/K336F in corn HSL1B for mesotrione is 19%, and the catalytic efficiency for BBC-OH is 100%, which are 11% and 5% higher than those of the wild type, respectively.
  • the double point mutation Q140H/Y205F of sorghum HSL1 had a catalytic efficiency of 98% for BBC-OH, 7% for furansuline, and 7% for Y16550, which were 77%, 7%, and 7% higher than those of the wild type, respectively;
  • the triple-point mutation Q140H/Y205F/K336F of sorghum HSL1 has a catalytic efficiency of 26% for mesotrione, 100% for BBC-OH, 15% for furansulfuron, and 10% for Y16550, which are 6%, 79%, 15% and 10% higher than those of the wild type, respectively.
  • the rice HIS1, rice HSL1, rice HSL2, rice HSL4, wheat HSL2, sorghum HSL1, corn HSL1A and corn HSL1 mutant provided by the present invention have enhanced hydroxylation activity on HPPD inhibitors, that is, the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase protein provided by the present invention can detoxify HPPD inhibitors. Therefore, the Fe(II)/ ⁇ -ketoglutarate-dependent dioxygenase provided by the present invention can be used to improve the herbicide resistance of crops.

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Abstract

La présente invention concerne une protéine dioxygénase dépendante de l'acide Fe (II)/alpha-cétoglutarique, un gène codant pour la dioxygénase dépendante de l'acide Fe (II)/alpha-cétoglutarique, un vecteur recombiné, une cellule transgénique, une composition et son utilisation dans l'amélioration de la résistance aux herbicides d'une culture. L'invention concerne en outre un procédé d'amélioration de la résistance aux herbicides d'une culture. La dioxygénase dépendante de l'acide Fe (II)/alpha-cétoglutarique et le gène codant pour celle-ci dans la présente invention peuvent être utilisés pour améliorer la résistance d'une culture à un inhibiteur de HPPD.
PCT/CN2023/126049 2023-10-23 2023-10-23 Protéine 2-odd, gène, vecteur, cellule, composition et utilisation associée Pending WO2025086073A1 (fr)

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CN110268069A (zh) * 2017-02-10 2019-09-20 日本史迪士生物科学株式会社 2-氧代戊二酸依赖性地氧化4-hppd抑制剂的催化活性提高了的hsl蛋白质的制造方法
CN113073088A (zh) * 2021-03-31 2021-07-06 四川天豫兴禾生物科技有限公司 具有三酮类除草剂抗性的hir突变体及其在植物育种中的应用

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CN105861524A (zh) * 2010-12-28 2016-08-17 日本史迪士生物科学株式会社 对4-hppd抑制剂的抵抗性或敏感性提高了的植物
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