WO2020221312A1 - 抗除草剂基因、多肽及其在植物育种中的应用 - Google Patents

抗除草剂基因、多肽及其在植物育种中的应用 Download PDF

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WO2020221312A1
WO2020221312A1 PCT/CN2020/087855 CN2020087855W WO2020221312A1 WO 2020221312 A1 WO2020221312 A1 WO 2020221312A1 CN 2020087855 W CN2020087855 W CN 2020087855W WO 2020221312 A1 WO2020221312 A1 WO 2020221312A1
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plant
herbicide
polypeptide
plants
resistant
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PCT/CN2020/087855
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English (en)
French (fr)
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钟英丽
李峰
谢洪涛
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山东舜丰生物科技有限公司
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Priority to CN202080003126.8A priority Critical patent/CN112639086B/zh
Publication of WO2020221312A1 publication Critical patent/WO2020221312A1/zh

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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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/123—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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

Definitions

  • the present invention relates to the field of botany, and more specifically to herbicide resistance genes, polypeptides and their applications in plant breeding.
  • HPPD 4-Hydroxyphenylpyruvate Dioxygenase
  • Tyrosine in organisms Tyrosine generates p-hydroxyphenylpyruvic acid (HPPA) under the action of tyrosine aminotransferase (TAT).
  • TAT tyrosine aminotransferase
  • HPPD can catalyze the conversion of HPPA into homogentisic acid. (homogentisate, HGA).
  • HGA homogentisate
  • the main function of HPPD is to promote the catabolism of tyrosine, aryl acid and phenylalanine. But the effect in plants is significantly different from that in animals.
  • Tocopherols play the role of membrane-related antioxidants. They are essential antioxidants for plant growth and can effectively enhance plant resistance. Plastoquinone is a key cofactor in the process of photosynthesis in plants and promotes the synthesis of carotenoids in plants. In plants, more than 60% of chlorophyll is bound to the light-harvesting antenna complex, which absorbs sunlight Energy and transfer excitation energy to the photosynthesis reaction center, and carotenoids are an important part of the chlorophyll binding protein and antenna system in the reaction center. They are responsible for the important function of light absorption auxiliary pigments in plant photosynthesis, and have absorption and transmission. The ability of electrons and plays an important role in scavenging free radicals.
  • HPPD has been identified as a herbicide target since the 1990s.
  • HPPD-inhibiting herbicides have been proven to be very effective selective herbicides. They have broad-spectrum herbicidal activity. They can be used before or after emergence. They have high activity, low residue, are safe for mammals, and are environmentally friendly.
  • five herbicides targeting HPPD have been developed according to the structure, which mainly include triketones, pyridones, isoxazolones, diketonitriles and benzophenones.
  • HPPD-inhibiting herbicides indiscriminately kill weeds and also bring certain damage to crops. Therefore, it is particularly important to obtain herbicide-tolerant crops.
  • the current strategy also includes overexpression of this enzyme to produce a large number of herbicide target enzymes in plants and reduce the inhibitory effect of herbicides.
  • the overexpression of HPPD makes plants have better pre-germination tolerance to the diketonitrile derivative (DKN) of isoxafluran, but the tolerance is not enough to resist herbicide treatment after germination.
  • DKN diketonitrile derivative
  • the purpose of the present invention is to provide an HPPD resistance gene with high resistance to HPPD inhibitors, coding polypeptides and applications thereof.
  • an isolated herbicide resistance polypeptide is provided, and the herbicide resistance polypeptide is a mutant HPPD polypeptide,
  • the mutant HPPD polypeptide there are one or more amino acid sequence differences between the mutant HPPD polypeptide and the parent HPPD polypeptide, and the differences include a mutation corresponding to the 342th amino acid of SEQ ID NO.:1:
  • the tyrosine (Y) at position 342 is mutated into one or more amino acids selected from the group consisting of histidine (H), asparagine (Asn), alanine (Ala), Lysine (Lys), Arginine (Arg), Cysteine (C) Phenylalanine (Phe).
  • the tyrosine (Y) at position 342 is mutated to histidine (H) or cysteine (C).
  • the tyrosine (Y) at position 342 is mutated to histidine (H).
  • the herbicide resistance polypeptide further includes other mutation sites, and the other mutation sites are mutated at one or more amino acids selected from the following group corresponding to SEQ ID NO.:1:
  • the serine (S) at position 214 is mutated to one or more amino acids selected from the group consisting of valine (V) and leucine (L).
  • the arginine (R) at position 349 is mutated to one or more amino acids selected from the group consisting of serine (S) and threonine (T).
  • the proline (P) at position 340 is mutated to one or more amino acids selected from the group consisting of alanine (A), serine (S), and leucine (L).
  • the threonine (T) at position 341 is mutated into one or more amino acids selected from the group consisting of histidine (H), arginine (R), lysine (K ).
  • the tyrosine (Y) at position 343 is mutated into one or more amino acids selected from the group consisting of histidine (H), cysteine (C), arginine ( R), Lysine (K), Phenylalanine (F).
  • the glutamine (Q) at position 344 is mutated to one or more amino acids selected from the group consisting of arginine (R) and tryptophan (W).
  • the asparagine (N) at position 345 is mutated to one or more amino acids selected from the group consisting of aspartic acid (D) and glycine (G).
  • the leucine (L) at position 346 is mutated to one or more amino acids selected from the group consisting of phenylalanine (F) and serine (S).
  • the lysine (K) at position 347 is mutated to one or more amino acids selected from the group consisting of glutamic acid (E) and glycine (G).
  • the lysine (K) at position 348 is mutated to one or more amino acids selected from the group consisting of glutamic acid (E) and glycine (G).
  • the valine (V) at position 350 is mutated to one or more amino acids selected from the group consisting of alanine (A), serine (S), and threonine (T).
  • the glycine (G) at position 351 is mutated to one or more amino acids selected from the group consisting of serine (S), aspartic acid (D), and asparagine (N).
  • the aspartic acid (D) at position 352 is mutated to one or more amino acids selected from the group consisting of aspartic acid (N), glycine (G), and serine (S).
  • glutamic acid (E) at position 433 is mutated to one or more amino acids selected from the group consisting of lysine (K) and arginine (R).
  • the mutation includes a combination of Y342H and one or more mutations selected from the following group: S214V, S214L, R349S, R349T, E433K, E433R.
  • the mutation is selected from the following group: Y342H, R349S, or a combination thereof.
  • the other mutation sites can maintain or enhance the tolerance or resistance of the mutant polypeptide to HPPD inhibitory herbicides or increase the application range of the mutant HPPD polypeptide to herbicides.
  • amino acid sequence of the herbicide resistance polypeptide is shown in SEQ ID NO.: 2 or 3.
  • the herbicide-resistant polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO.: 2 or 3, an active fragment thereof, or a conservative variant polypeptide thereof.
  • the herbicide-resistant polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO.: 4 or 5, an active fragment thereof, or a conservative variant polypeptide thereof.
  • the mutein except for the mutation (such as 214, 342, 349, 340, 341, 343, 344, 345, 346, 347, 348, 350, 351, 352, 433) ,
  • the rest of the amino acid sequence is the same or substantially the same as the sequence shown in SEQ ID NO.:1.
  • the said substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are not the same, wherein, The difference includes amino acid substitution, deletion or addition, and the mutant protein has herbicide tolerance activity.
  • the herbicide is an HPPD inhibitory herbicide, selected from the group consisting of triketones, diketonitriles, isoxazoles, pyrazoles, benzophenones, and quinazolines Diketones, or combinations thereof.
  • the triketone herbicide is selected from the group consisting of sulcotrione, mesotrione, cyclosulfone, sulcotrione, or a combination thereof.
  • the isoxazole herbicide is selected from the group consisting of isoxaflutole, clomazone, clomazone, or a combination thereof.
  • the quinazolindione herbicide includes quinazone, methaqualone, the compounds described in CN104557739A and CN110669016A.
  • the homology between the herbicide-resistant polypeptide and the sequence shown in SEQ ID NO.:1 is at least 80%, preferably at least 85% or 90%, and more preferably at least 95%. %, preferably at least 98% or 99%.
  • sequence of the parent HPPD polypeptide and the amino acid sequence shown in SEQ ID No. 1 have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, At least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the herbicide tolerance concentration V1 of the herbicide resistance polypeptide is compared with the tolerance concentration V2 of the parent HPPD polypeptide to the same herbicide, V1/V2 ⁇ 2, preferably V1/ V2 ⁇ 3, preferably V1/V2 ⁇ 4, preferably V1/V2 ⁇ 5, preferably V1/V2 ⁇ 6, preferably V1/V2 ⁇ 8, more preferably V1/V2 ⁇ 10.
  • the parental HPPD polypeptide is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the parental HPPD polypeptide is derived from one or more plants selected from the group consisting of gramineous, leguminous, and cruciferous plants.
  • the parental HPPD polypeptide is derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, soybean, Arabidopsis, potato, tomato, lettuce, Canola, cabbage, quinoa.
  • the parent HPPD polypeptide is derived from Arabidopsis thaliana.
  • the herbicide resistance polypeptide is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the herbicide resistance polypeptide is derived from one or more plants selected from the group consisting of gramineous, leguminous, and cruciferous plants.
  • the herbicide-resistant polypeptide is derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, soybean, Arabidopsis, potato, tomato, Lettuce, rape, cabbage, quinoa.
  • the herbicide resistance polypeptide is derived from Arabidopsis thaliana.
  • the herbicide resistance polypeptide can tolerate a herbicide with a concentration ⁇ 5 ⁇ M, preferably ⁇ 10 ⁇ M, preferably ⁇ 20 ⁇ M, preferably ⁇ 50 ⁇ M, more preferably ⁇ 100 ⁇ M .
  • the herbicide resistance polypeptide can tolerate a herbicide with a concentration of 10-400 ⁇ M, preferably ⁇ 20-300 ⁇ M, and more preferably 40-260 ⁇ M.
  • the herbicide resistance polypeptide is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 2 or 3 is formed by the substitution, deletion or addition of one or more (such as 2, 3, 4 or 5) amino acid residues, and has A polypeptide derived from (a) with herbicide tolerance activity.
  • the homology between the derived polypeptide and the sequence shown in SEQ ID NO.: 2 or 3 is at least 60%, preferably at least 70%, more preferably at least 80%, It is optimally at least 90%, such as 95%, 97%, 99%.
  • the herbicide resistance polypeptide is formed by mutation of the wild-type HPPD polypeptide shown in SEQ ID NO.:1.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the herbicide resistance polypeptide of the first aspect of the present invention.
  • polynucleotide is selected from the following group:
  • the polynucleotide is selected from the group consisting of genomic sequence, cDNA sequence, RNA sequence, or a combination thereof.
  • the polynucleotide additionally contains an auxiliary element selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or flanking the ORF of the herbicide resistance polypeptide. Its combination.
  • the polynucleotide further includes a regulatory element operably connected to it.
  • the regulatory element is selected from one or more of the following group: enhancer, transgenic
  • Transposon promoter, terminator, leader sequence, polyadenylic acid sequence, marker gene.
  • the polynucleotide further comprises a promoter operably linked to the ORF sequence of the herbicide resistance polypeptide.
  • the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.
  • the third aspect of the present invention provides a vector which contains the polynucleotide according to the second aspect of the present invention.
  • the vectors include expression vectors, shuttle vectors, and integration vectors.
  • the fourth aspect of the present invention provides a host cell containing the vector according to the third aspect of the present invention or the genome integrated with the polynucleotide according to the second aspect of the present invention.
  • the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.
  • the host cell is a prokaryotic cell, such as Escherichia coli.
  • the eukaryotic cells include plant cells.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperm is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cunninghamaceae, Cupressaceae, Tricuspidae Cunninghamaceae, Taxaceae, Ephedraceae, Maizeaceae, Monotypicaceae, Centipedeaceae, or combinations 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, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, legumes, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, tobacco, rice, cabbage, soybean, tomato, corn, tobacco, wheat, barley, millet, sorghum, potato, quinoa, lettuce, rapeseed , Strawberry, or a combination thereof.
  • the fifth aspect of the present invention provides a method for preparing a herbicide-resistant polypeptide.
  • the method includes the steps:
  • the sixth aspect of the present invention provides an enzyme preparation comprising the herbicide-resistant polypeptide of the first aspect of the present invention.
  • the enzyme preparations include injections and/or freeze-dried preparations.
  • the seventh aspect of the present invention provides a method for improving plants, the method comprising the steps:
  • step (b) Regenerate the plant cells in step (a) into plants.
  • the plant cell is modified by one or more methods selected from the following group: genetic engineering, natural mutation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis) , Chemical mutagenesis (such as nitrous acid, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus or bacteria-mediated mutagenesis).
  • genetic engineering natural mutation
  • physical mutagenesis such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis
  • Chemical mutagenesis such as nitrous acid, hydroxylamine, EMS, nitrosoguanidine, etc.
  • biological mutagenesis such as virus or bacteria-mediated mutagenesis
  • step (a) includes the steps:
  • step (1) (2) contacting the plant cell with the Agrobacterium in step (1), so that the DNA coding sequence of the herbicide resistance polypeptide is transferred into the plant cell and integrated into the chromosome of the plant cell;
  • step (a) gene editing technology is used to modify the plant cell, so that the plant cell expresses the herbicide resistance polypeptide of the first aspect of the present invention.
  • step (a) gene editing technology is used to transform the plant cell, so that the HPPD in the plant cell is at the tyrosine position corresponding to the 342th position of SEQ ID NO.:1 Mutation occurred.
  • step (a) it further includes using gene editing technology to transform the plant cell so that the HPPD in the plant cell is in the 214th, 349th, and 340th corresponding to SEQ ID NO.:1 341, 343, 344, 345, 346, 347, 348, 350, 351, 352, 433 amino acid mutations.
  • the gene editing technology is selected from the group consisting of CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the gene editing technology includes any technical method that can produce the mutation.
  • the method improves the herbicide tolerance of plants.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperm is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cunninghamaceae, Cupressaceae, Tricuspidae Cunninghamaceae, Taxaceae, Ephedraceae, Maizeaceae, Monotypicaceae, Centipedeaceae, or combinations 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, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, legumes, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, tobacco, rice, cabbage, soybean, tomato, corn, tobacco, wheat, barley, millet, sorghum, potato, quinoa, lettuce, rapeseed , Strawberry, or a combination thereof.
  • the method further includes the step of testing the herbicide resistance of the plant cell.
  • the plant has a tolerance concentration of ⁇ 50nM under the condition of the culture medium; preferably, ⁇ 100nM; preferably, ⁇ 200nM; preferably, ⁇ 250nM; preferably, ⁇ 300nM ; Preferably, ⁇ 350nM, preferably ⁇ 400nM; more preferably, ⁇ 450nM herbicide.
  • the plant can tolerate a herbicide with a concentration of ⁇ 5 ⁇ M under soil cultivation conditions; preferably, ⁇ 10 ⁇ M; preferably, ⁇ 15 ⁇ M; more preferably, ⁇ 20 ⁇ M herbicide.
  • the plant can tolerate a concentration of 5 ⁇ M-50 ⁇ M under soil cultivation conditions, preferably, 10 ⁇ M-30 ⁇ M, more preferably, 10 ⁇ M-25 ⁇ M, more preferably, 15 ⁇ M-20 ⁇ M weeding Agent.
  • the plant is a plant that has grown for 2-4 weeks.
  • the tolerance is treated by spraying herbicides.
  • the plants improved by the method can tolerate at least a concentration of 50 nM, preferably 100 nM, and more preferably 200 nM herbicides (mesotrione, sulcotrione, cyclosulfone, Bicyclic sulfone, isoxaflutole, quinazone and/or quinazone).
  • herbicides meotrione, sulcotrione, cyclosulfone, Bicyclic sulfone, isoxaflutole, quinazone and/or quinazone.
  • the eighth aspect of the present invention provides a use of the herbicide-resistant polypeptide of the first aspect of the present invention or its encoding gene, for cultivating plant herbicide-resistant strains, or for preparing and cultivating plant herbicide-resistant strains Reagents or kits.
  • the ninth aspect of the present invention provides a herbicide resistance sensitive site, the site includes:
  • the first resistance sensitive site corresponding to (i) the amino acid at position 342 of the wild-type HPPD polypeptide derived from Arabidopsis thaliana; (ii) the amino acid at position 339 of the wild-type HPPD polypeptide derived from rice; iii) The 334th amino acid of the wild-type HPPD polypeptide derived from corn; (iv) the 333rd amino acid of the wild-type HPPD polypeptide derived from sorghum; (v) the 329th amino acid of the wild-type HPPD polypeptide derived from wheat; Or (vi) the 341th amino acid of wild-type HPPD polypeptide derived from soybean.
  • the resistance sensitive site further includes:
  • the second resistance sensitive site corresponding to (i) the 349th amino acid of the wild-type HPPD polypeptide derived from Arabidopsis thaliana, and (ii) the 346th amino acid of the wild-type HPPD polypeptide derived from rice; iii) The amino acid at position 341 of the wild-type HPPD polypeptide derived from corn; (iv) the amino acid at position 340 of the wild-type HPPD polypeptide derived from sorghum; (v) the amino acid at position 336 of the wild-type HPPD polypeptide derived from wheat; Or (vi) the 348th amino acid of wild-type HPPD polypeptide derived from soybean.
  • the resistance sensitive site also includes other resistance sensitive sites, corresponding to (i) the 340, 341, 343, 344, 345, 345, 345, 345, 340, 341, 343, 344, 345, One or more amino acids at positions 346, 347, 348, 350, 351, 352, and 433; (ii) the 337th, 338, 339, 341, 342, 343, 344, 345 of wild-type HPPD polypeptide derived from rice , 346,348,349,430 amino acids; (iii) 332,333,335,336,337,338,339,340,342,343,344,432 amino acids of wild-type HPPD polypeptide derived from corn; (iv ) Amino acids 331,332,334,335,336,337,338,339,341,342,343,424 of wild-type HPPD polypeptide derived from sorghum; (v) 327,328th of wild-type HPPD polypeptid
  • the polypeptide has sensitive and insensitive types.
  • the site is tyrosine (Y)
  • the polypeptide is sensitive, and the polypeptide is sensitive to herbicides.
  • the site is histidine (H), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg) or cysteine (C)
  • H histidine
  • Asparagine Asn
  • Gln glutamine
  • lysine Lysine
  • Arg arginine
  • cysteine C
  • the tolerance concentration V1 of the insensitive polypeptide to the herbicide is compared with the tolerance concentration V2 of the sensitive polypeptide to the same herbicide, V1/V2 ⁇ 2, V1/V2 ⁇ 3, V1/V2 ⁇ 4, V1/V2 ⁇ 5, V1/V2 ⁇ 6, V1/V2 ⁇ 8, preferably V1/V2 ⁇ 5, more preferably V1/V2 ⁇ 10.
  • the polypeptide has sensitive and insensitive types, when the first resistance sensitive site is tyrosine (Y), and the second resistance sensitive site is arginine (R), the polypeptide is sensitive, and the polypeptide is sensitive to herbicides; when the first resistance sensitive site is histidine (H), asparagine (Asn), Alanine (Ala), lysine (Lys), arginine (Arg), cysteine (C) or phenylalanine (Phe), the second sensitive site for resistance is serine (S) , Threonine (T), the polypeptide is insensitive, and the polypeptide is resistant to herbicides.
  • the insensitive polypeptide is the herbicide-resistant polypeptide of claim 1, and the sensitive polypeptide is a wild-type HPPD polypeptide.
  • the tenth aspect of the present invention provides a fusion protein comprising the mutant polypeptide or its biologically active fragment, and other components fused with it, such as a tag peptide such as a histidine tag, for example, 6 ⁇ His, or
  • a tag peptide such as a histidine tag, for example, 6 ⁇ His
  • the plastid guide peptide is, for example, a peptide guided into the chloroplast.
  • the eleventh aspect of the present invention provides a plant cell, plant tissue, plant part, plant that is tolerant to or resistant to HPPD inhibitory herbicide, wherein the plant cell, plant tissue, plant Parts and plants contain the herbicide-resistant polypeptide or its polynucleotide sequence.
  • the twelfth aspect of the present invention provides a method for identifying or selecting transformed plant cells, plant tissues, plants or parts thereof, characterized in that it comprises: (i) providing transformed plant cells, plant tissues, plants or parts thereof , Wherein the transformed plant cell, plant tissue, plant or part thereof comprises the herbicide-resistant polypeptide according to the first aspect of the present invention or the polynucleotide according to the second aspect of the present invention or the third aspect of the present invention a;
  • the plant cell, plant tissue, plant or part thereof may contain another isolated polynucleotide.
  • the twelfth aspect of the present invention provides a method for identifying herbicide-tolerant plants, characterized in that it comprises:
  • step (i) it is determined by sequencing whether the plant sample has the herbicide-resistant polypeptide according to the first aspect of the present invention or the polynucleotide according to the second aspect of the present invention or The carrier described in the third aspect of the present invention.
  • the thirteenth aspect of the present invention provides a method for controlling unwanted plants in a plant cultivation site, characterized in that the method comprises:
  • the fourteenth aspect of the present invention provides a method for producing herbicide-resistant plants, characterized in that it comprises:
  • first plant is a herbicide-resistant plant, which contains the herbicide-resistant polypeptide of the first aspect of the present invention or the polynucleotide of the second aspect of the present invention Or the carrier described in the third aspect of the present invention.
  • the second plant is a plant that is not resistant to herbicides or has weak herbicide resistance.
  • the fifteenth aspect of the present invention provides a method for screening tolerant herbicides or identifying triketone herbicides, including the following steps:
  • test compound in the presence of the test compound, the test compound is applied to a plant expressing the mutant HPPD polypeptide of claim 1, and the growth or viability of the plant is analyzed;
  • test compound is tolerant to herbicides or triketone herbicides.
  • the growth or viability conditions include: leaf color, plant height, and survival rate.
  • Figure 1 shows a schematic diagram of AtHPPD base editing library construction.
  • Figure 2 shows the detection of single-base editing in the 2-21 heterozygous AtHPPD gene.
  • Figure 3 shows the phenotypic segregation of 2-21 heterozygous seeds on MST resistance selection medium.
  • FIG. 4 shows that the 2-21 progeny plants with MST resistance were all detected to contain T to C mutations.
  • Figure 5 shows that 2-21 seedlings have both normal growth and development and high MST resistance.
  • Figure 6 shows that 2-21 has a tolerance of 5 ⁇ M MST spraying under soil culture conditions.
  • Figure 7 shows that the AtHPPDY342H transgenic T1 generation exhibited MST resistance.
  • Figure 8 shows that the AtHPPDY342H transgenic T2 generation exhibited MST resistance.
  • Figure 9 shows the tolerance of AtHPPDY342H plants to different herbicides.
  • Figure 10 shows the sequence difference analysis of the edited rice plant after Sanger sequencing.
  • Figure 11 shows the difference in rice plants after spraying with 4g.a.i/mu quinazone for 7 days.
  • the present inventors unexpectedly screened the key amino acid sites with herbicide tolerance activity in plants for the first time.
  • the present invention found that after the key sites in the wild-type HPPD polypeptide are modified, the herbicide tolerance of plants can be significantly improved. On this basis, the present inventor completed the present invention.
  • AxxB means that amino acid A at position xx is changed to amino acid B
  • L87I means that amino acid L at position 87 is changed to I, and so on.
  • HPPD 4-Hydroxyphenylpyruvate Dioxygenase
  • HPPD EC 1.13.11.27
  • HPPD 4-Hydroxyphenylpyruvate Dioxygenase
  • HPPD EC 1.13.11.27
  • HPPD 4-hydroxyphenylpyruvate
  • HGA homogentisate
  • HPPD-inhibiting herbicides have been proven to be very effective selective herbicides. They have broad-spectrum herbicidal activity. They can be used before or after emergence. They have high activity, low residue, are safe for mammals, and are environmentally friendly. Features.
  • HPPD inhibitor As used herein, the terms “HPPD inhibitor”, “HPPD inhibitory herbicide”, and “HPPD inhibitory herbicide” are used interchangeably and refer to substances that have herbicidal activity by themselves or are combined with other herbicides that can change their effects. / Or a combination of additives, which inhibit HPPD, behave as a preparation that inhibits plant growth or even kills plants.
  • Substances capable of inhibiting HPPD and acting as herbicides are well known in the art, including many types, 1) three Ketones, for example, sulcotrione (Sulcotrione, CAS number: 99105-77-8); Mesotrione (Mesotrione, CAS number: 104206-82-8); bicyclopyrone (bicyclopyrone, CAS number: 352010- 68-5); tembotrione (tembotrione, CAS number: 335104-84-2); tefuryltrione (CAS number: 473278-76-1); Bicyclon (Benzobicyclon, CAS number: 156963- 66-5); 2) Diketone nitriles, for example, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)propane-1,3- Dione (CAS Number: 143701-75-1); 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-3,4
  • the herbicide is isoxazoles and triketones; preferably, the herbicide is isoxaflutole and mesotrione.
  • the herbicides can comprehensively consider the types of crops or weeds to which they are applicable, and control unwanted plants (such as weeds) before emergence, after emergence, before planting and during planting.
  • it is a triketone HPPD inhibitor, such as sulcotrione, mesotrione, cyclosulfone, and bicyclosulfone.
  • the term "effective amount” or “effective concentration” means an amount or concentration, respectively, which is sufficient to kill or inhibit the growth of non-target plants, plant tissues, plant cells or host cells, but the amount does not Kill the herbicide-resistant plants, plant tissues, plant cells and host cells of the present invention or not severely inhibit their growth (target plants).
  • the non-target plants can be similar parent (or wild-type) plants, plant tissues, plant cells or host cells, and can also be weeds, or wild-type plants that grow from the cultivation site and are not related to the target plant (such as soybeans grown in corn fields).
  • the effective amount of herbicide is the amount routinely used to kill the target weeds in agricultural production systems. Such amounts are known to those of ordinary skill in the art.
  • the herbicides of the present invention exhibit herbicidal activity when they are applied directly to plants or to the location of plants at any growth stage or before planting or emergence.
  • the observed effect depends on the plant species to be controlled, the growth stage of the plant, the application parameters of the dilution and the size of the spray droplets, the particle size of the solid components, the environmental conditions during use, the specific compounds used, and the specific adjuvants used And the carrier, soil type, etc., and the amount of chemicals applied. As is known in the art, these and other factors can be adjusted to promote non-selective or selective herbicidal effects.
  • the terms "herbicide resistance polypeptide”, “mutant HPPD polypeptide”, “mutant PaHPPD polypeptide”, “mutant HPPD protein”, “mutant HPPD enzyme”, “polypeptide of the present invention” and the like are used interchangeably, All refer to the polypeptide described in the first aspect of the present invention.
  • the herbicide resistance polypeptide is a protein or polypeptide having SEQ ID NO.: 2-3, or a derivative polypeptide or active fragment derived therefrom with the same herbicide tolerance activity.
  • herbicide resistance As used herein, the terms “herbicide resistance”, “herbicide tolerance” and “herbicide tolerance activity” are used interchangeably, and refer to herbicides, especially triketone HPPD inhibitors, such as sulcotrione , Mesotrione, Cyclosulfone or Bicyclic Sulcotrione is tolerant, and the tolerance of the herbicide resistance of the present invention can be characterized by the use concentration or use amount of the herbicide.
  • parental nucleotide or polypeptide refers to nucleic acid molecules or polypeptides (proteins) that can be found in nature, including wild-type nucleic acid molecules or proteins (polypeptides) that have not been artificially modified, and can also include artificial The nucleic acid molecule or protein (polypeptide) modified but not containing the content of the present invention.
  • the nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence can be deduced from the nucleotide sequence.
  • the "parental plant” refers to a plant containing the parental nucleotide or polypeptide.
  • the "parent nucleotide or polypeptide” can be extracted from the parent plant according to techniques well known to those skilled in the art, or can be obtained by chemical synthesis.
  • the amino acid sequence of the parent HPPD polypeptide, for example, is shown in SEQ ID No. 1.
  • the “tolerance” or “resistance” in the present invention refers to the ability of HPPD protein or cells, tissues or plants containing protein to withstand herbicides while maintaining enzyme activity or viability or plant growth , Generally can be characterized by parameters such as the amount or concentration of the herbicide.
  • the HPPD enzyme of "increased tolerance to HPPD inhibitory herbicides” or “increased resistance to HPPD inhibitory herbicides” refers to such HPPD enzymes that are maintained under the same conditions as the parent HPPD enzyme Under its activity of catalyzing the conversion of p-hydroxyphenylpyruvate to homogentisic acid, its tolerance (for example, using the maximum tolerance concentration as a characteristic parameter) is at least 1.5-10 times higher than that of the parent HPPD enzyme.
  • a plant with "enhanced tolerance to HPPD-inhibiting herbicides” or “enhanced resistance to HPPD-inhibiting herbicides” refers to a plant that has tolerance or resistance to the HPPD-inhibiting herbicide Compared with the same wild-type plant, its tolerance concentration is at least 2 to 16 times higher than that of the same wild-type plant.
  • the optimal degree of improving "tolerance” or “resistance” in the present invention is that at the same amount or concentration of herbicide, it can reduce or inhibit or kill unwanted plants without affecting the presence of the mutation of the present invention. The growth or viability of protein plants.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state of living cells are not separated and purified, but the same polynucleotides or polypeptides are separated and purified from other substances that exist in the natural state. .
  • isolated herbicide resistance polypeptide means that the herbicide resistance polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. Those skilled in the art can use standard protein purification techniques to purify the herbicide-resistant polypeptide. A substantially pure polypeptide can produce a single main band on a non-reducing polyacrylamide gel.
  • amino acid refers to a carboxylic acid containing an amino group.
  • Various proteins in organisms are composed of 20 basic amino acids. Except for glycine, all are L- ⁇ -amino acids (proline is a kind of L- ⁇ -imino acid), and its general structural formula is (R group is a variable group).
  • protein protein
  • polypeptide amino acids
  • Residue polymers include polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues.
  • the protein and polypeptide of the present invention can be produced recombinantly or chemically synthesized.
  • unwanted plants is understood as plants that affect the normal growth of desired plants (such as crops) and have no practical or application value, and may include weeds such as dicotyledonous and monocotyledonous weeds.
  • Dicotyledonous weeds include, but are not limited to, weeds of the following genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Spring Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium Genus (Xanthium), Convolvulus (Convolvulus), Ipomoea (Polygonum), Sesbania (Sesbania), Ragweed (Ambrosia), Cirsium, Carduus , Sonchus (Sonchus), Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica , Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Cornflower
  • Monocotyledonous weeds include, but are not limited to, weeds of the following genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Bluegrass (Poa), Fesuca (Festuca), Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus ), Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Ribes Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Agrostis Alopecurus) and Apera (Apera).
  • the undesired plants may also include other plants different from the plants to be cultivated, such as parts that grow naturally in rice cultivation areas or crops such as a small amount of soybeans;
  • plant tissue or “plant part” includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, Branches, seedlings, fruits, pits, spikes, roots, root tips, anthers, etc.
  • the term "gene editing” technology mainly includes CRISPR technology, TALEN technology, and ZFN technology.
  • the gene editing tools referred to in CRISPR technology include guideRNA and Cas protein (such as Cas9, Cpf1, Cas12b, etc.), and Cas protein can recognize and cut target DNA under the guidance of guideRNA.
  • the gene editing tool referred to in TALEN technology is a restriction enzyme that can cut specific DNA sequences, which includes a TAL effector DNA binding domain and a DNA cutting domain.
  • the gene editing tool referred to in the ZFN technology is also a restriction enzyme that can cut a specific DNA sequence, which includes a zinc finger DNA binding domain and a DNA cutting domain.
  • operably linked is intended to indicate that the nucleotide sequence of interest is linked to the one or more regulatory elements (e.g., , In an in vitro transcription/translation system or in the host cell when the vector is introduced into the host cell).
  • nucleic acid refers to DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.
  • the term "homology” or “identity” is used to refer to the sequence matching between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • each molecule is the same at that position.
  • the identity of two nucleotide sequences can be confirmed by the following method: BLAST algorithm (Altannet al., 1990, Mol. Biol. 215:403-10 obtained from the National Center for Biotechnology Information (NCBI), using The default parameters are determined.
  • mutant protein As used herein, the terms "mutant protein”, “mutant protein of the present invention”, “herbicide resistance polypeptide of the present invention”, and “mutant HPPD polypeptide” are used interchangeably, and all refer to non-naturally occurring HPPD polypeptides that are mutants.
  • the mutein is a protein artificially modified based on the protein shown in SEQ ID NO. 1, wherein the mutein contains core amino acids related to herbicide tolerance activity, and at least one of the core amino acids One is artificially modified.
  • core amino acid refers to SEQ ID NO.:1 and has at least 80% homology with SEQ ID NO.:1, such as 84%, 85%, 90%, 92%, 95%, 98% Or in 99% of the sequence, the corresponding position is the specific amino acid described herein. For example, based on the sequence shown in SEQ ID NO. 1, the core amino acid is:
  • Proline (P) at position 340 Proline (P) at position 340; and/or
  • Threonine (T) at position 341 At position 341; and/or
  • Lysine (K) at position 347 Lysine (K) at position 347;
  • Valine (V) at position 350 Valine (V) at position 350; and/or
  • Glycine (G) at position 351 Glycine (G) at position 351;
  • Aspartic acid (D) at position 352 is at position 352; and/or
  • Glutamic acid (E) at position 433 and the mutant protein obtained by mutating the above-mentioned core amino acids has herbicide tolerance activity.
  • the core amino acids of the present invention are mutated as follows:
  • Serine (S) at position 214 is mutated to valine (V) or leucine (L); and/or
  • Tyrosine (Y) at position 342 is mutated to histidine (H), asparagine (Asn), alanine (Ala), lysine (Lys), arginine (Arg), cysteine Acid (C) or phenylalanine (Phe); and/or
  • the arginine (R) at position 349 is mutated to threonine (T) or serine (S); and/or
  • Proline (P) at position 340 is mutated to alanine (A), serine (S), leucine (L);
  • Threonine (T) at position 341 is mutated to histidine (H), arginine (R) or lysine (K);
  • Tyrosine (Y) at position 343 is mutated to histidine (H), cysteine (C), arginine (R), lysine (K) or phenylalanine (F);
  • the asparagine (N) at position 345 is mutated to aspartic acid (D) or glycine (G);
  • Leucine (L) at position 346 is mutated to phenylalanine (F) or serine (S);
  • the lysine (K) at position 347 is mutated to glutamic acid (E) or glycine (G);
  • the lysine (K) at position 348 is mutated to glutamic acid (E) or glycine (G);
  • valine (V) at position 350 is mutated to alanine (A), serine (S) or threonine (T);
  • G glycine at position 351 to serine (S), aspartic acid (D) or asparagine (N);
  • Glutamic acid (E) at position 433 is mutated to lysine (K) or arginine (R).
  • the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO. 1.
  • the mutant protein When a specific mutant protein has 80% or more homology with the sequence shown in SEQ ID NO. 1, the mutant protein’s
  • the amino acid numbering may have a misalignment relative to the amino acid numbering of SEQ ID NO. 1, such as shifting 1-5 to the N-terminus or C-terminus of the amino acid.
  • the parent p-hydroxyphenylpyruvate dioxygenase protein can be derived from any plant, especially the aforementioned monocotyledonous or dicotyledonous plants.
  • the prior art documents have disclosed some parental (such as wild-type) p-hydroxyphenylpyruvate dioxidase protein sequences and coding sequences, and these prior art documents are hereby incorporated by reference.
  • the parent p-hydroxyphenylpyruvate dioxidase protein of the present invention is derived from Arabidopsis or Oryza. More preferably, the parent p-hydroxyphenylpyruvate dioxidase protein has the amino acid sequence shown in SEQ ID NO. 1, or has at least 80%, at least 85%, or at least the amino acid sequence shown in SEQ ID NO. An amino acid sequence of 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the mutant protein of the present invention is a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, plants) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, plants
  • the mutein of the present invention may be glycosylated or non-glycosylated.
  • the mutein of the present invention may also include or exclude the starting methionine residue.
  • the present invention also includes fragments, derivatives and analogs of the mutein.
  • fragment refers to a protein that substantially retains the same biological function or activity as the mutein.
  • the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
  • the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs fall within the scope of those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitutions according
  • the active mutant protein of the present invention has herbicide tolerance activity.
  • the mutant protein is shown in SEQ ID NO.: 2 or 3. It should be understood that compared with the sequence shown in SEQ ID NO.: 2 or 3, the mutein of the present invention generally has higher homology (identity). Preferably, the mutein of the present invention is similar to SEQ ID NO.: The homology of the sequence shown in 2 or 3 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98% or 99%.
  • mutant protein of the present invention can also be modified.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of mutein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein 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). It also includes mutant proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • polynucleotide encoding a mutein may include a polynucleotide encoding the mutein of the present invention, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • sequence of the polynucleotide encoding the mutant protein of the present invention is shown in SEQ ID NO.:6.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or muteins having the same amino acid sequence as the present invention.
  • 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 it will not substantially change the encoding of the mutant protein.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned 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 (or 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) formamide, 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, and more Fortunately, hybridization occurs when more than 95%.
  • the muteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention 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 cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
  • 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.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used.
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant herbicide-resistant polypeptides. Generally speaking, there are the following steps:
  • the 342, 349, 343, 346, 347, 348, 350, 351, 433 positions in the HPPD amino acid sequence of Arabidopsis thaliana source of the present invention are in rice (sequence reference genebank, XM_015770677, corresponding positions 339, 346 , 340, 343, 344, 345, 347, 348, 430), in sorghum (sequence reference UNIPROT: C5XVJ3, corresponding position 333,340,334,337,338,339,341,342,424), in wheat ( Sequence reference UNIPROT: Q45FE8,329,336,330,333,334,335,337,338,420), in soybean (sequence reference UNIPROT: A5Z1N7, corresponding to positions 341,348,342,345,346,347,349 , 350, 432), in maize (sequence reference UNIPROT: I7HIS1, 334, 341, 3
  • the wild-type amino acid sequence of rice OsHPPD is shown in SEQ ID NO.: 12.
  • amino acid sequence of the mutant of OsHPPD (Y339H) is shown in SEQ ID NO.:13.
  • the present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or herbicide resistance polypeptide coding sequences of the present invention, and methods for producing the polypeptides of the present invention through recombinant technology.
  • the polynucleotide sequence 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 or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, a terminator, and translation control elements.
  • the promoter of the present invention can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be homologous or heterologous to the host cell Of genes encoding extracellular or intracellular polypeptides.
  • As the promoter to be expressed in plant cells or plants it is preferable to use a natural promoter of p-hydroxyphenylpyruvate dioxygenase or a heterologous promoter active in plants.
  • the promoter may be constitutively expressed or may be inducible. Examples of promoters include, for example, histone promoter, rice actin promoter, plant virus promoters such as cauliflower mosaic virus promoter and the like.
  • the expression vector of the present invention also contains at least one origin of replication to realize self-replication.
  • the choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, and its replication does not depend on the replication of chromosomes, such as plasmids, extrachromosomal elements, mini-chromosomes or artificial chromosomes.
  • the vector may contain any elements that guarantee self-replication.
  • the vector may be a vector that is integrated into the genome when introduced into a host cell and replicates with the chromosome into which it is integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector may also be a vector for gene editing the endogenous HPPD gene of the host cell.
  • the vector can be, for example, a plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art and have been described in many ways in the art.
  • the expression vector in the present invention is a plasmid.
  • the expression vector may also contain one or more selectable marker genes for selecting host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase, genes conferring tolerance to neomycin, genes conferring tolerance to tetracycline or ampicillin, and the like.
  • More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the yield of the gene product.
  • the increase in the number of copies of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, in the latter case
  • the cells containing the amplified copy of the selectable marker gene and the resulting additional copy of the polynucleotide can be selected by artificially culturing the cells in the presence of a suitable selectable agent.
  • an expression vector containing a DNA sequence encoding a herbicide-resistant polypeptide and appropriate transcription/translation control signals can be used to construct an expression vector containing a DNA sequence encoding a herbicide-resistant polypeptide 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.
  • 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
  • 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 plant cell (such as a cell of crops and forestry plants).
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell (such as a cell of crops and forestry plants).
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, animal cells, etc.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription.
  • 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 prokaryote 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 host is a eukaryote, 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.
  • the transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain plants with changed herbicide tolerance.
  • the gene editing technology can also be used to directly edit the HPPD in the target plant genome, so that the plant cells can express the herbicide-resistant polypeptide of the present invention.
  • Representative gene editing technologies include CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • 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 cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are 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 physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. 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 protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultrafiltration treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption Chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Recombinant herbicide-resistant polypeptides have many uses. For example, it is used to screen compounds, polypeptides or other ligands that promote or counteract the functions of herbicide-resistant polypeptides. Using the expressed recombinant herbicide resistance polypeptide to screen the polypeptide library can be used to find valuable polypeptide molecules that can stimulate the function of the herbicide resistance polypeptide.
  • the present invention also includes polyclonal antibodies and monoclonal antibodies that are specific to herbicide-resistant polypeptides or their coding genes, especially monoclonal antibodies.
  • the present invention not only includes complete monoclonal or polyclonal antibodies, but also includes immunologically active antibody fragments or chimeric antibodies.
  • the antibody of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified herbicide-resistant polypeptide gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies.
  • the various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of herbicide-resistant polypeptide gene products. These fragments or functional regions can be prepared by recombinant methods or synthesized by a peptide synthesizer.
  • Antibodies that bind to the unmodified form of the herbicide-resistant polypeptide gene product can be produced by immunizing animals with the gene product produced in prokaryotic cells (such as E.
  • antibodies that bind to the post-translationally modified form can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).
  • Antibodies against herbicide-resistant polypeptides can be used to detect herbicide-resistant polypeptides in samples.
  • a method for detecting the presence of herbicide resistance polypeptide in a sample is to use the specific antibody of the herbicide resistance polypeptide for detection, which includes: contacting the sample with the specific antibody of the herbicide resistance polypeptide; observing whether an antibody complex is formed , The formation of an antibody complex indicates the presence of herbicide-resistant peptides in the sample.
  • a part or all of the polynucleotide of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing the differential expression of genes in tissues.
  • RNA-polymerase chain reaction (RT-PCR) in vitro amplification with herbicide-resistant polypeptide-specific primers can also detect the herbicide-resistant polypeptide transcript.
  • the present invention also provides a method for improving HPPD-inhibiting herbicide tolerance or resistance of plant cells, plant tissues, plant parts, or plants, which includes double oxidation with the mutant p-hydroxyphenylpyruvate containing the present invention
  • the enzyme protein or its biologically active fragment or the nucleic acid molecule encoding the nucleic acid sequence of the fusion protein is transformed into the plant or its part and expressed.
  • the nucleic acid molecule can be expressed as an extrachromosomal entity, or can be integrated into the genome of a plant cell to achieve expression, especially through homologous recombination into an endogenous gene position of the plant cell to achieve expression.
  • the present invention also provides a method for improving tolerance or resistance to HPPD-inhibiting herbicides in plants or parts thereof, which comprises expressing the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention or The plant of its biologically active fragment or fusion protein is crossed with another plant, and the plant or its part is screened for increased resistance or tolerance to HPPD inhibitory herbicides.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the present invention also provides a method for improving tolerance or resistance to HPPD-inhibiting herbicides in plant cells, plant tissues, plant parts or plants, which includes treatment of said plant cells, plant tissues, plant parts or plants
  • the endogenous HPPD protein is gene-edited to realize the expression of the mutant p-hydroxyphenylpyruvate dioxidase protein of the present invention or its biologically active fragment or fusion protein therein.
  • the present invention further relates to plant cells, plant tissues, plant parts and plants obtained by the above method, and their progeny.
  • the plant cell, plant tissue or plant part transformed with the polynucleotide of the present invention can be regenerated into a whole plant.
  • the present invention includes cell cultures, including tissue cell cultures, liquid cultures and solid plate cultures. Seeds produced by the plants of the present invention and/or used to regenerate the plants of the present invention are also included in the scope of the present invention. Other plant tissues and parts are also included in the present invention.
  • the present invention also includes methods for producing plants or cells containing the nucleic acid molecules of the present invention. A preferred method of producing such plants is by planting the seeds of the present invention. Plants transformed in this way can gain resistance to multiple herbicides with different modes of action.
  • the present invention also provides a method for controlling an effective amount of undesired plants in a plant cultivation site, which comprises applying an effective amount of one or more HPPD inhibitory herbicides to the cultivation site containing the plants or seeds of the present invention to control the undesired plants. Agent.
  • the term "cultivation site” includes the site where the plant of the present invention is cultivated, such as soil, and also includes, for example, plant seeds, plant seedlings, and grown plants.
  • the term "effective amount for controlling unwanted plants” refers to the amount of herbicide sufficient to affect the growth or development of unwanted plants, such as weeds, for example to prevent or inhibit the growth or development of unwanted plants, or to kill said unwanted plants. plant.
  • the effective amount for controlling unwanted plants does not significantly affect the growth and/or development of the plant seeds, plant seedlings or plants of the present invention. Those skilled in the art can determine such an effective amount for controlling unwanted plants through routine experiments.
  • the present invention provides a method for identifying triketone HPPD herbicides by using mutant HPPD, said mutant HPPD having the polypeptide or active fragment shown in SEQ ID NO. 2 or SEQ ID NO. 3.
  • the method includes the following steps: providing a mutant HPPD polypeptide, or a cell or plant expressing the mutant HPPD polypeptide (test group); transforming a mutant HPPD polypeptide, or a cell or plant expressing the mutant HPPD polypeptide, and the parent ( (Such as wild-type) protein, cell or plant control group to apply the test compound; determine the activity or growth or viability of the test group and the control group; select the test that causes the control group activity or growth or viability to decrease compared with the test group Compound.
  • the present invention finds for the first time that the amino acid at position 1024 of Arabidopsis HPPD is mutated from T to C, and the corresponding amino acid at position 342 is mutated from Y to R, which can impart herbicide resistance to plants; its amino acids in rice Homologous position 339 can also confer herbicide resistance to plants after mutation.
  • the herbicide-resistant polypeptide of the present invention can be used to cultivate new herbicide-tolerant plant varieties.
  • PU6 represents the Arabidopsis U6 promoter used in the vector, derived from Arabidopsis No. 3 stain (LR215054.1: 4569938-4570230); PUBQ represents the Arabidopsis AtUBQ1 promoter sequence is derived from Arabidopsis No. 3 stain ( CP002686.1: 19505047-19505665); the APOBEC1, nCAS9 and UGI sequences used in libraries 1 and 2 refer to the document of Gaudelli NM et al. [1].
  • the ABE7.10 used in Library 3 refers to the literature of Komor AC et al. [2].
  • T1 generation seeds were selected on 1/2MS+40 ⁇ g/L hygromycin (Hyg) medium and transplanted to soil. Harvest T2 generation seeds.
  • the T2 generation plants were screened, and the selection medium was 1/2MS+40 ⁇ g/L hygromycin (Hyg)+100nM mesotrione (MST), and 2-21 seedlings were obtained through screening.
  • the seedling contained a T to C mutation, and the amino acid sequence was converted from Y to R at position 342, that is, the 1024 base T of the AT1G06570.1 CDs sequence (Genebank accession: NM_100536) was converted to C.
  • the genetic testing diagram is shown in Figure 2.
  • the 2-21 mutation site can be stably inherited in the offspring
  • HPPD gene sequence of the herbicide-resistant progeny of 2-21 heterozygous plants was further detected by sequencing, and the sequencing map is shown in FIG. 4.
  • 4 plants were homozygous with cDNA 1024 T to C mutation, and 6 plants were heterozygous.
  • 2-21 base editing homozygotes have no effect on the normal growth of plants on 1/2MS medium, and in 1/2MS+MST herbicide medium, when the MST concentration is as high as 200nM, 2-21 homozygous leaves are still Green, the growth of the seedlings is shown in Figure 5, indicating that 2-21 homozygous plants have good tolerance to MST. Under the condition of germination and growth on the medium, the maximum tolerance concentration of 2-21 homozygotes to MST is about 200 nM.
  • the 2-21 mutant also has good tolerance to other HPPD inhibitory herbicides
  • Example 2 Y339H mutation in rice HPPD enhances herbicide resistance
  • Vector selection Choose a Crispr-ABE vector optimized for plants with high editing efficiency, use NG as the PAM domain of the recognition position, and design sgRNA for the Y-H site on the HPPD gene.
  • electrophoresis agarose concentration: 0.8%; electrophoresis voltage: 80V; electrophoresis time: 1.5h
  • Primer annealing will anneal the artificially synthesized DNA oligonucleotide double strands, as follows:
  • the T4 ligation vector uses a 10 ⁇ L ligation system, and the T4 DNA ligase system is ligated for 30 minutes at 25°C.
  • Reagent volume ddH 2 O Make up to 10 ⁇ L Vector 30ng 10 ⁇ T4 DNA ligase rection buffer 1 ⁇ L T4 Ligase 0.5 ⁇ L Add gRNA at the end of the variable 1 ⁇ L
  • This method uses electric transfer, the method is as follows:
  • Electrode cup After washing with ddH 2 O, soak in 75% ethanol for 20 minutes, soak in absolute ethanol for 20 minutes, blow dry in a fume hood, and pre-cool on ice;
  • the plasmid is extracted and sequenced to obtain the final gene editing vector.
  • Electrode cup After washing with ddH 2 O, soak in 75% ethanol for 20 minutes, soak in absolute ethanol for 20 minutes, blow dry in a fume hood, and pre-cool on ice;
  • Aspirate 500 ⁇ L of the bacterial solution (determined according to the size of the plate and the competent transformation efficiency) and spread it on the YEP plate containing screening resistance, and invert it for 2 days.
  • Induce callus of rice Xiushui 134 Peel the rice seeds, wash the seeds with sterile water until the water becomes clear after washing, sterilize with 70% alcohol for 30 seconds, then place 5% sodium hypochlorite on a horizontal shaker and shake for 20 minutes, and then sterilize with sodium hypochlorite After washing with sterile water 5 times, place on sterile absorbent paper, air-dry the surface water of seeds, inoculate them on induction medium and cultivate callus at 28°C.
  • Agrobacterium infection of rice callus In each batch of transformation, select about 100 Xiushui 134 callus subcultured for 14 days with a diameter of 2-3mm, and collect the callus into a triangular flask; Pour the Agrobacterium solution resuspended in the infection solution into the Erlenmeyer flask containing the callus, and place it in a shaker at 28°C, 200 rpm for 20 minutes; after the infection is complete, pour out the bacterial solution and remove the callus. Place the tissue on sterile filter paper to air dry for about 20 minutes, and place it on a common medium plate for co-cultivation.
  • the plate is covered with a piece of sterile filter paper soaked in AAM (acetosyringone AS) liquid medium; cleaned after 3 days of infection Agrobacterium was removed (washed with sterile water 5 times, and then washed with 500mg/L cephalosporin antibiotics for 20 minutes), and placed on 40mg/L hygromycin selection medium for selection and culture.
  • AAM acetosyringone AS
  • Screening, differentiation and rooting of resistant callus transfer the co-cultured callus to the screening medium for the first round of screening (2 weeks); after the first round of screening, move the newly grown callus to the screening Medium (containing 40mg/L hygromycin) for the second round of selection (2 weeks); after the selection is completed, pick yellow-white callus with good growth status for differentiation, and add 40mg/L hygromycin to the differentiation medium
  • seedlings of about 1 cm can be obtained after 3 to 4 weeks; the differentiated seedlings are transferred to the rooting medium (with 20mg/L hygromycin) for rooting culture; the rooted seedlings are refined After the seedlings are processed, they are moved to the pots with soil and placed in the greenhouse for cultivation.
  • Synthesize detection primers HPPD-F CACGAGTTCGCCGAGTTCA (SEQ ID NO.: 16) and HPPD-R: TTGACACCTTTCTGCGCCTA (SEQ ID NO.: 17).
  • the amplified fragment is 521bp, and the sgRNA site is approximately in the middle of the amplified fragment.
  • sanger sequencing was performed. The sequencing results showed that the 1015th position of the nucleotide sequence of OsHPPD was mutated to C, resulting in the 339th position of amino acid whose Y was mutated to H (see Figure 10).
  • the gene-editing positive plants were sprayed with an application rate of 4 g.a.i/mu (Quazone). Observe the state of the leaves after seven days.
  • OsHPPD Y339H
  • AtHPPD Y342H locus can also enhance the tolerance of plants to HPPD inhibitory herbicides.

Abstract

提供了一种突变的HPPD多肽，其与亲本HPPD多肽的相比存在一个或多个氨基酸序列的差异，所述差异包括在对应于SEQ ID NO.:1的第342位氨基酸发生突变，突变后的HPPD多肽对除草剂具有很强的耐受性。还提供了突变的HPPD多肽的编码基因及其在植物育种中的应用。

Description

抗除草剂基因、多肽及其在植物育种中的应用 技术领域
本发明涉及植物学领域，更具体地涉及抗除草剂基因、多肽及其在植物育种中的应用。
背景技术
对羟苯基丙酮酸双氧化酶(4-Hydroxyphenylpyruvate Dioxygenase，HPPD，EC 1.13.11.27)是生物体内酪氨酸代谢过程中的重要酶，几乎存在于所有需氧的生物体中，生物体内酪氨酸(Tyrosine)在酪氨酸氨基转移酶(Tyrosine aminotransferase,TAT)的作用下生成对羟基苯丙酮酸(p-hydroxyphenylpyruvic acid,HPPA),在氧气的参与下HPPD能够将HPPA催化转化成尿黑酸(homogentisate，HGA)。在动物体内，HPPD的主要作用是促进酪氨酸、芳氨酸、苯丙氨酸的分解代谢。但在植物体内的作用与动物体内显著不同，尿黑酸进一步形成质体醌(plastoquinones)和生育酚(tocopherols，维生素E)。生育酚起到膜相关抗氧化剂的作用，是植物生长必须的抗氧化剂，能有效地增强植物的抗逆性。质体醌是植物进行光合作用过程中关键的辅助因子,促进植物体内类胡萝卜素等的合成.在植物体中高于60％的叶绿素都结合于捕光天线复合物上,该复合物吸收太阳光能并将激发能传递给光合作用反应中心,而类胡萝卜素是反应中心的叶绿素结合蛋白和天线系统的重要组成部分,在植物光合作用中担负着光吸收辅助色素的重要功能,具有吸收和传递电子的能力,并在清除自由基方面起着重要的作用。
HPPD的抑制则会导致植物细胞内的光合作用解偶联、辅助捕光色素缺乏，同时由于缺乏通常由类胡萝卜素提供的光保护作用，活性氧中间体和光氧化导致叶绿素破坏，结果造成植物光合作用组织产生白化症状，生长受到抑制，直至死亡。因此，HPPD自20世纪90年代起，被确定为除草剂靶标。HPPD抑制类除草剂已证实是非常有效的选择性除草剂，具有广谱的除草活性，既可在芽前也可以在芽后使用，具有活性高、残留低、对哺乳动物安全和环境友好等特点。目前，按结构分已经开发出了5种以HPPD为靶标的除草剂，主要包括三酮类、吡□酮类、异噁唑酮类、二酮腈类和二苯酮类。
然而，这些HPPD抑制除草剂在其不加选择杀死杂草的同时也给作物带来 一定的伤害，因此获得耐受除草剂的作物尤为重要。目前的策略除了试图绕过HPPD介导的尿黑酸产生外，还包括进行该酶的过表达从而在植物中产生大量的除草剂靶酶，减轻除草剂的抑制作用。HPPD的过表达使得植物对异噁氟草的二酮腈衍生物(DKN)有更好的萌发前耐受性，但该耐受性不足以抵抗萌发后的除草剂处理。
因此，本领域迫切需要开发和改进对HPPD抑制剂的耐受性系统。
发明内容
本发明的目的在于提供一种对HPPD抑制剂具有高抗性的HPPD抗性基因、编码多肽及其应用。
在本发明的第一方面，提供了一种分离的除草剂抗性多肽，所述的除草剂抗性多肽为突变的HPPD多肽，
并且所述突变的HPPD多肽与亲本HPPD多肽的相比存在一个或多个氨基酸序列的差异，所述差异包括在对应于SEQ ID NO.:1的第342位氨基酸发生突变：
第342位的酪氨酸(Y)。
在另一优选例中，所述第342位的酪氨酸(Y)突变为选自下组的一种或多种氨基酸：组氨酸(H)、天冬酰胺(Asn)、丙氨酸(Ala)、赖氨酸(Lys)、精氨酸(Arg)、半胱氨酸(C)苯丙氨酸(Phe)。
在另一优选例中，所述第342位的酪氨酸(Y)突变为组氨酸(H)或半胱氨酸(C)。
在另一优选例中，所述第342位的酪氨酸(Y)突变为组氨酸(H)。
在另一优选例中，所述除草剂抗性多肽进一步包括其他突变位点，所述其他突变位点在对应于SEQ ID NO.:1的选自下组的一个或多个氨基酸发生突变：
第214位的丝氨酸(S)；
第349位的精氨酸(R)；
第340位的脯氨酸(P)；
第341位的苏氨酸(T)；
第343位的酪氨酸(Y)；
第344位的谷氨酰胺(Q)；
第345位的天冬酰胺(N)；
第346位的亮氨酸(L)；
第347位的赖氨酸(K)；
第348位的赖氨酸(K)；
第350位的缬氨酸(V)；
第351位的甘氨酸(G)；
第352位的天冬氨酸(D)；
第433位的谷氨酸(E)。
在另一优选例中，第214位的丝氨酸(S)突变为选自下组的一种或多种氨基酸：缬氨酸(V)、亮氨酸(L)。
在另一优选例中，第349位的精氨酸(R)突变为选自下组的一种或多种氨基酸：丝氨酸(S)、苏氨酸(T)。
在另一优选例中，第340位的脯氨酸(P)突变为选自下组的一种或多种氨基酸：丙氨酸(A)、丝氨酸(S)、亮氨酸(L)。
在另一优选例中，第341位的苏氨酸(T)突变为选自下组的一种或多种氨基酸：组氨酸(H)、精氨酸(R)、赖氨酸(K)。
在另一优选例中，第343位的酪氨酸(Y)突变为选自下组的一种或多种氨基酸：组氨酸(H)、半胱氨酸(C)、精氨酸(R)、赖氨酸(K)、苯丙氨酸(F)。
在另一优选例中，第344位的谷氨酰胺(Q)突变为选自下组的一种或多种氨基酸：精氨酸(R)、色氨酸(W)。
在另一优选例中，第345位的天冬酰胺(N)突变为选自下组的一种或多种氨基酸：天冬氨酸(D)、甘氨酸(G)。
在另一优选例中，第346位的亮氨酸(L)突变为选自下组的一种或多种氨基酸：苯丙氨酸(F)、丝氨酸(S)。
在另一优选例中，第347位的赖氨酸(K)突变为选自下组的一种或多种氨基酸：谷氨酸(E)、甘氨酸(G)。
在另一优选例中，第348位的赖氨酸(K)突变为选自下组的一种或多种氨基酸：谷氨酸(E)、甘氨酸(G)。
在另一优选例中，第350位的缬氨酸(V)突变为选自下组的一种或多种氨基酸：丙氨酸(A)、丝氨酸(S)、苏氨酸(T)。
在另一优选例中，第351位的甘氨酸(G)突变为选自下组的一种或多种氨基酸：丝氨酸(S)、天冬氨酸(D)、天冬酰胺(N)。
在另一优选例中，第352位的天冬氨酸(D)突变为选自下组的一种或多种氨基酸：天冬氨酸(N)、甘氨酸(G)、丝氨酸(S)。
在另一优选例中，第433位的谷氨酸(E)突变为选自下组的一种或多种氨基酸：赖氨酸(K)、精氨酸(R)。
在另一优选例中，所述的突变包括Y342H与选自下组的一个或多个突变的组合：S214V、S214L、R349S、R349T、E433K、E433R。
在另一优选例中，所述的突变选自下组：Y342H、R349S、或其组合。
在另一优选例中，所述的其他突变位点能够保持或增强突变多肽对HPPD抑制性除草剂的耐受性或抗性或增加突变型HPPD多肽对除草剂的适用范围。
在另一优选例中，所述除草剂抗性多肽的氨基酸序列如SEQ ID NO.:2或3所示。
在另一优选例中，所述的除草剂抗性多肽为具有SEQ ID NO.:2或3所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
在另一优选例中，所述的除草剂抗性多肽为具有SEQ ID NO.:4或5所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
在另一优选例中，所述的突变蛋白除所述突变(如214、342、349、340、341、343、344、345、346、347、348、350、351、352、433位)外，其余的氨基酸序列与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的突变蛋白具有除草剂耐受活性。
在另一优选例中，所述的除草剂为HPPD抑制性除草剂，选自下组：三酮类、二酮腈类、异噁唑类、吡唑类、二苯酮类、喹唑啉二酮类、或其组合。
在另一优选例中，所述的三酮类除草剂选自下组：磺草酮、硝磺草酮、环磺酮、双环磺草酮、或其组合。
在另一优选例中，所述异噁唑类除草剂选自下组：异噁唑草酮、异噁氯草酮、异恶草酮、或其组合。
在另一优选例中，所述的喹唑啉二酮除草剂包括喹草酮、甲基喹草酮、CN104557739A和CN110669016A中所记载的化合物。
在另一优选例中，所述除草剂抗性多肽与SEQ ID NO.:1所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％或99％。
在另一优选例中，所述亲本HPPD多肽的序列与SEQ ID No.1所示的氨基酸序列具有至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性。
在另一优选例中，所述的除草剂抗性多肽对除草剂的耐受浓度V1与亲本HPPD多肽对相同除草剂的耐受浓度V2相比，V1/V2≥2，较佳地V1/V2≥3，较佳地V1/V2≥4，较佳地V1/V2≥5，较佳地V1/V2≥6，较佳地V1/V2≥8，更佳地V1/V2≥10。
在另一优选例中，所述亲本HPPD多肽来源于单子叶植物或双子叶植物。
在另一优选例中，所述亲本HPPD多肽来源于选自下组的一种或多种植物：禾本科、豆科、十字花科植物。
在另一优选例中，所述亲本HPPD多肽来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述的亲本HPPD多肽来源于拟南芥(Arabidopsis thaliana)。
在另一优选例中，所述除草剂抗性多肽来源于单子叶植物或双子叶植物。
在另一优选例中，所述除草剂抗性多肽来源于选自下组的一种或多种植物：禾本科、豆科、十字花科植物。
在另一优选例中，所述除草剂抗性多肽来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述的除草剂抗性多肽来源于拟南芥(Arabidopsis thaliana)。
在另一优选例中，所述的除草剂抗性多肽能够耐受浓度≥5μM，较佳地，≥10μM，较佳地，≥20μM，较佳地≥50μM，更佳地≥100μM的除草剂。
在另一优选例中，所述的除草剂抗性多肽能够耐受浓度为10-400μM，较佳地≥20-300μM，更佳地40-260μM的除草剂。
在另一优选例中，所述的除草剂抗性多肽选自下组：
(a)具有SEQ ID NO.:2或3所示氨基酸序列的多肽；
(b)将SEQ ID NO.:2或3所示氨基酸序列经过一个或多个(如2个、3个、4个或5个)氨基酸残基的取代、缺失或添加而形成的，且具有除草剂耐受活性的由(a)衍生的多肽。
在另一优选例中，所述的衍生的多肽与SEQ ID NO.:2或3所示序列的同源性至少为60％，较佳地至少为70％，更佳地至少为80％，最佳地至少为90％，如95％、97％、99％。
在另一优选例中，所述除草剂抗性多肽为SEQ ID NO.:1所示的野生型的HPPD多肽经突变形成的。
本发明第二方面提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的除草剂抗性多肽。
在另一优选例中，所述多核苷酸选自下组：
(a)编码如SEQ ID NO.:2-3所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:6所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:6所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID NO.:2-3所示多肽的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述的多核苷酸选自下组：基因组序列、cDNA序列、RNA序列、或其组合。
在另一优选例中，所述的多核苷酸在所述除草剂抗性多肽的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸还包括与之可操作性连接的调控元件。
在另一优选例中所述的调控元件选自下组中的一种或多种：增强子、转
座子、启动子、终止子、前导序列、多腺苷酸序列、标记基因。
在另一优选例中，该多核苷酸还包含与所述除草剂抗性多肽的ORF序列操作性连接的启动子。
在另一优选例中，所述的启动子选自下组：组成型启动子、组织特异性启动子、诱导型启动子、或者强启动子。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第四方面提供了一种宿主细胞，所述的宿主细胞含有本发明第三方面所述的载体或基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞或植物细胞。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述真核细胞包括植物细胞。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述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、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、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、烟草、水稻、白菜、大豆、番茄、玉米、烟草、小麦、大麦、小米、高粱、马铃薯、藜麦、生菜、油菜、草莓、或其组合。
本发明第五方面提供了一种除草剂抗性多肽的制备方法，所述的方法包括步骤：
(a)在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达所述的除草剂抗性多肽；和
(b)分离所述的除草剂抗性多肽。
本发明第六方面提供了一种酶制剂，所述酶制剂包括本发明第一方面所述的除草剂抗性多肽。
在另一优选例中，所述的酶制剂包括注射剂、和/或冻干制剂。
本发明第七方面提供了一种改良植物的方法，所述的方法包括步骤：
(a)提供一植物细胞，对所述植物细胞进行改造，从而使所述植物细胞表达本发明第一方面所述的除草剂抗性多肽；和
(b)将步骤(a)中的植物细胞再生成植株。
在另一优选例中，用选自下组的一种或多种方法对所述植物细胞进行改造：基因工程、自然变异、物理诱变(如紫外线诱变、X射线或Y射线诱变)、化学诱变(如亚硝酸、羟胺、EMS、亚硝基胍等)、生物诱变(如病毒或细菌介导的诱变)。
在另一优选例中，所述的步骤(a)包括步骤：
(1)提供携带表达载体的农杆菌，所述的表达载体含有本发明第一方面所述的除草剂抗性多肽的DNA编码序列；
(2)将植物细胞与步骤(1)中的农杆菌接触，从而使所述除草剂抗性多肽的DNA编码序列转入植物细胞，并且整合到植物细胞的染色体上；和
(3)选择已转入所述除草剂抗性多肽的DNA编码序列的植物细胞。
在另一优选例中，在步骤(a)中，利用基因编辑技术改造所述植物细胞，从而使所述植物细胞表达本发明第一方面所述的除草剂抗性多肽。
在另一优选例中，在步骤(a)中，利用基因编辑技术改造所述植物细胞，从而使所述植物细胞中的HPPD在对应于SEQ ID NO.:1的第342位的酪氨酸发生突变。
在另一优选例中，在步骤(a)中，还包括利用基因编辑技术改造所述植物细胞，使所述植物细胞中的HPPD在对应于SEQ ID NO.:1的第214、349、340、341、343、344、345、346、347、348、350、351、352、433位中的一个或多个位点的氨基酸发生突变。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN。
在另一优选例中，所述的基因编辑技术包括可以产生所述突变的任何技术方法。
在另一优选例中，所述方法改良植物耐受除草剂的性能。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述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、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、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、烟草、水稻、白菜、大豆、番茄、玉米、烟草、小麦、大麦、小米、高粱、马铃薯、藜麦、生菜、油菜、草莓、或其组合。
在另一优选例中，所述方法还包括步骤：对所述植物细胞，测试其抗除草剂的性能。
在另一优选例中，所述的植物在培养基条件下耐受浓度为≥50nM；较佳地，≥100nM；较佳地，≥200nM；较佳地，≥250nM；较佳地，≥300nM；较佳地，≥350nM，较佳地，≥400nM；更佳地，≥450nM的除草剂。
在另一优选例中，所述的植物在土壤栽培条件下能够耐受浓度为≥5μM；较佳地，≥10μM；较佳地，≥15μM；更佳地，≥20μM的除草剂。
在另一优选例中，所述的植物在土壤栽培条件下能够耐受浓度为5μM－50μM，较佳地，10μM－30μM，更佳地，10μM－25μM，更佳地，15μM－20μM的除草剂。
在另一优选例中，所述的植物为生长2-4周的植物。
在另一优选例中，所述的耐受性通过喷施除草剂处理。
在另一优选例中，所述方法改良的植物在萌发先后至少能够耐受浓度为50nM，较佳地100nM，更佳地200nM的除草剂(硝磺草酮、磺草酮、环磺酮、双环磺酮、异噁唑草酮、喹草酮和/或甲基喹草酮)。
本发明第八方面提供了一种本发明第一方面所述的除草剂抗性多肽或其编码基因的用途，用于培育植物抗除草剂株系、或用于制备培育植物抗除草剂株系的试剂或试剂盒。
本发明第九方面提供了一种除草剂抗性敏感位点，所述的位点包括：
(I)第一抗性敏感位点，对应于(i)来源于拟南芥的野生型HPPD多肽的第342位氨基酸；(ii)来源于水稻的野生型HPPD多肽的第339位氨基酸；(iii)来源于玉米的野生型HPPD多肽的第334位氨基酸；(iv)来源于高粱的野生型HPPD多肽的第333位氨基酸；(v)来源于小麦的野生型HPPD多肽的第329位氨基酸；或(vi)来源于大豆的野生型HPPD多肽的第341位氨基酸。
在另一优选例中，所述抗性敏感位点还包括：
(II)第二抗性敏感位点，对应于(i)来源于拟南芥的野生型HPPD多肽的第349位氨基酸、(ii)来源于水稻的野生型HPPD多肽的第346位氨基酸；(iii)来源于玉米的野生型HPPD多肽的第341位氨基酸；(iv)来源于高粱的野生型HPPD多肽 的第340位氨基酸；(v)来源于小麦的野生型HPPD多肽的第336位氨基酸；或(vi)来源于大豆的野生型HPPD多肽的第348位氨基酸。
在另一优选例中，所述抗性敏感位点还包括其他抗性敏感位点，对应于(i)来源于拟南芥的野生型HPPD多肽的第340、341、343、344、345、346、347、348、350、351、352、433位中的一个或多个氨基酸；(ii)来源于水稻的野生型HPPD多肽的第337，338，339，341，342，343，344，345，346，348，349，430位氨基酸；(iii)来源于玉米的野生型HPPD多肽的第332，333，335，336，337，338，339，340，342，343，344,432位氨基酸；(iv)来源于高粱的野生型HPPD多肽的第331，332，334，335，336，337，338，339，341，342，343,424位氨基酸；(v)来源于小麦的野生型HPPD多肽的第327,328，330，331，332,333,334，335，337，338，339,420位氨基酸；或(vi)来源于大豆的野生型HPPD多肽的第339，340，342，343，344，345，346，347，349，350，351,432位氨基酸。
在另一优选例中，所述的多肽具有敏感型和不敏感型，当所述位点为酪氨酸(Y)时，所述的多肽为敏感型，并且所述的多肽对除草剂敏感；当所述的位点为组氨酸(H)、天冬酰胺(Asn)、谷氨酰胺(Gln)、赖氨酸(Lys)、精氨酸(Arg)或半胱氨酸(C)时，所述的多肽为不敏感型，并且所述的多肽对除草剂具有抗性，
较佳地，所述的不敏感型多肽对除草剂的耐受浓度V1与敏感型多肽对相同除草剂的耐受浓度V2相比，V1/V2≥2，V1/V2≥3，V1/V2≥4，V1/V2≥5，V1/V2≥6，V1/V2≥8，较佳地V1/V2≥5，更佳地V1/V2≥10。
在另一优选例中，所述的多肽具有敏感型和不敏感型，当所述第一位抗性敏感位点为酪氨酸(Y),第二位抗性敏感位点为精氨酸(R)时，所述的多肽为敏感型，并且所述的多肽对除草剂敏感；当所述的第一位抗性敏感位点为组氨酸(H)、天冬酰胺(Asn)、丙氨酸(Ala)、赖氨酸(Lys)、精氨酸(Arg)、半胱氨酸(C)或苯丙氨酸(Phe),第二位抗性敏感位点为丝氨酸(S)、苏氨酸(T)时，所述的多肽为不敏感型，并且所述的多肽对除草剂具有抗性。
在另一优选例中，所述的不敏感型多肽为权利要求1所述的除草剂抗性多肽，所述的敏感型多肽为野生型HPPD多肽。
本发明第十方面，提供一种融合蛋白，包含所述的突变多肽或其生物活性片段，以及与之融合的其它组分，例如标签肽如，组氨酸标签，例如，6×His，或者质体引导肽例如引导到叶绿体内的肽。
本发明第十一方面，提供一种耐受HPPD抑制性除草剂或对HPPD抑制性除草 剂具有抗性的植物细胞、植物组织、植物部分、植物，其中，所述植物细胞、植物组织、植物部分、植物含有所述的除草剂抗性多肽或其多核苷酸序列。
本发明第十二方面，提供一种鉴定或选择转化的植物细胞、植物组织、植物或其部分的方法，其特征在于，包括：(i)提供转化的植物细胞、植物组织，植物或其部分，其中所述转化的植物细胞、植物组织、植物或其部分包含本发明第一方面所述的除草剂抗性多肽或本发明第二方面所述的多核苷酸或本发明第三方面所述的载体；
(ii)使转化的植物细胞、植物组织、植物或其部份与除草剂接触；
(iii)确定植物细胞、植物组织、植物或其部分是否受除草剂影响；和
(iv)鉴定或选择转化的植物细胞、植物组织、植物或其部分。
在另一优选例中，所述的植物细胞、植物组织、植物或其部分可以包含另一种分离的多核苷酸。
本发明第十二方面，提供一种鉴定耐受除草剂植物的方法，其特征在于，包括：
(i)鉴定植物样本是否具有本发明第一方面所述的除草剂抗性多肽或本发明第二方面所述的多核苷酸或本发明第三方面所述的载体。
在另一优选例中，在步骤(i)中，通过测序确定所述植物样本中是否具有本发明第一方面所述的除草剂抗性多肽或本发明第二方面所述的多核苷酸或本发明第三方面所述的载体。
本发明第十三方面，提供一种在植物栽培地点控制不想要植物的方法，其特征在于，所述方法包括：
(1)在所述的栽培地点种植包含本发明第一方面所述的除草剂抗性多肽或本发明第二方面所述的多核苷酸或本发明第三方面所述的载体的植物；
(2)在所述的栽培地点对所述植物施用有效量的除草剂。
本发明第十四方面，提供一种用于产生除草剂抗性植物的方法，其特征在于，包括：
将第一植物与第二植物杂交，其中所述第一植物为抗除草剂的植物，其含有本发明第一方面所述的除草剂抗性多肽或本发明第二方面所述的多核苷酸或本发明第三方面所述的载体。
在另一优选例中，所述第二植物为不抗除草剂或除草剂抗性较弱的植物。
本发明第十五方面，提供一种筛选耐受除草剂或鉴定三酮类除草剂的方法， 包括以下步骤：
(a)在测试组中，在测试化合物的存在下，将测试化合物施用于表达权利要求1所述的突变型HPPD多肽的植物，对所述植物的生长或生存力情况进行分析；
并且在不施用所述测试化合物且其他条件相同的对照组中，对对照组的所述植物的生长或生存力情况进行分析；
(b)对测试组和对照组的植物的生长或生存力进行比较，其中，与对照组相比，如果施用了测试化合物的植物的生长或生存力不受影响或长势良好，则表示所述测试化合物是耐受除草剂或三酮类除草剂。
在另一优选例中，所述生长或生存力情况包括：叶片颜色、株高、存活率。
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AtHPPD碱基编辑文库构建示意图。
图2显示了2-21杂合子AtHPPD基因检测到单碱基编辑。
图3显示了2-21杂合子种子在MST抗性筛选培养基上出现表型分离。
图4显示了具有MST抗性的2-21子代植株均检测到含有T到C突变。
图5显示了2-21幼苗同时具有正常的生长发育和较高的MST抗性。
图6显示了在土壤培养条件下2-21具有5μM MST喷施耐受能力。
图7显示了AtHPPDY342H转基因T1代表现出MST抗性。
图8显示了AtHPPDY342H转基因T2代表现出MST抗性。
图9显示了AtHPPDY342H植株对不同除草剂的耐受性。
图10显示了水稻编辑植株进行Sanger测序后分析序列差异。
图11显示了使用4g.a.i/mu喹草酮喷施7天后水稻植株差异。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地筛到了具有植物中的除草剂耐受活性的关键氨基酸位点。本发明发现，对野生型HPPD多肽中的关键位点进行改造后，可以显著提高植物的除草剂耐受性。在此基础上，本发明人完成了本 发明。
术语
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如“L87I”表示第87位的氨基酸L突变为I，以此类推。
如本文所用，术语“HPPD”是指对羟苯基丙酮酸双氧化酶(4-Hydroxyphenylpyruvate Dioxygenase，HPPD，EC 1.13.11.27)，其存在于各种生物体中，是催化酪氨酸的降解产物-对羟苯基丙酮酸(4-hydroxyphenylpyruvate，HPP)加氧生成尿黑酸(homogentisate，HGA)反应的关键酶。HPPD的抑制会导致植物细胞内的光合作用解偶联、辅助捕光色素缺乏，同时由于缺乏通常由类胡萝卜素提供的光保护作用，活性氧中间体和光氧化导致叶绿素破坏，结果造成植物光合作用组织产生白化症状，生长受到抑制，直至死亡。HPPD抑制类除草剂已证实是非常有效的选择性除草剂，具有广谱的除草活性，既可在芽前也可以在芽后使用，具有活性高、残留低、对哺乳动物安全和环境友好等特点。
如本文所用，术语“HPPD抑制剂”、“HPPD抑制性除草剂”、“HPPD抑制类除草剂”可互换使用，是指本身有除草活性的物质或者与能改变其效果的其他除草剂和/或添加剂合用的物质，其通过抑制HPPD，表现为抑制植物生长甚至使植物死亡的制剂，本身能够通过抑制HPPD而起除草作用的物质在本领域中是熟知的，包括许多类型，1)三酮类，例如，磺草酮(Sulcotrione，CAS号：99105-77-8)；硝磺草酮(Mesotrione，CAS号：104206-82-8)；氟吡草酮(bicyclopyrone，CAS号：352010-68-5)；环磺酮(tembotrione，CAS号：335104-84-2)；呋喃磺草酮(tefuryltrione，CAS号：473278-76-1)；双环磺草酮(Benzobicyclon，CAS号：156963-66-5)；2)二酮腈类，例如，2-氰基-3-环丙基-1-(2-甲基磺酰基-4-三氟甲基苯基)丙-1,3-二酮(CAS号：143701-75-1)；2-氰基-3-环丙基-1-(2-甲基磺酰基-3,4-二氯苯基)丙-1,3-二酮(CAS号：212829-55-5)；2-氰基-1-[4-(甲基磺酰基)-2-三氟甲基苯基]-3-(1-甲基环丙基)丙-1,3-二酮(CAS号：143659-52-3)；3)异噁唑类，例如，异噁氟草(isoxaflutole，又称异噁唑草酮，CAS号：141112-29-0)；异噁氯草酮(isoxachlortole，CAS号：141112-06-3)；异恶草酮(clomazone，CAS号：81777-89-1)；4)吡唑类，例如，苯唑草酮(topramezone，CAS号：210631-68-8)；磺酰草吡唑(pyrasulfotole，CAS号：365400-11-9)；苄草唑(pyrazoxyfen，CAS 号：71561-11-0)；吡唑特(pyrazolate，CAS号：58011-68-0)；吡草酮(benzofenap，CAS号：82692-44-2)；双唑草酮(CAS号：1622908-18-2)；Tolpyralate(CAS号：1101132-67-5)；苯唑氟草酮(CAS号：1992017-55-6)；环吡氟草酮(CAS号：1855929-45-1)；三唑磺草酮(CAS号：1911613-97-2)；5)二苯酮类；6)喹唑啉二酮类，是指含有如图
Figure PCTCN2020087855-appb-000001
所示喹唑啉二酮母核结构的HPPD抑制剂类化合物，如公开号CN110669016A、CN104557739A、WO2019196904A1等专利中公开的化合物，如喹草酮(CAS号：1639426-14-4)、甲基喹草酮(CAS号1639426-42-8)。7)其他类：lancotrione(CAS号：1486617-21-3)；fenquinotrione(CAS号：1342891-70-6)。优选地，所述除草剂是异噁唑类、三酮类；优选地，所述除草剂是异噁唑草酮、硝磺草酮。所述的除草剂可以综合考虑所适用作物或杂草的类型，在于出苗前、出苗后、种植前和种植时控制不想要植物(如杂草)。优选地为三酮类HPPD抑制剂，如磺草酮、硝磺草酮、环磺酮、双环磺酮。
术语“有效量”或“有效浓度”分别意指这样的量或浓度，所述量或浓度足够杀死非目标的植物、植物组织、植物细胞或宿主细胞或抑制其生长，但是所述量不杀死本发明的抗除草剂植物、植物组织、植物细胞和宿主细胞或不严重抑制其生长(目标植物)。所述的非目标的植物可以为相似的亲本(或野生型)植物、植物组织、植物细胞或宿主细胞，还可以为杂草，或栽培地点长出的与目标植物不相关的野生型植物(如玉米田地所长出的大豆)。一般地，除草剂的有效量是农业生产系统中例行用来杀死目的杂草的量。这种量是本领域普通技术人员已知的。本发明所述的除草剂是在任何生长阶段或在种植或出苗之前直接施加至植物或施加至植物的地点时，它们显示除草活性。观察到的效果取决于待控制的植物物种、植物的生长阶段、稀释物的施加参数和喷雾液滴大小、固态组分的粒度、使用时的环境条件、所用的具体化合物、使用的具体辅助剂和载体、土壤类型等，以及施加的化学品的量。如本领域已知，可以调节这些因素和其他因素以促进非选择性或选择性除草作用。
如本文所用，术语“除草剂抗性多肽”、“突变的HPPD多肽”、“突变的PaHPPD多肽”、“突变HPPD蛋白”、“突变HPPD酶”、“本发明多肽”等可互换使用，都指本发明第一方面所述的多肽。
在另一优选例中，所述的除草剂抗性多肽为具有SEQ ID NO.:2-3的蛋白或多肽，或其衍生的具有相同除草剂耐受活性的衍生多肽或活性片段。
如本文所用，术语“除草剂抗性”、“除草剂耐受性”“除草剂耐受活性”可互换使用，是指对除草剂，尤其是三酮类HPPD抑制剂，如磺草酮、硝磺草酮、环磺酮或双环磺草酮具有耐受性，本发明的除草剂抗性的耐受性可以通过除草剂的使用浓度或使用量等特征进行表征。
术语“亲本核苷酸或多肽”指的是可以在自然界中被发现存在的核酸分子或多肽(蛋白质)，其包括未经人工改造的野生型核酸分子或蛋白质(多肽)，也可以包括经过人工改造但不含有本发明内容的核酸分子或蛋白质(多肽)。其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述“亲本植物”即含有亲本核苷酸或多肽的植物。所述“亲本核苷酸或多肽”可以根据本领域技术人员所熟知的技术从亲本植物中进行提取，亦可通过化学合成的方法获得。所述亲本HPPD多肽的氨基酸序列，例如SEQ ID No.1所示。
本发明所述的“耐受性”或“抗性”是指HPPD蛋白或含有蛋白的细胞、组织或植物体，在保持酶活性或生存力或植物生长情况下，所能承受除草剂的能力，一般可以用除草剂的使用量或使用浓度等参数进行表征。进一步的，本发明中“对HPPD抑制性除草剂的耐受性增强”或“对HPPD抑制性除草剂的抗性增强”的HPPD酶是指这样的HPPD酶，与亲本HPPD酶在同等条件保持其将对羟基苯丙酮酸催化转化为尿黑酸的活性下，其耐受能力(如以最大耐受浓度作为表征参数)表现出比亲本HPPD酶高至少1.5-10倍。“对HPPD抑制性除草剂的耐受性增强”或“对HPPD抑制性除草剂的抗性增强”的植物是指这样的植物，其对所述HPPD抑制性除草剂的耐受性或抗性与同种野生型植物相比提高，其耐受浓度相比同种野生型植物的耐受浓度高至少2倍-16倍。本发明所述的提高“耐受性”或“抗性”的最佳程度为在同等除草剂使用量或浓度下，可以减少或抑制或杀死不想要植物但不影响含有本发明所述突变蛋白的植物的生长或生存能力。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的除草剂抗性多肽”是指该除草剂抗性多肽基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化该除草剂抗性多肽。基本上纯的多肽在非还原聚丙烯酰胺凝胶 上能产生单一的主带。
如本文所用，所述“氨基酸”是指含有氨基的羧酸。生物体内的各种蛋白质是由20种基本氨基酸构成的。除甘氨酸外均为L－α－氨基酸(其中脯氨酸是一种L－α－亚氨基酸)，其结构通式为
Figure PCTCN2020087855-appb-000002
(R基为可变基团)。
术语“蛋白”、“多肽”和“肽”在本发明中可以互换使用，指的是氨基酸
残基聚合物，包括其中一个或多个氨基酸残基是天然氨基酸残基的化学类似物的聚合物。本发明的蛋白和多肽可以重组产生，也可以通过化学合成。
术语“不想要的植物”理解为影响所需植物(如农作物)正常生长的、没有实用或应用价值的植物，可以包括杂草，例如双子叶和单子叶杂草。双子叶杂草包括，但不限于以下属的杂草：白芥属(Sinapis)、独行菜属(Lepidium)、拉拉藤Galium)、繁缕属(Stellaria)、母菊属(Matricaria)、春黄菊属(Anthemis)、牛膝菊属(Galinsoga)、藜属(Chenopodium)、荨麻属(Urtica)、千里光属(Senecio)、苋属(Amaranthus)、马齿苋属(Portulaca)、苍耳属(Xanthium)、旋花属(Convolvulus)、番薯属(Ipomoea)、蓼属(Polygonum)、田菁属(Sesbania)、豚草属(Ambrosia)、蓟属(Cirsium)、飞廉属(Carduus)、苦苣菜属(Sonchus)、茄属(Solanum)、蔊菜属(Rorippa)、节节菜属(Rotala)、母草属(Lindernia)、野芝麻属(Lamium)、婆婆纳属(Veronica)、苘麻属(Abutilon)、三棘果属(Emex)、曼陀罗属(Datura)、堇菜属(Viola)、鼬瓣花属(Galeopsis)、罂粟属(Papaver)、矢车菊属(Centaurea)、车轴草属(Trifolium)、毛莨属(Ranunculus)和蒲公英属(Taraxacum)。单子叶杂草包括，但不限于以下属的杂草：稗属(Echinochloa)、狗尾草属(Setaria)、黍属(Panicum)、马唐属(Digitaria)、梯牧草属(Phleum)、早熟禾属(Poa)、羊茅属(Festuca)、穇属(Eleusine)、臂形草属(Brachiaria)、黑麦草属(Lolium)、雀麦属(Bromus)、燕麦属(Avena)、莎草属(Cyperus)、高粱属(Sorghum)、冰草属(Agropyron)、狗牙根属(Cynodon)、雨久花属(Monochoria)、飘拂草属(Fimbristyslis)、慈姑属(Sagittaria)、荸荠属(Eleocharis)、藨草属(Scirpus)、雀稗属(Paspalum)、鸭嘴草属(Ischaemum)、尖瓣花属(Sphenoclea)、龙爪茅属(Dactyloctenium)、剪股颖属(Agrostis)、看麦娘属(Alopecurus)和阿披拉草属(Apera)。所述的不想要植物还可以包括与所要栽培植物不同的其他植物，例如在水稻栽培地自然生长的部分或少量大豆等作物；
在本发明中，术语“植物组织”或“植物部分”包括植物细胞、原生质体、 植物组织培养物、植物愈伤组织、植物块以及植物胚、花粉、胚珠、种子、叶、茎、花、枝、幼苗、果实、核、穗、根、根尖、花药等。
在本发明中，术语“基因编辑”技术主要包括CRISPR技术、TALEN技术、ZFN技术。CRISPR技术中所指基因编辑工具包括guideRNA、Cas蛋白(如Cas9、Cpf1、Cas12b等)，Cas蛋白在guideRNA的引导下可识别并切割靶标DNA。TALEN技术中所指的基因编辑工具是可以切割特定DNA序列的限制酶，其包括一个TAL效应子DNA结合结构域和一个DNA切割结构域。ZFN技术中所指的基因编辑工具也是可以切割特定DNA序列的限制酶，其包括一个锌指DNA结合结构域与一个DNA切割结构域。本领域技术人员熟知，将编码基因编辑工具的核苷酸及其他调控元件构建于适宜的载体中，再转化细胞，可以实现对细胞内基因组的编辑，所述编辑的类型包括基因敲除、插入、碱基编辑。
如本文中所使用的，术语“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调节元件(例如，处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。
术语“多核苷酸”、“核苷酸序列”、“核酸序列”、“核酸分子”和“核酸”可以互换使用，包括DNA、RNA或者其杂交体，可以是双链或单链的。
术语“同源性”或“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。例如两个核苷酸序列的同一性可以通过以下方式确认：从美国国立生物技术信息中心(NCBI)获得的BLAST运算法则(Altschulet al.,1990,Mol.Biol.215:403-10)，使用默认参数确定。
本发明突变蛋白及其编码核酸
如本文所用，术语“突变蛋白”、“本发明突变蛋白”、“本发明的除草剂抗性多肽”、“突变型HPPD多肽”可互换使用，均指非天然存在的为突变的HPPD多肽，且所述突变蛋白为基于SEQ ID NO.:1所示蛋白进行人工改造的蛋白，其中，所述的突变蛋白含有与除草剂耐受活性相关的核心氨基酸，且所述核心氨基酸中至少有一个是经过人工改造的。
术语“核心氨基酸”指的是基于SEQ ID NO.:1，且与SEQ ID NO.:1同源性达至少80％，如84％、85％、90％、92％、95％、98％或99％的序列中，相应位点是本文所述的特定氨基酸，如基于SEQ ID NO.:1所示的序列，核心氨基酸为：
第214位的丝氨酸(S)；和/或
第342位的酪氨酸(Y)；和/或
第349位的精氨酸(R)；和/或
第340位的脯氨酸(P)；和/或
第341位的苏氨酸(T)；和/或
第343位的酪氨酸(Y)；和/或
第344位的谷氨酰胺(Q)；和/或
第345位的天冬酰胺(N)；和/或
第346位的亮氨酸(L)；和/或
第347位的赖氨酸(K)；和/或
第348位的赖氨酸(K)；和/或
第350位的缬氨酸(V)；和/或
第351位的甘氨酸(G)；和/或
第352位的天冬氨酸(D)；和/或
第433位的谷氨酸(E)，且对上述核心氨基酸进行突变所得到的突变蛋白具有除草剂耐受活性。
优选地，在本发明中，对本发明的所述核心氨基酸进行如下突变：
第214位的丝氨酸(S)突变为缬氨酸(V)或亮氨酸(L)；和/或
第342位的酪氨酸(Y)突变为组氨酸(H)、天冬酰胺(Asn)、丙氨酸(Ala)、赖氨酸(Lys)、精氨酸(Arg)、半胱氨酸(C)或苯丙氨酸(Phe)；和/或
第349位的精氨酸(R)突变为苏氨酸(T)或丝氨酸(S)；和/或
第340位的脯氨酸(P)突变为丙氨酸(A)、丝氨酸(S)亮氨酸(L)；
第341位的苏氨酸(T)突变为组氨酸(H)、精氨酸(R)或赖氨酸(K)；
第343位的酪氨酸(Y)突变为组氨酸(H)、半胱氨酸(C)、精氨酸(R)，赖氨酸(K)或苯丙氨酸(F)；
第344位的谷氨酰胺(Q)突变为精氨酸(R)或色氨酸(W)；
第345位的天冬酰胺(N)突变为天冬氨酸(D)或甘氨酸(G)；
第346位的亮氨酸(L)突变为苯丙氨酸(F)或丝氨酸(S)；
第347位的赖氨酸(K)突变为谷氨酸(E)或甘氨酸(G)；
第348位的赖氨酸(K)突变为谷氨酸(E)或甘氨酸(G)；
第350位的缬氨酸(V)突变为丙氨酸(A)、丝氨酸(S)或苏氨酸(T)；
第351位的甘氨酸(G)突变为丝氨酸(S)、天冬氨酸(D)或天冬酰胺(N)；
第352位的天冬氨酸(D)突变为天冬氨酸(N)、甘氨酸(G)或丝氨酸(S)；和/或
第433位的谷氨酸(E)突变为赖氨酸(K)或精氨酸(R)。
应理解，本发明突变蛋白中的氨基酸编号基于SEQ ID NO.:1作出，当某一具体突变蛋白与SEQ ID NO.:1所示序列的同源性达到80％或以上时，突变蛋白的氨基酸编号可能会有相对于SEQ ID NO.:1的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似的除草剂耐受活性的突变蛋白不在本发明突变蛋白的范围内。
在本发明中，亲本对羟苯基丙酮酸双氧化酶蛋白可以来源于任何植物，特别是前述单子叶或双子叶植物。现有技术文献中已经公开了一些来源的亲本(如野生型)对羟苯基丙酮酸双氧化酶蛋白序列以及编码序列，这些现有技术文献在此引入本文作为参考。
优选地，本发明的亲本对羟苯基丙酮酸双氧化酶蛋白来源于拟南芥或稻属。更优选地，所述亲本对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO.1所示的氨基酸序列，或者与SEQ ID NO.1所示氨基酸序列有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
本发明突变蛋白是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述突变蛋白的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述突变蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、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)被取代的突变蛋白，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突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，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，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
Figure PCTCN2020087855-appb-000003
本发明的活性突变蛋白具有除草剂耐受活性。
优选地，所述的突变蛋白如SEQ ID NO.:2或3所示。应理解，本发明突变蛋白与SEQ ID NO.:2或3所示的序列相比，通常具有较高的同源性(相同性)， 优选地，所述的突变蛋白与SEQ ID NO.:2或3所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％或99％。
此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变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。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
术语“编码突变蛋白的多核苷酸”可以是包括编码本发明突变蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
在一优选实施方式中，本发明的编码突变蛋白的多核苷酸的序列如SEQ ID NO.:6所示。
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肽或突变蛋白的片段、类似物和衍生物。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突变蛋白的功能。
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(或严紧条件)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)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的突变蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过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cDNA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。这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。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
通过常规的重组DNA技术(Science，1984；224：1431)，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除草剂抗性多肽。一般来说有以下步骤：
(1).用本发明的编码除草剂抗性多肽的多核苷酸(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。
应注意，本发明的来源拟南芥的HPPD氨基酸序列中的342、349、343、346、347、348、350、351、433位点在水稻(序列参考genebank，XM_015770677，对应位点339、346、340、343、344、345、347、348、430)、在高粱(序列参考UNIPROT：C5XVJ3，对应位点333，340，334，337，338，339，341，342，424)、在小麦(序列参考UNIPROT：Q45FE8，329，336，330，333，334，335，337，338，420)、在大豆(序列参考UNIPROT：A5Z1N7，对应位点341，348，342，345，346，347，349，350，432)、在玉米(序列参考UNIPROT：I7HIS1，334、341、335、338、339、340、342、343、432)中均为保守位点。因此，上述位点在作物中对除草剂的抗性具有至关重要的作用。
在一优选实施方式中，水稻OsHPPD野生型氨基酸序列如SEQ ID NO.:12所示。
在一优选实施方式中，OsHPPD(Y339H)的突变体氨基酸序列如SEQ ID NO.:13所示。
载体、植物改良
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除草剂抗性多肽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。
本发明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、哺乳动物细胞病毒或其他载体。总之，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、终止子和翻译控制元件。
本发明所述的启动子可以是在选定宿主细胞内显示转录活性的任何核苷酸序列，包括突变的、截断的和杂合的启动子，并且可能获子与宿主细胞同源或异源的编码细胞外或细胞内多肽的基因。作为在植物细胞或植物中表达的启动子，优选使用对羟苯基丙酮酸双氧化酶天然的启动子，或者在植物中具有活性的异源启动子。所述启动子可以是组成型表达的，或者可以是诱导型表达的。启动子的实例包括例如组蛋白启动子，水稻肌动蛋白启动子，植物病毒启动子例如花椰菜花叶病毒启动子等。
在某些实施方式中，本发明所述表达载体中还至少含有一个复制起点，以实现自我复制。载体的选择通常取决于载体与该载体待引入之宿主细胞的相容性。载体可能是自主复制载体，即作为染色体外实体存在的载体，它的复制不依赖于染色体的复制，例如质粒、染色体外元件、微型染色体或人工染色体。该载体可能包含保证自我复制的任何元件。或者，在某些实施方式中，所述载体可能是当引入宿主细胞时被整合入基因组中并与其所整合入的染色体一起复制的载体。此外，可使用单个载体或质粒或者一起包含待引入宿主细胞基因组之总DNA的两个或更多个载体或质粒，或者转座子。或者，所述载体也可以是对宿主细胞内源性的HPPD基因进行基因编辑的载体。
载体可以是例如质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的，在本领域中众多描述。优选地，本发明中的表达载体是质粒。表达载体中也可以含有一个或多个可选择标记基因以便用于选择包含载体的宿主细胞。这种可选择的标记包括编码二氢叶酸还原酶的基因，或赋予新霉素耐受性的基因，赋予对四环素或氨苄青霉素耐受性的基因等。
可将一拷贝以上的本发明之多核苷酸插入宿主细胞中以提高基因产物的产 量。可通过将至少一个额外拷贝的序列整合入宿主细胞基因组中或者通过将可扩增的可选择标记基因与所述多核苷酸包含在一起来达到多核苷酸拷贝数目的增加，在后一情形下，包含扩增拷贝的选择标记基因以及由此而来的附加拷贝的多核苷酸的细胞可通过在适当的可选择制剂存在的条件下人工培养所述细胞进行选择。
本领域的技术人员熟知的方法能用于构建含除草剂抗性多肽编码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。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，如植物细胞(如农作物和林业植物的细胞)。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；植物细胞、动物细胞等。
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。
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。
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。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，从而获得除草剂耐受性改变的植物。
也可以利用基因编辑技术直接对目标植物基因组中的HPPD进行编辑，从而使植物细胞表达本发明的除草剂抗性多肽。代表性的基因编辑技术包括CRISPR基因 编辑体系、易错PCR、基因重组、TALEN和ZFN。
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、超滤处理、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。
重组的除草剂抗性多肽有多方面的用途。例如用于筛选促进或对抗除草剂抗性多肽功能的化合物、多肽或其它配体。用表达的重组除草剂抗性多肽筛选多肽库可用于寻找有价值的能刺激除草剂抗性多肽功能的多肽分子。
另一方面，本发明还包括对除草剂抗性多肽或是其编码基因具有特异性的多克隆抗体和单克隆抗体，尤其是单克隆抗体。本发明不仅包括完整的单克隆或多克隆抗体，而且还包括具有免疫活性的抗体片段、或嵌合抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如，纯化的除草剂抗性多肽基因产物或者其具有抗原性的片段，可被施用于动物以诱导多克隆抗体的产生。本发明的各类抗体可以利用除草剂抗性多肽基因产物的片段或功能区，通过常规免疫技术获得。这些片段或功能区可以利用重组方法制备或利用多肽合成仪合成。与除草剂抗性多肽基因产物的未修饰形式结合的抗体可以用原核细胞(例如E.Coli)中生产的基因产物来免疫动物而产生；与翻译后修饰形式结合的抗体(如糖基化或磷酸化的蛋白或多肽)，可以用真核细胞(例如酵母或昆虫细胞)中产生的基因产物来免疫动物而获得。抗除草剂抗性多肽的抗体可用于检测样品中的除草剂抗性多肽。
一种检测样品中是否存在除草剂抗性多肽的方法是利用除草剂抗性多肽的特异性抗体进行检测，它包括：将样品与除草剂抗性多肽特异性抗体接触；观察是否形成抗体复合物，形成了抗体复合物就表示样品中存在除草剂抗性多肽。
本发明多核苷酸的一部分或全部可作为探针固定在微阵列(microarray)或DNA芯片(又称为“基因芯片”)上，用于分析组织中基因的差异表达分析。用除草 剂抗性多肽特异的引物进行RNA-聚合酶链式反应(RT-PCR)体外扩增也可检测除草剂抗性多肽的转录产物。
本发明还提供了一种提高植物细胞、植物组织、植物部分或植物的HPPD抑制性除草剂耐受性或抗性的方法，其中包括用包含本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白的编码核酸序列的核酸分子转化所述植物或其部分，并使之表达。所述核酸分子可以作为染色体外实体存在而进行表达，或者可以整合到植物细胞的基因组中实现表达，特别是通过同源重组整合到植物细胞的内源基因位置处实现表达。
本发明还提供了一种提高植物或其部分的HPPD抑制性除草剂耐受性或抗性的方法，其中包括将表达本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白的植物与另一植物杂交，以及筛选具有提高的HPPD抑制性除草剂抗性或耐受性的植物或其部分。
本发明还提供了一种提高植物细胞、植物组织、植物部分或植物中的HPPD抑制性除草剂耐受性或抗性的方法，其中包括对所述植物细胞、植物组织、植物部分或植物的内源性HPPD蛋白进行基因编辑，以实现在其中表达本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白。
本发明进一步涉及通过上述方法获得的植物细胞、植物组织、植物部分和植物，及其后代。优选地，可以将转化了本发明多核苷酸的植物细胞、植物组织或植物部分再生为整个植株。本发明包括细胞培养物，包括组织细胞培养物、液体培养物和固体平板培养物。由本发明植物所产生和/或用于再生本发明植物的种子也包括在本发明范围内。其他植物组织和部分也包括在本发明中。本发明同样包括产生含有本发明核酸分子的植物或细胞的方法。产生这类植物的一种优选方法为通过种植本发明的种子。以这种方式转化的植物可以获得对多种具有不同作用模式的除草剂的抗性。
本发明还提供了一种在植物栽培地控制不想要植物有效量的方法，其中包括对包含本发明的植物或种子的栽培地施用控制不想要植物有效量的一种或多种HPPD抑制性除草剂。
在本发明中，术语“栽培地”包括栽培本发明植物的场地例如土壤，也包括例如植物种子、植物苗以及长成的植物。术语“控制不想要植物有效量”指的是除草剂的量足以影响不想要植物，如杂草，的生长或发育，例如阻止或抑制不想要植物的生长或发育，或者杀灭所述不想要植物。有利地，所述控制不想要植物有效量 不会显著影响本发明植物种子、植物苗或植物的生长和/或发育。本领域技术人员可以通过常规实验确定这样的控制不想要植物有效量。
本发明提供了一种通过使用突变型HPPD鉴定三酮类HPPD除草剂的使用方法，所述的突变型HPPD具有SEQ ID NO.2或SEQ ID NO.3所示的多肽或活性片段。所述的方法包括以下步骤：提供一种突变型HPPD多肽，或表达突变型HPPD多肽的细胞或植物(测试组)；向突变型HPPD多肽，或表达突变型HPPD多肽的细胞或植物及亲本(如野生型)蛋白、细胞或植物的对照组施加所述测试化合物；测定测试组及对照组的活性或生长或生存力；选择与测试组相比引起对照组活性或生长或生存力减少的测试化合物。
本发明的主要优点包括：
(a)本发明首次发现，拟南芥HPPD第1024位碱基由T突变为C，相应的342位氨基酸由Y突变为R后，可赋予植株抗除草剂的特性；其在水稻中的氨基酸同源位点339，突变后同样可以赋予植株除草剂抗性。
(b)通过导入除草剂抗性多肽的编码基因，可以增强拟南芥对除草剂的抗性或耐受性。
(c)通过导入本发明除草剂抗性多肽的编码基因，可以增强植物对除草剂的抗性或耐受性。
(d)本发明的除草剂抗性多肽可以用于培育除草剂耐受性植物新品种。
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，例如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。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1 抗除草剂突变位点的筛选
1、sgRNA库的构建
在AtHPPD基因组序列范围内设计267个gRNA(以GACCCGTTTCTTGAGATTC SEQ ID NO.:7)；TACCAGAATCTCAAGAAAC(SEQ ID NO.:8)；TCTGGTAGTAAGTAGGCGG (SEQ ID NO.:9)为例)，如表1，并且将这些gRNA克隆至3种碱基编辑载体上，如图1，构建三种碱基编辑表达文库，构建文库1和2的是碱基C转变为T的编辑器，文库3载体是碱基A转变为G的编辑器。三种文库构建质粒的骨架均为pCambia1300。PU6表示载体中所用拟南芥U6启动子，来源于拟南芥第3号染色(LR215054.1：4569938-4570230)；PUBQ表示拟南芥AtUBQ1启动子序列来源于拟南芥第3号染色(CP002686.1：19505047-19505665)；文库1和2中所用到的APOBEC1，nCAS9和UGI序列参考Gaudelli NM等人的文献[1]。文库3中用到的ABE7.10参考Komor AC等人的文献[2]。
2、碱基编辑器表达载体的构建涉及的实验材料及方法步骤；
1)BSA1酶切碱基编辑器载体37℃，4小时
Figure PCTCN2020087855-appb-000004
2)sgRNA互补双链退火与磷酸化
Figure PCTCN2020087855-appb-000005
3)链接反应室温10min:
Figure PCTCN2020087855-appb-000006
Figure PCTCN2020087855-appb-000007
4)将连接产物转化大肠杆菌DH5α
5)涂布至加入30μg/L卡纳霉素LB平板
6)刮取克隆提质粒文库
3、载体遗传转化
1)将1μg文库质粒转化农杆菌GV1301，涂布至加入25μg/L利福平和30μg/L卡纳霉素的LB平板。
2)刮板收集菌落。
3)浸花法转染拟南芥Col-0。每个文库转染不少于100株苗。
4)收集T1代种子。在1/2MS+40μg/L潮霉素(Hyg)培养基上筛选T1代阳性苗，移栽至土壤。收获T2代种子。
4、T2代植株的筛选
对T2代植株进行筛选，筛选培养基为1/2MS+40μg/L潮霉素(Hyg)+100nM硝磺草酮(MST)，通过筛选获得2-21幼苗。经测序检测，该幼苗含有T到C突变，氨基酸序列在342位由Y转化为R，即AT1G06570.1 CDs序列(Genebank accession:NM_100536)1024位碱基T转变为C。测序引物seq2:GCTCTTGTCGTTCCTTCTTC(SEQ ID NO.:10)；seq3:CGGAACAAAGAGGAAGAGTC(SEQ ID NO.:11)。基因检测图如图2所示。
5、2-21杂合子植株表现出除草剂抗性
2-21杂合子植株的种子在含有除草剂的培养基(1/2MS+40μg/L Hyg+100nM MST)上萌发生长有表型分离，如图3所示。考虑抗潮霉素基因的性状分离，对于显性的抗除草剂突变，正常的抗性出苗率9/16。我们的抗性苗是平均21株/36株，与理论数据比较接近。表明在Y342R杂合或纯合突变的条件下植物都有除草剂抗性。
6、2-21突变位点在子代中可以稳定遗传
进一步通过测序检测2-21杂合子植物的抗除草剂的子代HPPD基因序列，测序图谱如图4所示。10株被检测的MST抗性植株，含有cDNA 1024位T到C突变的纯合子为4株，杂合子有6株。
7、2-21突变体植株在培养基条件下对MST的耐受性实验
2-21碱基编辑纯合子在1/2MS培养基上对植株正常生长无影响，而且在1/2MS+MST除草剂培养基中，当MST浓度高达200nM时，2-21纯合子叶片仍为绿色，幼苗生长情况见图5，说明2-21纯合子植株对MST具有很好的耐受性。在培养基上萌发生长条件下，2-21纯合子对MST的最大耐受浓度约为200nM。
8、2-21突变体植株在土壤条件下对MST具有较好的耐受性
在土壤栽培条件下，生长间培养条件22℃，16L/8D，2-21纯合子植物与野生型Col-0在抽苔时期分别喷施0μM、1μM、5μM、20μM MST两周，2-21纯合子可耐受20μM MST喷施，植株生长情况见图6，说明-21纯合子对高浓度的MST具有较好的耐受性。
9、Y342R突变体转基因植株比野生型植物具有更好的耐受性
(1)构建pCambia1305-PAtHPPD：AtHPPDY342R-HA与pCambia1305-PAtHPPD：AtHPPD-HA载体，AtHPPDY342R转基因T1代植物在1/2MS+40μg/L Hyg+100nM MST筛选培养基上就出现MST抗体表型，而转入对照未突变的AtHPPD基因的植物T1代植物没有表现出明显抗性，幼苗生长情况参见图7，说明Y342R突变可以赋予植株很好的抗除草剂活性。
(2)扩增拟南芥AtHPPD基因内源启动子和基因组序列，用PCR方式体外突变Y342H位点，构建拟南芥野生型AtHPPD基因(HPDWT)和Y342H突变的AtHPPD基因(HPDY342H)的转基因载体。转化野生型拟南芥Col-0。筛选得到的T2代转基因植物进行除草剂抗性检测，如图8。将萌发11天的幼苗移栽至土壤，生长10天后喷施5μM MST，两周后拍照观察表型如图8A，第二次继续喷施5μM MST，两周后再拍照观察表型如图8B。结果显示转入Y342H突变的AtHPPD基因的转基因植物HPDY342H T2-19和T2-18株系，比转入野生型AtHPPD基因的HPDWT T2-2和T2-21株系对MST有更强的耐受性。
10、2-21突变体对其他HPPD抑制性除草剂同样具有很好的耐受性
将2-21(AtHPPDY342H)和Col-0种子分别在添加浓度为50nM和100nM ISO(isoxaflutole异恶唑草酮)，2μg/L甲基喹草酮的1/2MS培养基上萌发，生长14天记录表型，表明2-21对不同类型HPPD抑制剂均表现出抗性(如图9)。
实施例2 水稻HPPD中Y339H突变增强除草剂抗性
1、载体构建
载体选择：选择经过植物优化的编辑效率较高的Crispr-ABE载体，以NG 作为识别位置的PAM结构域，在HPPD基因上针对Y-H位点设计sgRNA。设计sgRNA-F：GGTAGTAGTTGGGCGGCGGC(SEQ ID NO.:14)和sgRNA-R：GCCGCCGCCCAACTACTACC(SEQ ID NO.:15)，在序列两侧添加合适粘性末端，构建到水稻Crispr-ABE载体上。
酶切Crispr-ABE载体：30μL酶切体系：用枪反复吸取混合，或是用手指轻弹管壁混合，然后再快速离心一下即可。
试剂 体积
ddH 2O 补足至30μL
Crispr-ABE 2μg
CutSmart 3μL
BsaI-HF 1μL
反应条件：
温度 时间
37℃ 4h
12℃ ∞
使用胶回收法，电泳：琼脂糖浓度：0.8％；电泳电压：80V；电泳时间：1.5h
引物退火将将人工合成的DNA寡核苷酸双链退火，具体如下：
将每条DNA寡核苷酸用1×Taq buffer稀释到10μM，上下游引物各吸取1μL混合退火。退火后，放冰上或者-20℃保存。
试剂 体积
Primer-F 1μL
Primer-R 1μL
1×Taq buffer 8μL
Figure PCTCN2020087855-appb-000008
Figure PCTCN2020087855-appb-000009
T4连接载体 使用10μL连接体系，T4DNA连接酶体系25℃连接30min。
10μL体系：
试剂 体积
ddH 2O 补至10μL
Vector 30ng
10×T4 DNA ligase rection buffer 1μL
T4 Ligase 0.5μL
变量最后加gRNA 1μL
反应条件：
Figure PCTCN2020087855-appb-000010
转E.coli(电转/化转)
本方法使用电转，方法如下：
电极杯：ddH 2O洗后，用75％乙醇浸泡20分钟，无水乙醇浸泡20分钟，通风橱内吹干，冰上预冷；
用1.5mL离心管分装LB，28℃预热；
将感受态细胞在冰上溶化5min；
取10μL连接产物加入50μL大肠杆菌电转感受态混匀。
吸取混合液于电极杯中，轻弹混匀，电击；
将预热的LB 700μL倒入电极杯内，将菌体冲洗到2ml管内。洗净电极杯并用75％乙醇浸泡；
将菌液在37℃下200rpm摇培1h；
将菌液全部涂布于含有筛选抗性的LB平板上，37℃倒置过夜培养。
提取质粒，测序后，获得最终基因编辑载体。
转农杆菌(电转)
电极杯：ddH 2O洗后，用75％乙醇浸泡20分钟，无水乙醇浸泡20分钟，通风橱内吹干，冰上预冷；
用1.5mL离心管分装YEP，28℃预热；
将感受态细胞在冰上溶化5min；
吸取1μl质粒与感受态细胞混匀；
吸取混合液于电极杯中，轻弹混匀，电击；
将预热的LB 700μL倒入电极杯内，将菌体冲洗到2ml管内。洗净电极杯并用75％乙醇浸泡；
将菌液在28℃下200rpm摇培1.5～2h；
将菌液吸取500μL(根据平板大小以及感受态转化效率决定)涂布于含有筛选抗性的YEP平板上，倒置培养2d。
2、愈伤组织转化
诱导水稻秀水134愈伤组织：剥取水稻种子，无菌水清洗种子，直至洗后的水变清澈，70％酒精消毒30秒，之后5％次氯酸钠置于水平摇床摇晃培养20分，次氯酸钠消毒后无菌水清洗5次，置于无菌吸水纸，风干种子表面水分，接种于诱导培养基上在28℃下培养愈伤。
农杆菌侵染水稻愈伤：在每一批转化中，选取继代培养14天，直径为2-3mm的秀水134愈伤组织约100个，将愈伤组织收集到三角瓶中；将已用侵染液重悬浮的农杆菌菌液倒入含有愈伤组织的三角瓶中，置于28℃、200转/分摇床中侵染20分；侵染完毕，倒掉菌液，将愈伤组织放置于无菌滤纸上风干20min左右，置于共同培养基平板上共同培养，平板上铺有一张AAM(添加乙酰丁香酮AS)液体培养基浸湿的无菌滤纸；侵染3天后，清洗去除农杆菌(先用无菌水洗5遍，再用500mg/L的头孢抗生素清洗20分钟)，置于40mg/L潮霉素筛选培养基上筛选培养。
抗性愈伤的筛选、分化和生根：将共培养后的愈伤组织移至筛选培养基进行第一轮筛选(2周)；第一轮筛选完毕后将新长出的愈伤移至筛选培养基(含40mg/L潮霉素)进行第二轮筛选(2周)；筛选完成后，挑取生长状态良好的黄白色愈伤组织进行分化，分化培养基中添加40mg/L潮霉素进行除草剂抗性筛选，3～4周后可以获得1cm左右的幼苗；将分化出的幼苗移至生根培养基(添加有20mg/L潮霉素)进行生根培养；将生根完成的幼苗进行炼苗处理后，移至装有土壤的花盆中置温室进行培养。
3、检测
合成检测引物HPPD-F:CACGAGTTCGCCGAGTTCA(SEQ ID NO.:16)和HPPD-R：TTGACACCTTTCTGCGCCTA(SEQ ID NO.:17)。扩增片段521bp，sgRNA位点大约位于扩增片段中间位置。对分化出的苗子取少量叶片以CTAB法提取基因组genome DNA。用上述检测引物对genome DNA进行PCR。扩增产物以1％琼脂糖电泳检测后，进行sanger测序。测序结果显示OsHPPD的核苷酸序列1015位T突变为C，导致氨基酸第339位Y突变为H(见图10)。
4、抗性鉴定
温室土壤栽培一个月后，利用4g.a.i/mu的施用量(喹草酮)喷洒基因编辑阳性植株。七天后观察叶片状态。
5、实验结果
喷施除草剂7天后，编辑植株状态叶片保持绿色，野生型植株叶片出现黄化(见图11)，即编辑植株的生长状态优于野生型植株。
6、实验结论
与AtHPPD Y342H位点相应的OsHPPD(Y339H)突变后，同样可以增强植物对HPPD抑制性除草剂的耐受性。
参考文献
1.Gaudelli NM,Komor AC,Rees HA,Packer MS,Badran AH,Bryson DI,Liu DR:Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.Nature 2017,551(7681):464-471.
2.Komor AC,Kim YB,Packer MS,Zuris JA,Liu DR:Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature  2016,533(7603):420-424.
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 (15)

  1. 一种分离的除草剂抗性多肽，其特征在于，所述的除草剂抗性多肽为突变的HPPD多肽，
    并且所述突变的HPPD多肽与亲本HPPD多肽的相比存在一个或多个氨基酸的差异，所述差异包括在对应于SEQ ID NO.:1的第342位氨基酸发生突变：
    第342位的酪氨酸(Y)。
  2. 如权利要求1所述的除草剂抗性多肽，其特征在于，所述第342位的酪氨酸(Y)突变为选自下组的一种或多种氨基酸：组氨酸(H)、天冬酰胺(Asn)、丙氨酸(Ala)、赖氨酸(Lys)、精氨酸(Arg)、半胱氨酸(C)苯丙氨酸(Phe)。
  3. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1所述的除草剂抗性多肽。
  4. 一种载体，其特征在于，所述的载体含有权利要求3所述的多核苷酸。
  5. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求4所述的载体或基因组中整合有权利要求3所述的多核苷酸。
  6. 一种除草剂抗性多肽的制备方法，其特征在于，所述的方法包括步骤：
    (a)在适合表达的条件下，培养权利要求5所述的宿主细胞，从而表达所述的除草剂抗性多肽；和
    (b)分离所述的除草剂抗性多肽。
  7. 一种酶制剂，其特征在于，所述酶制剂包权利要求1所述的除草剂抗性多肽。
  8. 一种改良植物的方法，其特征在于，所述的方法包括步骤：
    (a)提供一植物细胞，对所述植物细胞进行改造，从而使所述植物细胞表达权利要求1所述的除草剂抗性多肽；和
    (b)将步骤(a)中的植物细胞再生成植株。
  9. 一种权利要求1所述的除草剂抗性多肽或其编码基因的用途，其特征在于，用于培育植物抗除草剂株系、或用于制备培育植物抗除草剂株系的试剂或试剂盒。
  10. 一种除草剂抗性敏感位点，其特征在于，所述的位点包括：
    (I)第一抗性敏感位点，对应于(i)来源于拟南芥的野生型HPPD多肽的第342位氨基酸；(ii)来源于水稻的野生型HPPD多肽的第339位氨基酸；(iii)来源于玉米的野生型HPPD多肽的第334位氨基酸；(iv)来源于高粱的野生型HPPD多肽的第 333位氨基酸；(v)来源于小麦的野生型HPPD多肽的第329氨基酸；或(vi)来源于大豆的野生型HPPD多肽的第341位氨基酸。
  11. 一种鉴定或选择转化的植物细胞、植物组织、植物或其部分的方法，其特征在于，包括：(i)提供转化的植物细胞、植物组织，植物或其部分，其中所述转化的植物细胞、植物组织、植物或其部分包含权利要求1所述的除草剂抗性多肽或权利要求2所述的多核苷酸或权利要求3所述的载体；
    (ii)使转化的植物细胞、植物组织、植物或其部份与除草剂接触；
    (iii)确定植物细胞、植物组织、植物或其部分是否受除草剂影响；和
    (iv)鉴定或选择转化的植物细胞、植物组织、植物或其部分。
  12. 一种鉴定耐受除草剂植物的方法，其特征在于，包括：
    (i)鉴定植物样本是否具有权利要求1所述的除草剂抗性多肽或权利要求2所述的多核苷酸或权利要求3所述的载体。
  13. 一种在植物栽培地点控制不想要植物的方法，其特征在于，所述方法包括：
    (1)在所述的栽培地点种植包含权利要求1所述的除草剂抗性多肽或权利要求2所述的多核苷酸或权利要求3所述的载体的植物；
    (2)在所述的栽培地点对所述植物施用有效量的除草剂。
  14. 一种用于产生除草剂抗性植物的方法，其特征在于，包括：
    将第一植物与第二植物杂交，其中所述第一植物为抗除草剂的植物，其含有本发明权利要求1所述的除草剂抗性多肽或权利要求2所述的多核苷酸或权利要求3所述的载体。
  15. 一种筛选耐受除草剂或鉴定三酮类除草剂的方法，其特征在于，包括以下步骤：
    (a)在测试组中，在测试化合物的存在下，将测试化合物施用于表达权利要求1所述的突变型HPPD多肽的植物，对所述植物的生长或生存力情况进行分析；
    并且在不施用所述测试化合物且其他条件相同的对照组中，对对照组的所述植物的生长或生存力情况进行分析；
    (b)对测试组和对照组的植物的生长或生存力进行比较，其中，与对照组相比，如果施用了测试化合物的植物的生长或生存力不受影响或长势良好，则表示所述测试化合物是耐受除草剂或三酮类除草剂。
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