WO2021155753A1 - 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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WO2021155753A1
WO2021155753A1 PCT/CN2021/074052 CN2021074052W WO2021155753A1 WO 2021155753 A1 WO2021155753 A1 WO 2021155753A1 CN 2021074052 W CN2021074052 W CN 2021074052W WO 2021155753 A1 WO2021155753 A1 WO 2021155753A1
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polypeptide
wild
amino acid
herbicide
type als
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French (fr)
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王木桂
牛小牧
王丽梅
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山东舜丰生物科技有限公司
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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
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)

Definitions

  • the present invention relates to the field of botany, and more specifically to herbicide resistance genes, polypeptides and their applications in plant breeding.
  • Acetolactate synthase is a key enzyme in the synthesis of branched chain amino acids.
  • Sulfonylurea and imidazolinone herbicides inhibit the synthesis of branched chain amino acids by inhibiting the ALS enzyme activity in plants, thereby hindering cells
  • the DNA synthesis in the division phase eventually stops the plant from growing and gradually wither and die.
  • ALS inhibitor herbicides include sulfonylureas, imidazolinones, pyrimidotriazoles, salicylic acid pyrimidines (pyrimidinoxybenzoic acid), and sulfonamide carbonyl triazolinones. These compounds are collectively referred to as ALS inhibitor herbicides. These herbicides have the characteristics of strong selectivity, broad bactericidal spectrum, low toxicity and high efficiency. This kind of herbicide has the characteristics of strong selectivity, wide bactericidal spectrum, low toxicity and high efficiency, and has been widely used in large areas.
  • ALS resistance sites have been reported in rice, but the resistance capacity of mutants and the range of applicable herbicides are limited.
  • the level of ALS mutation herbicide resistance is related to the position of ALS amino acid mutation, and also related to the type of amino acid after mutation and the number of amino acid mutation. Therefore, to cultivate crops with high herbicide resistance and a wide range of applications, there is an urgent need in this field to develop and improve tolerance systems to ALS-inhibiting herbicides.
  • the purpose of the present invention is to provide ALS resistance genes with high resistance to ALS inhibitory herbicides and applications thereof.
  • an isolated herbicide resistance polypeptide is provided, and the herbicide resistance polypeptide is a mutant ALS polypeptide,
  • mutant ALS polypeptide is mutated at the 170th and/or 627th amino acid of the wild-type ALS polypeptide corresponding to SEQ ID NO.:1:
  • the valine (V) at position 170 is mutated into one or more amino acids selected from the group consisting of alanine (A), glycine (G), isoleucine ( I), Leucine (L).
  • valine (V) at position 170 is mutated to alanine (A).
  • the serine (S) at position 627 is mutated into one or more amino acids selected from the group consisting of glycine (G), alanine (A), and isoleucine (I) , Leucine (L), Valine (V).
  • the serine (S) at position 627 is mutated to glycine (G).
  • the mutation is selected from the following group: V170A, S627G, or a combination thereof.
  • 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 an amino acid sequence shown in SEQ ID NO.: 2 or 3, an active fragment thereof, or a conservative variant polypeptide thereof.
  • the remaining amino acid sequence of the mutant protein is the same or substantially the same as the sequence shown in SEQ ID NO.:1.
  • the said substantially identical is that at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are different, wherein, The difference includes amino acid substitution, deletion or addition, and the mutant protein has herbicide tolerance activity (preferably, ALS inhibitory herbicide resistance activity).
  • the ALS-inhibiting herbicide is selected from the group consisting of sulfonylureas, imidazolinones, pyrimidotriazoles, salicylic acid pyrimidines (pyrimidinoxybenzoic acid), and sulfonamides. Carbonyl triazolinones, or combinations thereof.
  • the imidazolinone herbicide is selected from the group consisting of imazethapyr (imidazolium nicotinic acid), imazaquin, imazethapyr, imidazolin, imazapyr, or a combination thereof.
  • the homology between the mutant protein and the sequence shown in SEQ ID NO.: 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, most Preferably, it is at least 98% or 99%.
  • the herbicide tolerance concentration V1 of the herbicide resistance polypeptide is compared with the tolerance concentration V2 of the wild-type ALS 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 ⁇ 16.
  • the ALS polypeptide is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the ALS polypeptide is derived from one or more plants selected from the group consisting of gramineous, leguminous, and cruciferous plants.
  • the ALS polypeptide is derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, soybean, Arabidopsis, potato, tomato, rape, and quinoa.
  • the ALS polypeptide is derived from Oryza sativa.
  • the herbicide is an ALS inhibitory herbicide, preferably an imidazolinone herbicide.
  • the imidazolinone herbicide is selected from the group consisting of imazethapyr (imidazolium nicotinic acid), imazaquin, imazethapyr, imidazolin, imazapyr, or a combination thereof.
  • 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%, Optimally, it is at least 90%, such as 95%, 97%, 99%.
  • the herbicide resistance polypeptide is formed by mutation of the wild-type ALS 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 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 containing the polynucleotide according to the second aspect of the present invention.
  • the vector includes an expression vector, a shuttle vector, and an integration vector.
  • 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 a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, 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, rice, cabbage, soybean, tomato, corn, tobacco, wheat, potato, tomato, rape, quinoa, sorghum 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.
  • step (a) includes the steps:
  • step (1) 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 ALS in the plant cell is at position 170 corresponding to the valine of SEQ ID NO.:1 , And/or Serine at position 627 is mutated.
  • 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 a base editor.
  • 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 a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, 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, rice, cabbage, soybean, tomato, corn, tobacco, wheat, potato, tomato, rape, quinoa, sorghum or a combination thereof.
  • the method further includes the step of testing the herbicide resistance of the plant cell.
  • the plant seedlings can tolerate a concentration (mass fraction) of ⁇ 0.03%; preferably, ⁇ 0.05%; preferably, ⁇ 0.08%; preferably, ⁇ 0.1%, more Preferably, ⁇ 0.2%, preferably, ⁇ 0.3%, preferably ⁇ 0.5%, preferably, ⁇ 1%, preferably, ⁇ 5%, preferably, ⁇ 10%, preferably, ⁇ 15%, more preferably, ⁇ 20% herbicide.
  • the plant seedlings can tolerate a concentration (mass fraction) of 0.03%-20%, preferably, 1%-20%, more preferably, 10%-20% of the herbicide.
  • the plants improved by the method can tolerate at least a concentration (mass fraction) of at least 5%, preferably 10%, more preferably 15% of ALS inhibitory herbicides (for example, imidazole) during germination. Linone herbicides).
  • 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 170th amino acid of the wild-type ALS polypeptide derived from rice; (ii) the 196th amino acid of the wild-type ALS polypeptide derived from Arabidopsis; iii) The 334th amino acid of the wild-type ALS polypeptide derived from sorghum; (iv) the 124th amino acid of the wild-type ALS polypeptide derived from wheat; (v) the 164th amino acid of the wild-type ALS polypeptide derived from corn; Or (vi) the 181st amino acid of the wild-type ALS polypeptide derived from rape; or (vii) the 169th position of the wild-type ALS polypeptide derived from soybean; or (viii) the 183rd position of the wild-type ALS polypeptide derived from potato Position; or (ix) position 185 of a wild-type ALS polypeptide derived from tomato; or (x) position 172 of a
  • the second resistance sensitive site corresponding to (i) the 627th amino acid of the wild-type ALS polypeptide derived from rice, (ii) the 653th amino acid of the wild-type ALS polypeptide derived from Arabidopsis; iii) The amino acid at position 624 of the wild-type ALS polypeptide derived from sorghum; (iv) the amino acid at position 581 of the wild-type ALS peptide derived from wheat; (v) the amino acid at position 621 of the wild-type ALS polypeptide derived from corn; Or (vi) the 638th amino acid of the wild-type ALS polypeptide derived from rape; or (vii) the 628th amino acid of the wild-type ALS polypeptide derived from soybean; or (viii) the amino acid of the wild-type ALS polypeptide derived from potato The amino acid at position 640; or (ix) the amino acid at position 642 of the wild-type ALS polypeptide derived from tomato;
  • the polypeptide has sensitive and insensitive types, when the first resistance sensitive site is valine (V), and/or the second resistance sensitive site is Serine (S), the polypeptide is sensitive, and the polypeptide is sensitive to herbicides; when the first resistance sensitive site is alanine (A), glycine (G), iso Leucine (I) or Leucine (L), and/or the second resistance sensitive site is glycine (G), alanine (A), isoleucine (I), leucine ( L) or valine (V), the polypeptide is insensitive, and the polypeptide is resistant to herbicides;
  • 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 ⁇ 16.
  • the insensitive polypeptide is the herbicide resistance polypeptide of claim 1, and the sensitive polypeptide is a wild-type ALS polypeptide.
  • Figure 1 shows the Anc689BE4max-nCas9 base editor.
  • Figure 2 shows the ABEmax-nCas9 base editor.
  • Figure 3 shows the phenotype of ALS-ABE-sg2 transgenic plants after spraying with 0.03% imidazolium for 35 days. The surviving plants were identified as ALS (V170A) mutants.
  • Figure 4 shows that the ALS-ABE-sg2 transgenic plants survived after herbicide treatment showed a T509->C base substitution in the ALS target region (indicated by the arrow), resulting in an amino acid mutation V170A.
  • the underline indicates the PAM sequence.
  • Figure 5 shows the phenotype of ALS-ABE-sg1 transgenic plants after spraying with 0.03% imidazole nicotinic acid for 35 days.
  • the surviving plants (indicated by the arrow) were identified as ALS (S627G) mutants.
  • Figure 6 shows that the ALS-ABE-sg1 transgenic plants that survived after herbicide treatment have a base substitution of A1879->C (indicated by the arrow) in the ALS target region, resulting in an amino acid mutation S627G.
  • the underline indicates the PAM sequence (the sequencing peak diagram is reverse sequencing).
  • 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 ALS polypeptide are modified, the herbicide tolerance of plants can be significantly improved. On this basis, the 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.
  • ALS refers to a key enzyme in the synthesis of branched chain amino acids.
  • Sulfonylurea and imidazolinone herbicides inhibit the synthesis of branched chain amino acids by inhibiting the ALS enzyme activity in plants, thereby hindering cells
  • the DNA synthesis in the division phase eventually stops the plant from growing and gradually wither and die.
  • ALS inhibitor As used herein, the terms "ALS inhibitor”, “ALS inhibitory herbicide”, and “ALS inhibitory herbicide” are used interchangeably and refer to a formulation that inhibits ALS, inhibits plant growth or even kills plants, preferably Imidazolinone herbicides, such as imazethapyr (imidazolium nicotinic acid), imazaquin, imazethapyr, imazapyr, and imazapyr.
  • Imidazolinone herbicides such as imazethapyr (imidazolium nicotinic acid), imazaquin, imazethapyr, imazapyr, and imazapyr.
  • polypeptide of the present invention As used herein, the terms “herbicide resistance polypeptide”, “mutant ALS polypeptide”, “mutant ALS protein”, “mutant ALS enzyme”, “polypeptide of the present invention” and the like are used interchangeably, and all refer to the first aspect of the present invention.
  • the polypeptide The polypeptide.
  • the herbicide resistance polypeptide is a protein or polypeptide having SEQ ID NO.: 2-3, or a derivative polypeptide or active fragment derived therefrom that has 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 that inhibit ALS, especially imidazolinone herbicides, such as Imazethapyr (imidazolium nicotinic acid), imazaquin, imazaquin, imazapyr, or imazapyr is tolerant, and the tolerance of the herbicide-resistant polypeptide of the present invention can be determined by the concentration or amount of the herbicide used. Characterization.
  • 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 in 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 (The R group is a variable group).
  • mutant protein As used herein, the terms “mutant protein”, “mutant protein of the present invention”, and “herbicide resistance polypeptide of the present invention” are used interchangeably, and all refer to non-naturally occurring ALS polypeptides that are mutants, and the mutant protein is An artificially modified protein based on the protein shown in SEQ ID NO.: 1, wherein the mutant protein contains core amino acids related to herbicide tolerance activity, and at least one of the core amino acids 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:
  • Valine (V) at position 170 Valine (V) at position 170; and/or
  • mutant protein obtained by mutating the above-mentioned core amino acid has herbicide tolerance activity.
  • the core amino acids of the present invention are subjected to the following mutations:
  • valine (V) at position 170 is mutated to alanine (A), glycine (G), isoleucine (I) or leucine (L); and/or
  • Serine (S) at position 627 is mutated to glycine (G), alanine (A), isoleucine (I), leucine (L) or valine (V).
  • 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.
  • a misalignment is within a reasonable range. Due to the misalignment of amino acid numbering, the homology reaches 80% (such as 90%, 95%, 98%), and the mutation has the same or similar herbicide tolerance activity. Proteins are also within the protection scope of the present invention.
  • the mutein 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, and plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the mutein of the present invention may be glycosylated or non-glycosylated. The mutein of the present invention may also include or not include the initial 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 belong to the scope well known to those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitution
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the active mutant protein of the present invention has herbicide tolerance activity.
  • the mutant protein is shown in SEQ ID NO.: 2 or 3.
  • the mutant protein of the present invention generally has higher homology (identity).
  • the mutant protein 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: in vivo or in vitro chemically derived forms of mutein 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 increase 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.: 4 or 5.
  • the present invention also relates to variants of the above-mentioned 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 does not substantially change the mutation protein encoded by it. Function.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (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 an 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, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available 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 is usually done by 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 fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA end rapid amplification method
  • 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 170 and 627 positions in the ALS amino acid sequence of the source rice of the present invention are in Arabidopsis (sequence accession number P17597, corresponding to positions 196 and 653) and in sorghum (sequence accession number LN898467.1, corresponding to positions).
  • nucleotide sequence encoding the mutant protein of the present invention is shown in SEQ ID NO.: 4 or 5.
  • wild-type ALS polypeptide refers to a naturally occurring, non-artificially modified ALS polypeptide whose nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., The amino acid sequence can be deduced from the nucleotide sequence.
  • the amino acid sequence of the wild-type ALS polypeptide is shown in SEQ ID NO.:1.
  • the full-length sequence or fragments of the polynucleotide encoding the herbicide-resistant polypeptide of the present invention can usually be obtained by PCR amplification, recombination, or artificial synthesis.
  • 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 is usually done by 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 fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • 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-resistant 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 used to express or produce recombinant herbicide-resistant polypeptides. Generally speaking, there are the following steps:
  • 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, and translation control elements.
  • 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 also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell may 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 include: 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 prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be carried out 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 the leaf disc method.
  • the transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain plants with altered herbicide tolerance.
  • the gene editing technology can also be used to directly edit the ALS 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 CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system is an acquired immune system in bacteria and archaea that resist the invasion of foreign viruses or plasmid DNA.
  • the nuclease of this system recognizes and degrades foreign DNA under the guidance of crRNA.
  • the type II CRISPR/Cas system is simple in composition, and only includes a nuclease Cas9 and tracrRNA:crRNA dimer to complete the recognition and cleavage function.
  • the CRISPR/Cas9 system has quickly become a new generation of genome editing technology due to its advantages of easy design and manipulation, efficient editing and wide versatility. It has been widely used in humans, mice, rats, zebrafish, Caenorhabditis elegans, plants, Different species such as fungi and bacteria.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the 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 sterilization, 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-resistant polypeptide in a sample is to use the specific antibody of the herbicide-resistant polypeptide for detection, which includes: contacting the sample with the specific antibody of the herbicide-resistant 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 known as 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 transcripts of herbicide-resistant polypeptides.
  • the herbicide-resistant polypeptide of the present invention can be used to cultivate new herbicide-tolerant plant varieties.
  • the base editor can realize the base conversion of C/G->T/A(CBE) or A/T->G/C(ABE) within a certain sequence window ( Komor et al., 2016), and Anc689BE4max-nCas9 (Figure 1) and ABEmax-nCas9 ( Figure 2) are optimized on the basis of the first generation of CBE and ABE base editors.
  • the results of their application in rice show that they can greatly improve the efficiency of base conversion ( Wang et al, 2019).
  • the present invention uses Anc689BE4max-nCas9 and ABEmax-nCas9 base editors as carriers, designs several sgRNAs in rice endogenous ALS genes (taking the sgRNA shown in Table 1 as an example), and clones them into Anc689BE4max-nCas9 or ABEmax-nCas9 vectors respectively , The formation of several base editors targeting rice endogenous ALS genes.
  • Table 1 sgRNA sequence targeting rice ALS gene
  • sgRNA number guide-PAM sequence (5’-3’) SEQ ID NO.: ALS-ABE-sg2 GCGGGGGACCTGGCCCGTGA TGG 7 ALS-ABE-sg1 GATCCCAAGTGGGGGCGCATTCA AGG 8
  • the base editor constructed above was transformed into the rice japonica rice variety Nipponbare or Xiushui 134 by Agrobacterium to obtain the T0 generation transgenic plants. After 15 days of planting in the greenhouse, the above plants were treated with 0.03% imidazole ("Dou Shuhao" brand imidazole herbicide, effective concentration of 10%, diluted at a ratio of 3:1000), sprayed, and the plant growth phenotype was recorded 35 days later Analyze the survival of plants.
  • imidazole Dou Shuhao brand imidazole herbicide, effective concentration of 10%, diluted at a ratio of 3:1000
  • the ALS gene of the plant was identified by PCR and sequencing. The results showed that the dead individual in the ALS-ABE-sg2 transgenic plant was the wild-type sequence (SEQ ID NO.: 6), and the surviving plant was relatively wild-type.
  • the base substitution of T509->C in the target region resulted in an amino acid mutation V170A, indicating that this mutation confers resistance to the imidazolium herbicide ( Figure 3, Figure 4).
  • the amino acid sequence of the mutant ALS is shown in SEQ ID NO.: Shown in 2.

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Abstract

提供了一种抗除草剂基因、该基因编码的多肽及其在植物育种中的应用，该多肽是一种突变的ALS多肽，其在SEQ ID NO：1所示野生型ALS多肽的第170和/或627位氨基酸发生突变，对除草剂具有耐受性，可用于培育ALS抑制性除草剂耐受性植物。

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
乙酰乳酸合成酶(acetolactate synthase，ALS)是支链氨基酸合成过程中的一个关键酶，磺酰脲和咪唑啉酮类除草剂通过抑制植物体内的ALS酶活性阻止支链氨基酸的合成，进而阻碍细胞分裂期的DNA合成，最终使植物停止生长并逐渐枯萎死亡。
目前，以ALS靶标开发了多种除草剂，包括磺酰脲类、咪唑啉酮类、嘧啶并三唑类、水杨酸嘧啶类(嘧啶氧苯甲酸类)、磺酰胺基羰基三唑啉酮类等化合物，这些化合物统称为ALS抑制剂类除草剂，这些除草剂具有选择性强、杀菌谱广、低毒高效等特点。这类除草剂具有选择性强、杀菌谱广、低毒高效等特点，目前已大面积推广使用。这类除草剂在杀草的同时，也会对一般不具有抗除草剂特性的农作物本身产生要害，限制了其使用空间；而且随着除草剂使用时间的延长，越来越多的杂草对除草剂产生抗性，使得除草剂的效力降低，缩短了其市场寿命，限制了其使用时间。因此，培育抗除草剂的作物品种是解决上述问题的方法之一，可以扩大除草剂的使用范围，延长其使用寿命。
目前，水稻中报道了一些ALS抗性位点，但突变体的抗性能力和适用的除草剂种类范围有限。ALS突变抗除草剂水平与ALS氨基酸突变位置有关，还与突变后的氨基酸种类及突变氨基酸数目有关。因此，若培育具有除草剂抗性高、适用范围广的农作物，本领域还迫切需要开发和改进对ALS抑制性除草剂的耐受性系统。
发明内容
本发明的目的在于提供对对ALS抑制性除草剂具有高抗性的ALS抗性基因及其应用。
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ALS多肽，
并且所述突变的ALS多肽在野生型ALS多肽的对应于SEQ ID NO.:1的第170位、和/或第627位氨基酸发生突变：
第170位的缬氨酸(V)；
第627位的丝氨酸(S)。
在另一优选例中，所述第170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)、甘氨酸(G)、异亮氨酸(I)、亮氨酸(L)。
在另一优选例中，所述第170位的缬氨酸(V)突变为丙氨酸(A)。
在另一优选例中，所述第627位的丝氨酸(S)突变为选自下组的一种或多种氨基酸：甘氨酸(G)、丙氨酸(A)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)。
在另一优选例中，所述第627位的丝氨酸(S)突变为甘氨酸(G)。
在另一优选例中，所述的突变选自下组：V170A、S627G、或其组合。
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，所述的突变蛋白除所述突变(如170、627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(较佳地，耐ALS抑制性除草剂的活性)。
在另一优选例中，所述ALS抑制性除草剂选自下组：磺酰脲类、咪唑啉酮类、嘧啶并三唑类、水杨酸嘧啶类(嘧啶氧苯甲酸类)、磺酰胺基羰基三唑啉酮类、或其组合。
在另一优选例中，所述的咪唑啉酮类除草剂选自下组：咪草烟(咪唑乙烟酸)、灭草喹、咪草酯、烟咪唑草、灭草烟、或其组合。
在另一优选例中，所述突变蛋白与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，所述的除草剂抗性多肽对除草剂的耐受浓度V1与野生型ALS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≥16。
在另一优选例中，所述ALS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ALS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ALS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、大豆、拟南芥、马铃薯、番茄、油菜、藜麦。
在另一优选例中，所述的ALS多肽来源于水稻(Oryza sativa)。
在另一优选例中，所述的除草剂为ALS抑制性除草剂，较佳地为咪唑啉酮类除草剂。
在另一优选例中，所述的咪唑啉酮类除草剂选自下组：咪草烟(咪唑乙烟酸)、灭草喹、咪草酯、烟咪唑草、灭草烟、或其组合。
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ALS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或3所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:4或5所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:4或5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或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，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、水稻、白菜、大豆、番茄、玉米、烟草、小麦、马铃薯、番茄、油菜、藜麦、高粱或其组合。
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，利用基因工程改造所述植物细胞，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；和
(b)将步骤(a)中的植物细胞再生成植株。
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ALS在对应于SEQ ID NO.:1的第170位的缬氨酸、和/或第627位的丝氨酸发生突变。
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，所述基因编辑技术包括碱基编辑器。
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、水稻、白菜、大豆、番茄、玉米、烟草、小麦、马铃薯、番茄、油菜、藜麦、高粱或其组合。
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，所述的植物幼苗能够耐受浓度(质量分数)为≥0.03％；较佳地，≥0.05％；较佳地，≥0.08％；较佳地，≥0.1％，较佳地，≥0.2％，较佳地，≥0.3％，较佳地≥0.5％，较佳地，≥1％，较佳地，≥5％，较佳地，≥10％，较佳地，≥15％，更佳地，≥20％的除草剂。
在另一优选例中，所述的植物幼苗能够耐受浓度(质量分数)为0.03％－20％，较佳地，1％－20％，更佳地，10％－20％的除草剂。
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(质量分数)为5％，较佳地10％，更佳地15％的ALS抑制性除草剂(比如，咪唑啉酮类除草剂)。
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)来源于水稻的野生型ALS多肽的第170位氨基酸；(ii)来源于拟南芥的野生型ALS多肽的第196位氨基酸；(iii)来源于高粱的野生型ALS多肽的第334位氨基酸；(iv)来源于小麦的野生型ALS多肽的第124位氨基酸；(v)来源于玉米的野生型ALS多肽的第164位氨基酸；或(vi)来源于油菜的野生型ALS多肽的第181位氨基酸；或(vii)来源于大豆的野生型ALS多肽的第169位；或(viii)来源于马铃薯的野生型ALS多肽的第183位；或(ix)来源于番茄的野生型ALS多肽的第185位；或(x)来源于大麦的野生型ALS多肽的第172位；和/或
(II)第二抗性敏感位点，对应于(i)来源于水稻的野生型ALS多肽的第627位氨基酸、(ii)来源于拟南芥的野生型ALS多肽的第653位氨基酸；(iii)来源于高粱的野生型ALS多肽的第624位氨基酸；(iv)来源于小麦的野生型ALS肽的第581位氨基酸；(v)来源于玉米的野生型ALS多肽的第621位氨基酸；或(vi)来源于油菜的野生型ALS多肽的第638位氨基酸；或(vii)来源于大豆的野生型ALS多肽的第628位氨基酸；或(viii)来源于马铃薯的野生型ALS多肽的第640位氨基酸；或(ix)来源于番茄的野生型ALS多肽的第642位氨基酸；或(x)来源于大麦的野生型ALS多肽的第629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，当所述第一位抗性敏感位点为缬氨酸(V),和/或第二位抗性敏感位点为丝氨酸(S)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丙氨酸(A)、甘氨酸(G)、异亮氨酸(I)或亮氨酸(L)，和/或第二位抗性敏感位点为甘氨酸(G)、丙氨酸(A)、异亮氨酸(I)、亮氨酸(L)或缬氨酸(V)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≥16。
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ALS多肽。
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Anc689BE4max-nCas9碱基编辑器。
图2显示了ABEmax-nCas9碱基编辑器。
图3显示了ALS-ABE-sg2的转基因植株用0.03％咪唑乙烟酸喷施处理35天后的表型，存活的植株经鉴定为ALS(V170A)突变体。
图4显示了ALS-ABE-sg2的转基因植株经除草剂处理后存活的植株在ALS靶点区出现T509->C的碱基替换(箭头所示)，导致一个氨基酸突变V170A。下划线标示PAM序列。
图5显示了ALS-ABE-sg1的转基因植株用0.03％咪唑乙烟酸喷施处理35天后的表型，存活的植株(箭头所示)经鉴定为ALS(S627G)突变体。
图6显示了ALS-ABE-sg1的转基因植株经除草剂处理后存活的植株在ALS靶点区出现A1879->C的碱基替换(箭头所示)，导致一个氨基酸突变S627G。下划线标示PAM序列(该测序峰图为反向测序)。
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ALS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“ALS”是指是支链氨基酸合成过程中的一个关键酶，磺酰脲和咪唑啉酮类除草剂通过抑制植物体内的ALS酶活性阻止支链氨基酸的合成，进而阻碍细胞分裂期的DNA合成，最终使植物停止生长并逐渐枯萎死亡。
如本文所用，术语“ALS抑制剂”、“ALS抑制性除草剂”、“ALS抑制类除草剂”可互换使用，是指通过抑制ALS，抑制植物生长甚至使植物死亡的制剂，优选地为咪唑啉酮类除草剂，如咪草烟(咪唑乙烟酸)、灭草喹、咪草酯、烟咪唑草、灭草烟。
如本文所用，术语“除草剂抗性多肽”、“突变的ALS多肽”、“突变ALS蛋白”、“突变ALS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ALS抑制性除草剂，尤其是咪唑啉酮类除草剂，如咪草烟(咪唑乙烟酸)、灭草 喹、咪草酯、烟咪唑草或灭草烟具有耐受性，本发明的除草剂抗性多肽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。
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 PCTCN2021074052-appb-000001
(R基为可变基团)。
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”可互换使用，均指非天然存在的为突变的ALS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170位的缬氨酸(V)；和/或
第627位的丝氨酸(S)；
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170位的缬氨酸(V)突变为丙氨酸(A)、甘氨酸(G)、异亮氨酸(I)或亮氨酸(L)；和/或
第627位的丝氨酸(S)突变为甘氨酸(G)、丙氨酸(A)、异亮氨酸(I)、亮氨酸(L)或缬氨酸(V)。
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，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也在本发明的保护范围内。
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
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明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。
SEQ ID NO.:2 OsALS突变体氨基酸序列(V170A)
Figure PCTCN2021074052-appb-000002
SEQ ID NO.:3 OsALS突变体氨基酸序列(S627G)
Figure PCTCN2021074052-appb-000003
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.:4或5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。
应注意，本发明的来源水稻的ALS氨基酸序列中的170、627位点在拟南芥(序列登录号P17597，对应位点196、653)、在高粱(序列登录号LN898467.1，对应位点167、624)、在小麦(序列号AAO53549.1，对应位点124、581)、在大麦(序列号，KAE8811959.1对应位点172、629)、在玉米(序列号PWZ20335.1，对应位点164、621)、在油菜(序列号AJF23173.1，对应位点181、638)、在大豆(序列号NP001341804.1，对应位点171、628)、在马铃薯(序列号XP_006348357.1，对应位点183、640)、在番茄(序列号XP_004234664.1，对应位点185、642)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，本发明的编码突变蛋白的核苷酸序列如SEQ ID NO.:4或5所示。
SEQ ID NO.：4 突变体(V170A)(T 509->C)
Figure PCTCN2021074052-appb-000004
Figure PCTCN2021074052-appb-000005
SEQ ID NO.：5 突变体(S627G)(A 1879->C)
Figure PCTCN2021074052-appb-000006
Figure PCTCN2021074052-appb-000007
野生型ALS多肽
如本文所用，“野生型ALS多肽”是指天然存在的、未经过人工改造的ALS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。所述野生型ALS多肽的氨基酸序列如SEQ ID NO.:1所示。
重组技术和植物改良
本发明的编码除草剂抗性多肽的多核苷酸的全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的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。
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。
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。
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、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含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。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
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ALS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。
CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)系统为细菌与古生菌中抵御外源病毒或质粒DNA入侵的获得性免疫系统。该系统的核酸酶在crRNA的指导下识别并降解外源DNA。其中，II型CRISPR/Cas系统组成简单，仅包括一个核酸酶Cas9与tracrRNA:crRNA二聚体便可完成识别和切割功能。CRISPR/Cas9系统以其设计操纵简便、编辑高效与通用性广等优势迅速成为新一代的基因组编辑技术，已被广泛应用于人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物、真菌与细菌等不同物种。
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。
本发明的主要优点包括：
(a)本发明首次发现，水稻ALS的第509位碱基由T突变为C，相应的170位氨基酸由V突变为A后，可赋予植株相对野生型植株对除草剂的抗性。
(b)本发明首次发现，水稻ALS的第1879位碱基由A突变为C，相应的627位氨基酸由S突变为G后，可赋予植株相对野生型植株对除草剂的抗性。
(c)通过导入除草剂抗性多肽的编码基因，可以增强植物(如水稻)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。
具体实施方式
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 碱基编辑载体构建及抗除草剂突变位点的筛选
1、构建靶向水稻内源ALS基因的Anc689BE4max-nCas9和ABEmax-nCas9碱基编辑器
碱基编辑器可以在一定的序列窗口范围内实现C/G->T/A(CBE)或A/T->G/C(ABE)的碱基转换(Komor et al.,2016)，而Anc689BE4max-nCas9(图1)和ABEmax-nCas9(图2)是在第一代CBE和ABE碱基编辑器的基础上优化而来，在水稻中应用的结果表明能大幅提高碱基转换的效率(Wang et al,2019)。本发明以Anc689BE4max-nCas9和ABEmax-nCas9碱基编辑器为载体，在水稻内源ALS基因中设计若干sgRNA(以表1所示的sgRNA为例)，分别克隆至Anc689BE4max-nCas9或ABEmax-nCas9载体，形成若干靶向水稻内源ALS基因的碱基编辑器。
表1 靶向水稻ALS基因的sgRNA序列
sgRNA编号 guide-PAM序列(5’-3’) SEQ ID NO.:
ALS-ABE-sg2 GCGGGGGACCTGGCCCGTGA TGG 7
ALS-ABE-sg1 GATCCCAAGTGGGGGCGCATTCA AGG 8
ALS-CBE-sg4 GAGGTCCCCCGCCGCATGAT CGG 9
2、水稻遗传转化及除草剂抗性植株的筛选鉴定
把以上构建好的碱基编辑器分别通过农杆菌转化水稻粳稻品种日本晴或秀水134，获得T0代转基因植株。以上植株在温室种植15天后用0.03％咪唑乙烟酸(“豆说好”牌咪唑乙烟酸类除草剂，有效浓度为10％，以3：1000的比例稀释使用)，喷施处理，35天后记录植株生长表型，分析植株存活情况。
3、实验结果及结论
通过PCR及测序对植株的ALS基因进行鉴定，结果表明ALS-ABE-sg2的转基因植株中死亡的个体为野生型序列(SEQ ID NO.:6)，而存活的一株植株相对野生型序列在靶点区出现T509->C的碱基替换，导致一个氨基酸突变V170A，表明该突变赋予了植株对咪唑乙烟酸除草剂的抗性(图3，图4)，突变后的ALS的氨基酸序列如SEQ ID NO.:2所示。
ALS-ABE-sg1的转基因植株经0.03％咪唑乙烟酸除草剂喷施处理后有3株植株存活，经PCR及测序鉴定发现存活的植株在ALS靶点区均出现A1879->C的碱基替换，导致一个氨基酸突变S627G，表明该突变赋予了植株对咪唑乙烟酸除草剂的抗性(图5，图6)，突变后的ALS的氨基酸序列如SEQ ID NO.:3所示。
参考文献
Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,and Liu,D.R.(2016).Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.NATURE 533:420–424.
Mugui Wang,Zhidan Wang,Yanfei Mao,Yuming Lu,Ruifang Yang,Xiaoping Tao and Jian-Kang Zhu(2019).Optimizing base editors for improved efficiency and expanded editing scope in rice.PLANT BIOTECHNOLOGY JOURNAL 17:1697-1699.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种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ALS多肽，
    并且所述突变的ALS多肽在野生型ALS多肽的对应于SEQ ID NO.:1的第170位、和/或第627位氨基酸发生突变：
    第170位的缬氨酸(V)；
    第627位的丝氨酸(S)；
    所述第170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)、甘氨酸(G)、异亮氨酸(I)、亮氨酸(L)；
    所述第627位的丝氨酸(S)突变为选自下组的一种或多种氨基酸：甘氨酸(G)、丙氨酸(A)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)。
  2. 如权利要求1所述的多肽，其特征在于，所述的除草剂抗性多肽对除草剂的耐受浓度V1与野生型ALS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≥16。
  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，所述酶制剂包括权利要求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，利用基因工程改造所述植物细胞，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)来源于水稻的野生型ALS多肽的第170位氨基酸；(ii)来源于拟南芥的野生型ALS多肽的第196位氨基酸；(iii)来源于高粱的野生型ALS多肽的第334位氨基酸；(iv)来源于小麦的野生型ALS多肽的第124位氨基酸；(v)来源于玉米的野生型ALS多肽的第164位氨基酸；或(vi)来源于油菜的野生型ALS多肽的第181位氨基酸；或(vii)来源于大豆的野生型ALS多肽的第169位；或(viii)来源于马铃薯的野生型ALS多肽的第183位；或(ix)来源于番茄的野生型ALS多肽的第185位；或(x)来源于大麦的野生型ALS多肽的第172位；和/或
    (II)第二抗性敏感位点，对应于(i)来源于水稻的野生型ALS多肽的第627位氨基酸、(ii)来源于拟南芥的野生型ALS多肽的第653位氨基酸；(iii)来源于高粱的野生型ALS多肽的第624位氨基酸；(iv)来源于小麦的野生型ALS肽的第581位氨基酸；(v)来源于玉米的野生型ALS多肽的第621位氨基酸；或(vi)来源于油菜的野生型ALS多肽的第638位氨基酸；或(vii)来源于大豆的野生型ALS多肽的第628位氨基酸；或(viii)来源于马铃薯的野生型ALS多肽的第640位氨基酸；或(ix)来源于番茄的野生型ALS多肽的第642位氨基酸；或(x)来源于大麦的野生型ALS多肽的第629位氨基酸。
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