WO2019223676A1 - Clals蛋白、其编码基因及它们在预测西瓜除草剂抗性中的应用 - Google Patents

Clals蛋白、其编码基因及它们在预测西瓜除草剂抗性中的应用 Download PDF

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WO2019223676A1
WO2019223676A1 PCT/CN2019/087744 CN2019087744W WO2019223676A1 WO 2019223676 A1 WO2019223676 A1 WO 2019223676A1 CN 2019087744 W CN2019087744 W CN 2019087744W WO 2019223676 A1 WO2019223676 A1 WO 2019223676A1
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protein
watermelon
clals
sequence
amino acid
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PCT/CN2019/087744
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French (fr)
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许勇
田守蔚
姜临建
张海英
宫国义
郭邵贵
张洁
任毅
孙宏贺
李茂营
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北京市农林科学院
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Priority to US16/923,838 priority Critical patent/US20200340007A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • C12N15/8278—Sulfonylurea
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Y—ENZYMES
    • 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)
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to CLALS protein, its coding gene, and its application in watermelon prediction of watermelon herbicide resistance, variety improvement, and especially in breeding herbicide resistance traits.
  • Watermelon field weeds are one of the important factors limiting watermelon yield, quality and cost effectiveness. Due to the low planting density of watermelon, the area of the bare ground in the field is large before the stems are spread. In addition, the growing environment of watermelon is hot and humid, and weeds are prone to occur. Therefore, compared with other crops, watermelons are particularly harmful to grass. Artificial weeding in watermelon fields is laborious and laborious. Chemical herbicide application often causes medicinal harm to watermelons due to improper selection and improper application doses, resulting in reduced yield and quality of watermelons. Weeds caused watermelon production to be reduced by about 20% and accounted for more than 30% of field labor costs.
  • ALS Acetolactate synthase
  • ALS inhibitor herbicides inhibit the activity of ALS in plants, thereby preventing the synthesis of branched chain amino acids, which in turn affects protein synthesis and plant growth, and eventually causes plant death.
  • ALS inhibitor herbicides have the advantages of high activity, strong selectivity, broad herbicidal spectrum, and low toxicity, and have become the most active class of commercial herbicides in the 1990s.
  • nCas9-PBE can replace the C of the target site DNA with T, and the window of C base deamination covers 7 nucleotides of the target sequence (positions 3-9 from the far end of PAM).
  • the double-strand break (DSB) is generated at the target site without the involvement of donor DNA. It has the characteristics of simplicity, adaptability and high efficiency.
  • the successful establishment and application of the nCas9-PBE single base editing system provides a reliable solution for the efficient and large-scale creation of single base mutants, and provides important technical support for crop genetic improvement and new breed cultivation.
  • the object of the present invention is to cultivate watermelon varieties with herbicide resistance.
  • the present invention first protects the CLALS protein.
  • the CLALS protein may be the following W1) or W2):
  • Section N, Section II, and Section III may be included from the N-terminal to the C-terminal in order;
  • the segment II may be an amino acid residue
  • the segment I may be a1) or a2) or a3) as follows:
  • amino acid sequence is the polypeptide shown in sequence 2 in the sequence listing from position 1 to position 189 from the N-terminus;
  • a2) a polypeptide related to herbicide resistance obtained by replacing the polypeptide shown in a1) with one or several amino acid residues;
  • a3) a polypeptide having 80% or more identity with the polypeptide shown in a1) or a2), a polypeptide derived from watermelon and related to herbicide resistance;
  • the section III may be b1) or b2) or b3) as follows:
  • amino acid sequence is the polypeptide shown at sequence 191 to 662 from the N-terminus in Sequence Listing 2;
  • b2) a polypeptide related to herbicide resistance obtained by replacing the polypeptide shown in b1) with one or several amino acid residues;
  • b3) a polypeptide having 80% or more identity with the polypeptide shown in b1) or b2), a polypeptide derived from watermelon and related to herbicide resistance;
  • W2 A fusion protein obtained by attaching a tag to the N-terminus or / and C-terminus of W1).
  • identity refers to a sequence similarity to a natural amino acid sequence. “Identity” includes that the amino acid sequence shown from the 1st to the 189th position from the N-terminus of Sequence 2 in the Sequence Listing of the present invention has 80%, or 85% or higher, or 90% or higher, or 95% or Higher identity amino acid sequences.
  • identity is used to refer to sequence similarity to a natural amino acid sequence. “Identity” includes that the amino acid sequence shown at position 191 to 662 from the N-terminus of Sequence 2 in the Sequence Listing of the present invention has 80%, or 85% or higher, or 90% or higher, or 95% or Higher identity amino acid sequences.
  • the segment II may be a proline residue or a non-proline residue.
  • the non-proline residue may specifically be a serine residue or a leucine residue.
  • the CLALS protein may be composed of the segment I, the segment II, and the segment III in this order from the N-terminus to the C-terminus.
  • the CLALS protein may specifically be c1) or c2) or c3) or c4) or c5):
  • amino acid sequence is a protein shown in sequence 2 in the sequence listing;
  • amino acid sequence is a protein shown in sequence 4 in the sequence listing;
  • amino acid sequence is a protein shown in sequence 6 in the sequence listing;
  • c5) The protein having 80% or more identity with the protein shown in c1) or c2) or c3) or c4), a protein derived from watermelon and related to herbicide resistance.
  • identity refers to a sequence similarity to a natural amino acid sequence. “Identity” includes 80%, or 85% or higher, or 90% or higher, or 95% or higher identical to the amino acid sequence shown in sequence 2, sequence 4 or sequence 6 in the sequence listing of the present invention. Sexual amino acid sequence.
  • a nucleic acid molecule encoding the CLALS protein also falls within the protection scope of the present invention.
  • the nucleic acid molecule encoding the CLALS protein may be a DNA molecule shown by the following d1) or d2) or d3) or d4) or d5):
  • the nucleotide sequence is a DNA molecule shown in sequence 1 in the sequence listing;
  • nucleotide sequence is a DNA molecule shown in sequence 3 in the sequence listing;
  • nucleotide sequence is a DNA molecule shown in sequence 5 in the sequence listing;
  • d4) a DNA molecule having 75% or more identity with a nucleotide sequence defined by d1) or d2) or d3), and encoding the CLALS protein;
  • d5) a DNA molecule that hybridizes to a nucleotide sequence defined by d1) or d2) or d3) under stringent conditions and encodes the CLALS protein.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • the term “identity” is used to refer to a sequence similarity to a natural nucleic acid sequence. “Identity” includes the nucleotide sequence of a protein consisting of the amino acid sequence shown in sequence 2 in the coding sequence table of the present invention, or the core of the protein consisting of the amino acid sequence shown in sequence 4 in the coding sequence table of the present invention.
  • the nucleotide sequence of the protein consisting of the amino acid sequence shown in Sequence 6 in the coding sequence table of the present invention has a nucleotide sequence of 75%, or 80% or higher, or 85% or higher, or 90% or A higher, or 95% or higher, nucleotide sequence.
  • Sequence 1 in the Sequence Listing is composed of 1989 nucleotides.
  • the nucleotide of Sequence 1 in the Sequence Listing encodes the amino acid sequence shown in Sequence 2 in the Sequence Listing.
  • Sequence 3 in the Sequence Listing is composed of 1989 nucleotides.
  • the nucleotide of Sequence 3 in the Sequence Listing encodes the amino acid sequence shown in Sequence 4 in the Sequence Listing.
  • Sequence 5 in the Sequence Listing consists of 1989 nucleotides.
  • the nucleotide of Sequence 5 in the Sequence Listing encodes the amino acid sequence shown in Sequence 6 in the Sequence Listing.
  • identity can be evaluated with the naked eye or computer software.
  • identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
  • An expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic cell line containing the nucleic acid molecule also fall within the protection scope of the present invention.
  • the invention also protects Z1) or Z2):
  • the invention also protects a method for predicting the resistance of a watermelon herbicide to be tested.
  • the method for predicting the resistance of the tested watermelon herbicide protected by the present invention may specifically be S1): detecting the type of amino acid residues at position 190 of the CLALS protein of the tested watermelon from the N-terminus; "CLALS protein from the N-terminus” The type of amino acid residues starting at position 190 is only non-proline residues.
  • the tested watermelon or "CLALS protein has the type of amino acid residues at position 190 from the N-terminus of non-proline residues and proline residues.
  • the herbicide resistance of the tested watermelon was stronger than that of the tested watermelon in which the type of amino acid residues at position 190 of the CLALS protein from the N-terminus was only proline residues.
  • the amino acid type of the non-proline residue in "the amino acid residue type of the 190th position of the CLALS protein from the N-terminus is only a non-proline residue” may be one or two .
  • the method for predicting the resistance of the tested watermelon herbicide protected by the present invention may specifically be S2): detecting the nucleotide sequence of the 190th codon in a specific transcript of the total RNA of the watermelon to be tested; the specific transcript is The RNA transcribed from the gene encoding the CLALS protein, the first codon of which is the start codon; "the nucleotide sequence of the 190th codon in a specific transcript only encodes non-proline", the watermelon to be tested Or "The nucleotide sequence of the 190th codon in a specific transcript encodes non-proline and proline”
  • the herbicide resistance of the tested watermelon is stronger than the "nucleoside of the 190th codon in a specific transcript
  • the acid sequence encodes only "proline” for the test watermelon.
  • the type of non-proline amino acid in "the nucleotide sequence of the 190th codon in a specific transcript only encodes non-proline” may be one type or two types.
  • the method for predicting the resistance of the tested watermelon herbicide protected by the present invention may specifically be S3): detecting the nucleus at positions 568 and 569 of the gene encoding the CLALS protein in the total DNA of the tested watermelon from the 5 ′ end Glycylic acid species;
  • the herbicide resistance of the tested watermelon whose "CLALS protein-encoding gene has 568th and 569th nucleotide types from the 5 'end is only c" is weaker than F1 or F2 or F3;
  • F1 is The CLALS protein coding gene has 568th and 569th nucleotide types from the 5 'end of the tested watermelon without c;
  • F2 is the CLALS protein coding gene at the 569th nucleotide from the 5' end
  • the type of test watermelon containing c and the 568th nucleotide type does not contain c;
  • F3 is the CLALS protein encoding gene.
  • the method for predicting the resistance of a watermelon herbicide to be tested protected by the present invention may specifically include the following steps: detecting whether the total DNA of the watermelon to be tested has a DNA molecule shown in sequence 1 of the sequence listing, and a sequence shown in sequence 3 of the sequence listing A DNA molecule and a DNA molecule shown in Sequence 5 of the Sequence Listing;
  • the herbicide resistance of the watermelon to be tested is the DNA molecule shown in Sequence 3 and / or the DNA molecule of Sequence 5 in the Sequence Listing in the total DNA of the watermelon to be tested" is stronger than "total watermelon DNA to be tested” "Only the DNA molecule shown in Sequence 1 of the Sequence Listing" in the test watermelon.
  • the invention also protects the application of substance A, substance B or substance C in predicting the resistance of watermelon herbicides to be tested.
  • the substance A may be a substance for detecting the type of the amino acid residue at the 190th position from the N-terminus of the CLALS protein.
  • the substance B may be a substance for detecting a nucleotide sequence of a 190th codon in a specific transcript; the specific transcript is RNA transcribed from a gene encoding the CLALS protein, and the first codon Is the start codon.
  • the substance C may be a substance for detecting the nucleotide species at positions 568 and 569 from the 5 'end of the gene encoding the CLALS protein.
  • the invention also protects the application of complete product A, complete product B, or complete product C in predicting the resistance of watermelon herbicide to be tested.
  • the kit A may be the substance A and a carrier recorded with the method A; the method A may be: "The type of amino acid residues at position 190 of the CLALS protein from the N-terminus is only non-proline residues.
  • the tested watermelon or "CLALS protein with 190th amino acid residues from the N-terminus is a non-proline residue and a proline residue” has higher herbicide resistance than the "CLALS protein”
  • the type of amino acid residue at the 190th position from the N-terminus is only a proline residue ".
  • the product set B may be the substance B and a carrier recorded with method B; the method B may be: "the nucleotide sequence of the 190th codon in a specific transcript only encodes non-proline"
  • the herbicide resistance of the tested watermelon or "testine of the 190th codon in a specific transcript encodes non-proline and proline” is stronger than that of the test watermelon of "the 190th codon in a specific transcript”
  • the nucleotide sequence encodes only "proline” for the watermelon to be tested.
  • the kit C may be the substance C and a carrier on which the method C is described.
  • the method C may be: "The CLALS protein-encoding gene has 568th and 569th nucleotide types from the 5 'end only with c"
  • the herbicide resistance of the tested watermelon is weaker than F1 or F2 Or F3;
  • F1 is the watermelon encoding the CLALS protein from the 5 'end at 568th and 569th nucleotide types without c;
  • F2 is the CLALS protein encoding gene from the 5' end at 569
  • the watermelon species to be tested contains c and the 568th nucleotide type does not contain c;
  • F3 is the CLALS protein coding gene.
  • the 568th nucleotide type from the 5 'end contains c and 569th
  • the nucleotide species at position 8 does not contain the watermelon under test.
  • the amino acid type of the non-proline residue in "the amino acid residue type of the 190th position from the N-terminus of the CLALS protein is only a non-proline residue" may be one or two.
  • the type of non-proline amino acid in "the nucleotide sequence of the 190th codon in a specific transcript only encodes non-proline" may be one or two.
  • the invention also protects B1) or B2) or B3).
  • the specific transcript is an RNA transcribed from a gene encoding the CLALS protein,
  • the first codon is the start codon.
  • the herbicide according to any one of the above may be a herbicide that targets the CLALS protein (ie, an ALS inhibitor herbicide).
  • Any of the ALS inhibitor-type herbicides mentioned above may be Y1) or Y2) or Y3) or Y4) or Y5): Y1) sulfonylurea herbicide; Y2) triazopyrimidine herbicide; Y3) triazole Pyrrolidone herbicides; Y4) pyrimidine salicylic acid herbicides; Y5) imidazolinones.
  • the sulfonylurea herbicide may be besulfuron-methyl, closulfuron-methyl, bensulfuron-methyl, pyrimsulfuron-methyl, nicosulfuron-methyl, disulfuron-methyl, thiensulfuron-methyl, or sulfsulfuron-methyl .
  • the triazosin-type herbicide can be specifically saflufenacil, penoxsulam, saflufenacil, or disulfachlor.
  • the triazolinone herbicide may be fluazosulfuron.
  • the pyrimidinesalicylic acid herbicide may be bisoxafen.
  • the imidazolinones may be mimidazonic acid.
  • Any of the non-prolines mentioned above may be specifically serine or leucine.
  • a double-site or multiple-site mutation gene formed by a mutation of the amino acid residue at the 190th position of the CLALS protein from other amino acid residues in the CLALS protein also belongs to the protection scope of the present invention.
  • the inventors obtained the P190L mutant heterozygous and P190S mutant heterozygous through the plant single base editing system nCas9-PBE, and further obtained the P190L homozygous mutant strain (CLALS protein 190th from the N-terminus Amino acid residues are only leucine residues), P190S homozygous mutant strains (CLALS protein has 190th amino acid residues from N-terminal only serine residues), P190L hybrid mutant strains (CLALS protein from The amino acid residues at position 190 from the N-terminus are proline residues and leucine residues) and the P190S hybrid mutant strain (CLALS protein has amino acid residues at position 190 from the N-terminus as proline residues And serine residues).
  • CLALS protein 190th from the N-terminus Amino acid residues are only leucine residues
  • P190S homozygous mutant strains CLALS protein has 190th amino acid residues from N-terminal only serine residues
  • the seedlings of the above mutants and the seedlings of non-transgenic watermelon were sprayed with bensulfuron-methyl, and the results showed that the seedlings of the non-transgenic watermelon were very Near death (3-7 days after bensulfuron-methyl spraying), seedlings of the P190L heterozygous mutant strain, seedlings of the P190S heterozygous mutant strain, seedlings of the P190L homozygous mutant strain, and seedlings of the P190S homozygous mutant strain all grew normally. Moreover, the seedlings of the P190L homozygous mutant strain and the P190S homozygous mutant strain had a better growth status than the seedlings of the P190L heterozygous mutant strain and the P190S heterozygous mutant strain.
  • Figure 1 shows the results of herbicide resistance identification.
  • Figure 2 shows the results of identification of the herbicide resistance spectrum.
  • the following examples are for better understanding of the present invention, but they are not limited to the present invention.
  • the experimental methods in the following examples are conventional methods.
  • the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. For the quantitative experiments in the following examples, three repeated experiments are set, and the results are averaged.
  • the pBSE901 plasmid is described in the following literatures: Chen Y, Wang Z, Ni H, et al. CRISPR / Cas9-mediated base-editing system efficiently-generates gain-of-function mutations in Arabidopsis [J]. Science China Life Sciences, 2017, 60 (5): 520-523.
  • BM culture medium 0.44 g of MS culture medium, 3 g of sucrose and 0.8 g of agar were dissolved in 100 mL of deionized water, the pH value was adjusted to 5.8, and autoclaving was performed for 15 minutes.
  • MS medium is a product of PhytoTech.
  • Co-culture medium BM medium containing 1.5 mg / L 6-BA.
  • Selection medium 1 a co-culture medium containing 100 mg / L Timentin and 1.5 mg / L Basta.
  • Selection medium 2 a co-culture medium containing 100 mg / L Timentin and 2.0 mg / L Basta.
  • Bud elongation medium BM medium containing 0.1 mg / L 6-BA, 0.01 mg / L NAA, 100 mg / L Timentin, and 1.5 mg / L Basta.
  • Rooting medium BM medium containing 1 mg / L IBA.
  • the amino acid sequence of the CLALS protein is shown as sequence 2 in the sequence listing.
  • the gene encoding the CLALS protein ie, the CLALS gene
  • the target sequence is selected based on the nucleotide sequence of the CLALS gene, and the target sequence has a nucleotide sequence of 5'-AAGTTCCGAGAAGAATGAT-3 '.
  • Synthetic primer ALS-190F 5'-ATTG -3 '(double underlined as the target sequence) and primer ALS-190R: 5'-AAAC -3 '(double underlined is the reverse complementary sequence of the target sequence).
  • An annealing reaction is then performed to form the DNA molecule I.
  • Annealing procedure 95 ° C water bath for 10min, and naturally cooled to room temperature.
  • the recombinant plasmid pBSE901-ALS was sequenced. Based on the sequencing results, the structure of the recombinant plasmid pBSE901-ALS is described as follows: Insert the DNA molecule II into the recognition sequence of restriction enzyme Bsa I of pBSE901 plasmid.
  • the DNA molecule II is 5'-GAAGTTCCGAGAAGAATGAT-3 '.
  • the recombinant plasmid pBSE901-ALS constructed in step 1 is used to transform Agrobacterium tumefaciens EHA105 competent cells to obtain a recombinant Agrobacterium named EHA105-pBSE901-ALS.
  • EHA105-pBSE901-ALS monoclonal was inoculated into 20 mL of YEB liquid medium containing kanamycin 50mg / L and rifampicin 50mg / L, and cultured with shaking at 220 ° C and 28 ° C until the OD 600nm value reached 0.8-1.0. Agrobacterium infection.
  • step 2 After completing step 1, take healthy and sprouting seed kernels, slice from the coximal paraxial proximal end (size 1.5mm ⁇ 1.5mm) to obtain explants, and place the explants in a culture containing 10mL MS liquid medium. Dish (9cm).
  • step 3 take the culture dish, discard the bacterial solution, blot the excess bacterial solution with sterile filter paper, and then place it on a co-culture medium and co-cultivate for 4 days under dark conditions at 28 ° C.
  • step 4 transfer the explants to the selection medium 1, and alternately culture at 25 ° C with light and dark (14h light / 10h dark; light intensity is about 2000lx) for 2-4 weeks (subgenerations once a week).
  • step 5 transfer the explants to selection medium 2, and alternately culture at 25 ° C with light and dark (14h light / 10h dark; light intensity is about 2000lx) for 2-4 weeks (subsequent generations once a week), and get green bud.
  • step 6 the green shoots are transferred to a shoot elongation medium, and alternately cultured at 25 ° C with light and dark (14h light / 10h dark; light intensity is about 2000lx) for 4 weeks to obtain resistant seedlings. During the period, it will be replaced once a week.
  • step 7 transfer the resistant seedlings to the rooting medium, and alternately cultivate them at 25 ° C under light and dark (14h light / 10h dark; light intensity is about 2000lx) for 7 days to obtain regenerated plants, that is, the T 0 generation pseudo-transgenic plants .
  • PCR amplification was performed to obtain PCR amplification products.
  • Agarose gel electrophoresis was performed on the PCR amplified products, and the To 0 generation transgenic plants with a target band of about 500 bp were obtained as the T 0 generation transgenic plants.
  • the genomic DNA of the leaves of the To 0 generation transgenic plant was replaced with water, the recombinant plasmid pBSE901-ALS, and the genomic DNA of the leaves of the non-transgenic watermelon plant respectively.
  • the other steps were the same.
  • step (1) vertically insert a Bar immunoassay test strip (a product of Beijing Aochuang Jinbiao Biotechnology Co., Ltd.) into the centrifuge tube, and the test strip end will be submerged in the sample solution to a depth of about 0.5cm. Read the test results.
  • a Bar immunoassay test strip (a product of Beijing Aochuang Jinbiao Biotechnology Co., Ltd.)
  • the detection line and control line can generally appear within 1-2 minutes.
  • the detection standard is: only one purple-red quality control line appears on the test strip as a negative result; two purple-red bands appear on the detection strip, and one is purple-red
  • the detection line, a purple-red quality control line, is a positive result.
  • T 0 of the proposed generation of transgenic plants is the blade-positive T 0 generation of transgenic plants.
  • PCR was performed using genomic DNA from the leaves of the T 0 -positive transgenic plants as templates, and ALS-190-IDF: 5'-CGTCACCAATGTCTTCGCTTA-3 'and ALS-190-IDR: 5'-CAGGCTTCTTAGATTCAGATACCA-3' as primers for PCR amplification. PCR products were obtained and sequenced.
  • mutation gene 1 shown in sequence 3 in the sequence listing
  • sequence Sequence 1 namely, the CLALS gene
  • mutant gene 2 shown in sequence 5 in the sequence listing
  • sequence 1 that is, CLALS
  • Mutations from position 5 to end 568 and position 569 from the 5 ′ end are obtained by T.
  • Mutant gene 1 encodes mutein 1 shown in sequence 4 in the sequence listing
  • mutant gene 2 encodes mutein 2 shown in sequence 6 in sequence listing.
  • mutein 1 mutates the 190th proline to serine
  • mutein 2 mutates the 190th proline to leucine.
  • the heterozygous mutation with the mutant gene 1 was named as P190L mutant heterozygous.
  • the heterozygous mutant strain with the mutant gene 2 was named as P190S mutant heterozygous.
  • step 2 plant the hybrids to obtain plants.
  • Plants were identified as transgenic and contain P190L mutations. Untransgenic plants containing P190L mutations accounted for about 25%.
  • step 3 self-cross the non-transgenic plants containing the P190L mutation to harvest seeds. The seed was planted to obtain a plant, and the plant genotype was analyzed.
  • Untransgenic plants containing P190L homozygous mutations ie, P190L homozygous mutants accounted for about 25%.
  • step 3 the P190L homozygous mutant strain is selfed to breed a large number of offspring with the P190L homozygous mutation.
  • step 4 the P190L homozygous mutant and the non-transgenic watermelon plant are crossed, and the hybrid is the P190L heterozygous mutant.
  • the tested watermelon seeds are untransgenic watermelon seeds, seeds of P190L homozygous mutants, seeds of P190S homozygous mutants, seeds of P190L heterozygous mutants, or seeds of P190S heterozygous mutants.
  • Plant 20 test watermelon seeds in the field and routinely cultivate to obtain test watermelon seedlings in a two-leaf one-heart period.
  • step 2 After completing step 1, take the watermelon seedlings to be tested, spray the leaves with bensulfuron (the spraying dose is 17g ai / ha; g is grams, ai is the active ingredient, ha is hectares), and then culture for 7 days. Observe the growth status of the watermelon seedlings to be tested.
  • Seedlings, seedlings of the P190L homozygous mutant strain and seedlings of the P190S homozygous mutant strain all grew normally, and the seedlings of the seed of the P190L homozygous mutant strain and the seed of the P190S homozygous mutant strain grew better than Seedlings of the seeds of the P190L heterozygous mutant and seedlings of the P190S heterozygous mutant.
  • ALS inhibitor herbicides can be divided into five categories according to their chemical structure: 1) Sulfonylurea herbicides, such as methylsulfuron, bensulfuron, clopisulfuron, bensulfuron, bensulfuron, and pyrimsulfuron 2, nicosulfuron; 2) imidazolinones, such as memidazole nicotinic acid; 3) triazosin-type herbicides, such as penoxsulam, saflufenacil, diflusulfuron, and saflufenacil Amines, etc .; 4) pyrimidine salicylic acid herbicides, such as bispyrifen; 5) triazolinone herbicides, such as fluazosulfuron.
  • Sulfonylurea herbicides such as methylsulfuron, bensulfuron, clopisulfuron, bensulfuron, bensulfuron, and
  • the tested watermelon seeds are untransgenic watermelon seeds, seeds of P190L homozygous mutants, seeds of P190S homozygous mutants, seeds of P190L heterozygous mutants, or seeds of P190S heterozygous mutants.
  • step 2 After completing step 1, take the test watermelon seedlings, and spray the leaves (benzenesulfuron, clofsulfuron, bensulfuron-methyl, pyrazosulfuron, flusulfuron, and flusulfuron-methyl) on the leaves (benzene
  • the spray doses of Sulfonuron, Clossulfuron-methyl, Bensulfuron-methyl, Pyrisulfuron-methyl, Flusulfuron, and Flusulfuron were 15, 33.75, 22.5, 24, 48, and 31.5 g / ha; g is gram, ai is the active ingredient, ha is hectare), and then cultured for 7 days to observe the growth state of the watermelon seedling to be tested.
  • step 1 After step 1 is completed, take the watermelon seedlings to be tested, spray the leaves with the same volume of water as the herbicide in step 2, and then cultivate for 7 days. Observe the growth status of the watermelon seedlings as a control.
  • P190L homozygous mutants, P190S homozygous mutants, P190L heterozygous mutants and P190S heterozygous mutants have broad-spectrum resistance to ALS inhibitor herbicides.
  • a P190L mutant heterozygosity and a P190S mutant heterozygosity are obtained through a plant single base editing system nCas9-PBE, and a P190L homozygous mutant strain is further obtained
  • CLALS protein has a 190th amino acid residue type only from the N-terminus only bright Amino acid residues
  • P190S homozygous mutant strain CLALS protein is the only amino acid residue at position 190 from the N-terminus
  • P190L heterozygous mutant strain (CLALS protein is located at the 190th position from the N-terminus amino acid residue)
  • the types of residues are proline residues and leucine residues) and P190S hybrid mutants (the amino acid residues of the CLALS protein at the 190th position from the N-terminus are proline residues and serine residues).
  • the seedlings of the above mutants and the seedlings of non-transgenic watermelon (the 190th amino acid residue of the CLALS protein from the N-terminus is only a proline residue) were sprayed with bensulfuron-methyl, and the results showed that the seedlings of the non-transgenic watermelon were very Near death (3-7 days after bensulfuron-methyl spraying), seedlings of the P190L heterozygous mutant strain, seedlings of the P190S heterozygous mutant strain, seedlings of the P190L homozygous mutant strain, and seedlings of the P190S homozygous mutant strain all grew normally.
  • the seedlings of the P190L homozygous mutant strain and the P190S homozygous mutant strain had a better growth status than the seedlings of the P190L heterozygous mutant strain and the P190S heterozygous mutant strain. It can be seen that the type of amino acid residue at position 190 from the N-terminus of CLALS protein can be used as a detection object to predict the resistance of watermelon herbicides to be tested. The invention has great application value.

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Abstract

本发明公开了CLALS蛋白、其编码基因及它们在预测西瓜除草剂抗性中的应用。"CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基"的待测西瓜或"CLALS蛋白自N末端起第190位的氨基酸残基种类为非脯氨酸残基和脯氨酸残基"的待测西瓜的除草剂抗性强于"CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基"的待测西瓜。CLALS蛋白自N末端起第190位的氨基酸残基种类可以作为检测对象，预测待测西瓜除草剂抗性。

Description

CLALS蛋白、其编码基因及它们在预测西瓜除草剂抗性中的应用 技术领域
本发明属于生物技术领域，具体涉及CLALS蛋白、其编码基因及它们在西瓜预测西瓜除草剂抗性、品种改良尤其是培育抗除草剂性状中的应用。
背景技术
西瓜田杂草是限制西瓜产量、品质及成本效益的重要因素之一。西瓜由于种植密度低，在其茎蔓展开前，田间裸露地表的面积大，加之西瓜生长环境较为湿热，易于杂草发生，因此相较于其它作物，西瓜的草害尤为严重。西瓜田人工除草费事费力，施用化学除草剂常因选择不当、施用剂量不当等原因对西瓜造成药害，导致西瓜减产及品质降低。杂草造成西瓜减产约20％，占田间人工成本的30％以上，因此西瓜田除草成本及除草剂安全问题已成为影响西瓜产业发展的限制性因素。培育具有除草剂抗性的西瓜品种，使其在种植期间使用1-2次除草剂杀死杂草，但不影响西瓜本身的生长，不仅可以解决困扰西瓜生产的草害问题，而且符合西瓜简约化生产的要求。乙酰乳酸合成酶(acetolactate synthase，ALS)是植物和微生物支链氨基酸合成中的一个关键酶。ALS抑制剂类除草剂通过抑制植物体内ALS的活性，从而阻止支链氨基酸的合成，进而影响蛋白质合成及植株生长，最后造成植物死亡。ALS抑制剂类除草剂具有活性高、选择性强、杀草谱广、毒性低等优点，成为20世纪90年代商品化除草剂最活跃的一类。
2016年4月，Komor等人采用Cas9变体、胞嘧啶脱氨酶(cytidine deaminase，CD)和尿嘧啶糖基化酶抑制剂(uracil DNA glycosylase inhibitor，UGI)相融合的办法，在大鼠体内实现了高效的单碱基定点突变。依据相同原理，利用Cas9变体(nCas9-D10A)融合大鼠胞嘧啶脱氨酶(rAPOBEC1)和尿嘧啶糖基化酶抑制剂(UGI)，构建的高效的植物单碱基编辑系统nCas9-PBE已在水稻、小麦、玉米、拟南芥等作物实现了目标基因的单碱基定点突变。nCas9-PBE可将靶位点DNA的C替换为T，C碱基脱氨化的窗口覆盖靶序列的7个核苷酸(距离PAM远端的第3-9位)，该技术无需在基因组的靶位点产生DNA双链断裂(DSB)，也无需供体DNA的参与，具有简单、广适、高效的特点。nCas9-PBE单碱基编辑系统成功建立和应用，为高效和大规模创制单碱基突变体提供了一个可靠方案，为作物遗传改良和新品种培育提供了重要技术支撑。
发明公开
本发明的目的在于培育具有除草剂抗性的的西瓜品种。
本发明首先保护CLALS蛋白，CLALS蛋白可为如下W1)或W2)：
W1)自N端至C端依次可包括区段Ⅰ、区段Ⅱ和区段Ⅲ；
所述区段Ⅱ可为一个氨基酸残基；
所述区段Ⅰ可为如下a1)或a2)或a3)：
a1)氨基酸序列是序列表中序列2自N末端起第1至189位所示的多肽；
a2)将a1)所示的多肽经过一个或几个氨基酸残基的替换得到的与除草剂抗性相关的多肽；
a3)与a1)或a2)所示的多肽具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的多肽；
所述区段Ⅲ可为如下b1)或b2)或b3)：
b1)氨基酸序列是序列表中序列2自N末端起第191至662位所示的多肽；
b2)将b1)所示的多肽经过一个或几个氨基酸残基的替换得到的与除草剂抗性相关的多肽；
b3)与b1)或b2)所示的多肽具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的多肽；
W2)在W1)的N端或/和C端连接标签得到的融合蛋白质。
上述a3)中，使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明的序列表中序列2自N末端起第1至189位所示的氨基酸序列具有80％，或85％或更高，或90％或更高，或95％或更高同一性的氨基酸序列。
上述b3)中，使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明的序列表中序列2自N末端起第191至662位所示的氨基酸序列具有80％，或85％或更高，或90％或更高，或95％或更高同一性的氨基酸序列。
所述CLALS蛋白中，所述区段Ⅱ可为脯氨酸残基或非脯氨酸残基。所述非脯氨酸残基具体可为丝氨酸残基或亮氨酸残基。
所述CLALS蛋白自N端至C端依次可由所述区段Ⅰ、所述区段Ⅱ和所述区段Ⅲ组成。
所述CLALS蛋白具体可为如下c1)或c2)或c3)或c4)或c5)：
c1)氨基酸序列是序列表中序列2所示的蛋白质；
c2)氨基酸序列是序列表中序列4所示的蛋白质；
c3)氨基酸序列是序列表中序列6所示的蛋白质；
c4)将c1)或c2)或c3)所示的蛋白质的区段Ⅰ和/或区段Ⅲ经过一个或几个氨基酸残基的替换和/或缺失和/或添加得到的与除草剂抗性相关的蛋白质；
c5)与c1)或c2)或c3)或c4)所示的蛋白质具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的蛋白质。
上述c5)中，使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明的序列表中序列2、序列4或序列6所示的氨基酸序列具有80％，或85％或更高，或90％或更高，或95％或更高同一性的氨基酸序列。
编码所述CLALS蛋白的核酸分子也属于本发明的保护范围。
编码所述CLALS蛋白的核酸分子可为如下d1)或d2)或d3)或d4)或d5)所示的DNA分子：
d1)核苷酸序列是序列表中序列1所示的DNA分子；
d2)核苷酸序列是序列表中序列3所示的DNA分子；
d3)核苷酸序列是序列表中序列5所示的DNA分子；
d4)与d1)或d2)或d3)限定的核苷酸序列具有75％或75％以上同一性，且编码所述CLALS蛋白的DNA分子；
d5)在严格条件下与d1)或d2)或d3)限定的核苷酸序列杂交，且编码所述CLALS蛋白的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
上述d4)中，使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列表中序列2所示的氨基酸序列组成的蛋白质的核苷酸序列、或、本发明的编码序列表中序列4所示的氨基酸序列组成的蛋白质的核苷酸序列、或、本发明的编码序列表中序列6所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％，或80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。
序列表中序列1由1989个核苷酸组成，序列表中序列1的核苷酸编码序列表中序列2所示的氨基酸序列。序列表中序列3由1989个核苷酸组成，序列表中序列3的核苷酸编码序列表中序列4所示的氨基酸序列。序列表中序列5由1989个核苷酸组成，序列表中序列5的核苷酸编码序列表中序列6所示的氨基酸序列。
上文中，同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系也属于本发明的保护范围。
本发明还保护Z1)或Z2)：
Z1)所述CLALS蛋白或编码所述CLALS蛋白的核酸分子，在调控西瓜除草剂抗性中的应用；
Z2)所述CLALS蛋白或编码所述CLALS蛋白的核酸分子，在培育除草剂抗性改变的西瓜中的应用。
本发明还保护预测待测西瓜除草剂抗性的方法。
本发明所保护的预测待测西瓜除草剂抗性的方法，具体可为S1)：检测待测西瓜的所述CLALS蛋白自N末端起第190位的氨基酸残基种类；“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”的待测西瓜或“CLALS蛋白自N末端起第190位的氨基酸残基种类为非脯氨酸残基和脯氨酸残基”的待测西瓜的除草剂抗性强于“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基”的待测西瓜。
所述S1)中，“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”中非脯氨酸残基的氨基酸种类可以为一种，也可以为两种。
本发明所保护的预测待测西瓜除草剂抗性的方法，具体可为S2)：检测待测西瓜总RNA的特定转录本中第190个密码子的核苷酸序列；所述特定转录本为所述CLALS蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子；“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”的待测西瓜或“特定转录本中第190个密码子的核苷酸序列编码非脯氨酸和脯氨酸”的待测西瓜的除草剂抗性强于“特定转录本中第190个密码子的核苷酸序列仅编码脯氨酸”的待测西瓜。
所述S2)中，“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”中非脯氨酸的氨基酸种类可以为一种，也可以为两种。
本发明所保护的预测待测西瓜除草剂抗性的方法，具体可为S3)：检测待测西瓜总DNA中所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类；“CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类仅为c”的待测西瓜的除草剂抗性弱于F1或F2或F3；F1为CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类均不含有c的待测西瓜；F2为CLALS蛋白的编码基因自5’末端起第569位的核苷酸种类含有c且第568位的核苷酸种类不含有c的待测西瓜；F3为CLALS蛋白的编码基因自5’末端起第568位的核苷酸种类含有c且第569位的核苷酸种类不含有c的待测西瓜。
本发明所保护的预测待测西瓜除草剂抗性的方法，具体可包括如下步骤：检测待测西瓜总DNA中是否具有序列表的序列1所示的DNA分子、序列表的序列3所示的DNA分子和序列表的序列5所示的DNA分子；
“待测西瓜总DNA中具有序列表的序列3所示的DNA分子和/或具有序列表的序列5所示的DNA分子”的待测西瓜的除草剂抗性强于“待测西瓜总DNA中仅具有序列表的序列1所示的DNA分子”的待测西瓜。
本发明还保护物质甲、物质乙或物质丙在预测待测西瓜除草剂抗性中的应用。
所述物质甲可为用于检测所述CLALS蛋白自N末端起第190位的氨基酸残基种类的物质。
所述物质乙可为用于检测特定转录本中第190个密码子的核苷酸序列的物质；所述特定转录本为所述CLALS蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子。
所述物质丙可为用于检测所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类的物质。
本发明还保护成套产品甲、成套产品乙或成套产品丙在预测待测西瓜除草剂抗性中的应用。
所述成套产品甲可为所述物质甲和记载有方法甲的载体；所述方法甲可为：“所述CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”的待测西瓜或“CLALS蛋白自N末端起第190位的氨基酸残基种类为非脯氨酸残基和脯氨酸残基”的待测西瓜的除草剂抗性强于“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基”的待测西瓜。
所述成套产品乙可为所述物质乙和记载有方法乙的载体；所述方法乙可为：“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”的待测西瓜或“特定转录本中第190个密码子的核苷酸序列编码非脯氨酸和脯氨酸”的待测西瓜的除草剂抗性强于“特定转录本中第190个密码子的核苷酸序列仅编码脯氨酸”的待测西瓜。
所述成套产品丙可为所述物质丙和记载有方法丙的载体。所述方法丙可为：“所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类仅为c”的待测西瓜的除草剂抗性弱于F1或F2或F3；F1为CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类均不含有c的待测西瓜；F2为CLALS蛋白的编码基因自5’末端起第569位的核苷酸种类含有c且第568位的核苷酸种类不含有c的待测西瓜；F3为CLALS蛋白的编码基因自5’末端起第568位的核苷酸种类含有c且第569位的核苷酸种类不含有c的待测西瓜。
上述应用中，“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”中非脯氨酸残基的氨基酸种类可以为一种，也可以为两种。
上述应用中，“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”中非脯氨酸的氨基酸种类可以为一种，也可以为两种。
本发明还保护B1)或B2)或B3)。
B1)所述CLALS蛋白自N末端起第190位的氨基酸残基种类作为检测对象在预测待测西瓜除草剂抗性中的应用。
B2)特定转录本中第190个密码子的核苷酸序列作为检测对象在预测待测西瓜除草剂抗性中的应用；所述特定转录本为所述CLALS蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子。
B3)所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类作为检测对象在预测待测西瓜除草剂抗性中的应用。
上述任一所述除草剂可为以所述CLALS蛋白作为靶标的除草剂(即ALS抑制剂类除草剂)。
上述任一所述ALS抑制剂类除草剂可为Y1)或Y2)或Y3)或Y4)或Y5)：Y1)磺酰脲类除草剂；Y2)三唑嘧啶类除草剂；Y3)三唑啉酮类除草剂；Y4)嘧啶水杨酸类除草剂；Y5)咪唑啉酮类。
所述磺酰脲类除草剂具体可为苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、烟嘧磺隆、甲基二磺隆、噻吩磺隆或砜嘧磺隆。
所述三唑嘧啶类除草剂具体可为唑嘧磺草胺、五氟磺草胺、啶磺草胺或双 氟磺草胺。
所述三唑啉酮类除草剂具体可为氟唑磺隆。
所述嘧啶水杨酸类除草剂可为双草醚。
所述咪唑啉酮类可为甲咪唑烟酸。
上述任一所述非脯氨酸具体可为丝氨酸或亮氨酸。
CLALS蛋白自N末端起第190位的氨基酸残基的突变与CLALS蛋白中其它氨基酸残基的突变形成的双位点或多位点突变基因也属于本发明的保护范围。
CLALS蛋白自N末端起第190位的氨基酸残基的突变与CLALS蛋白中其它氨基酸残基的突变形成的双位点或多位点突变基因在调控西瓜除草剂抗性中的应用也属于本发明的保护范围。
在本发明的实施例中，发明人通过植物单碱基编辑系统nCas9-PBE获得了P190L突变杂合和P190S突变杂合，进一步获得了P190L纯合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为亮氨酸残基)、P190S纯合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为丝氨酸残基)、P190L杂合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类为脯氨酸残基和亮氨酸残基)和P190S杂合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类为脯氨酸残基和丝氨酸残基)。用苯磺隆喷施上述突变株的幼苗和未转基因西瓜(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基)的幼苗，结果表明，未转基因西瓜的幼苗很快死亡(苯磺隆喷施后3-7天)，P190L杂合突变株的幼苗、P190S杂合突变株的幼苗、P190L纯合突变株的幼苗和P190S纯合突变株的幼苗均正常生长，且P190L纯合突变株的幼苗和P190S纯合突变株的幼苗的生长状态优于P190L杂合突变株的幼苗和P190S杂合突变株的幼苗。
实验证明，CLALS蛋白自N末端起第190位的氨基酸残基种类可以作为检测对象，预测待测西瓜除草剂抗性。本发明具有重大的应用价值。
附图说明
图1为除草剂抗性鉴定结果。
图2为除草剂的抗性谱鉴定结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量实验，均设置三次重复实验，结果取平均值。
pBSE901质粒记载与如下文献中：Chen Y，Wang Z，Ni H，et al.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis[J].Science China Life Sciences，2017，60(5):520-523.
BM培养基：将0.44g MS培养基、3g蔗糖和0.8g琼脂溶于100mL去离子水，调节pH值为5.8，高温高压灭菌15min。MS培养基为PhytoTech公司的产品。
共培养基：含1.5mg/L 6-BA的BM培养基。
选择培养基1：含100mg/L Timentin和1.5mg/L Basta的共培养基。
选择培养基2：含100mg/L Timentin和2.0mg/L Basta的共培养基。
芽伸长培养基：含0.1mg/L 6-BA、0.01mg/L NAA、100mg/L Timentin和1.5mg/L Basta的BM培养基。
生根培养基：含1mg/L IBA的BM培养基。
实施例1、抗除草剂西瓜突变株的获得及其验证
CLALS蛋白的氨基酸序列如序列表中序列2所示。编码CLALS蛋白的基因(即CLALS基因)如序列表中序列1所示。根据CLALS基因的核苷酸序列选择靶序列，靶序列的核苷酸序列为：5’-AAGTTCCGAGAAGAATGAT-3’。
一、重组质粒pBSE901-ALS的构建
1、用限制性内切酶Bsa Ⅰ酶切pBSE901质粒，回收约15Kb的载体骨架。
2、人工合成引物ALS-190F：5’-ATTG
Figure PCTCN2019087744-appb-000001
-3’(双下划线为靶序列)和引物ALS-190R：5’-AAAC
Figure PCTCN2019087744-appb-000002
-3’(双下划线为靶序列的反向互补序列)，用去离子水分别将引物ALS-190F和引物ALS-190R稀释至100μM，得到引物ALS-190F稀释液和引物ALS-190R稀释液；然后进行退火反应，形成DNA分子Ⅰ。
退火程序：95℃水浴10min，自然冷却至室温。
3、将载体骨架和DNA分子Ⅰ连接，得到重组质粒pBSE901-ALS。
将重组质粒pBSE901-ALS进行测序。根据测序结果，对重组质粒pBSE901-ALS进行结构描述如下：向pBSE901质粒的限制性内切酶Bsa Ⅰ识别序列插入DNA分子Ⅱ。DNA分子Ⅱ为5’-GAAGTTCCGAGAAGAATGAT-3’。
二、农杆菌侵染液的制备
1、将步骤一构建的重组质粒pBSE901-ALS转化根癌农杆菌EHA105感受态细胞，得到重组农杆菌，命名为EHA105-pBSE901-ALS。
2、将EHA105-pBSE901-ALS单克隆接种于20mL含卡那霉素50mg/L和利福平50mg/L的YEB液体培养基，28℃、220rpm震荡培养至OD 600nm值达到0.8-1.0，得到农杆菌侵染液。
三、T 0代拟转基因植株的获得
1、取饱满的西瓜种子，小心剥去种皮(尽量避免伤及种仁)，先用10％(m/v)的次氯酸钠水溶液消毒15min，然后用无菌水洗涤3次，轻轻置于盛有BM培养基(经高压灭菌)的培养皿，28℃暗培养3天。
2、完成步骤1后，取健康萌动的种仁，自子叶近轴端切片(大小为1.5mm×1.5mm)，获得外植体，将外植体放入盛有10mL MS液体培养基的培养皿(规格为9cm)中。
3、取所述培养皿，加入50μL农杆菌侵染液，浸泡10min。
4、完成步骤3后，取所述培养皿，弃菌液，用无菌滤纸吸干多余的菌液，然后置于共培养基，28℃黑暗条件下共培养4天。
5、完成步骤4后，将外植体转移至选择培养基1，25℃光暗交替培养(14h光照/10h黑暗；光照强度约为2000lx)2-4周(每周继代1次)。
6、完成步骤5后，将外植体转移至选择培养基2，25℃光暗交替培养(14h光照/10h黑暗；光照强度约为2000lx)2-4周(每周继代1次)，得到绿色幼芽。
7、完成步骤6后，将绿色幼芽转移至芽伸长培养基，25℃光暗交替培养(14h光照/10h黑暗；光照强度约为2000lx)4周，得到抗性幼苗。期间，每周继代1次。
8、完成步骤7后，将抗性幼苗转移至生根培养基，25℃光暗交替培养(14h光照/10h黑暗；光照强度约为2000lx)7天，得到再生植株，即T 0代拟转基因植株。
四、T 0代拟转基因植株的鉴定
1、分子鉴定
以步骤三获得的T 0代拟转基因植株叶片的基因组DNA为模板，以BE3-IDF：5’-CATACCTCCCAGAACACAAATAAGC-3’和BE3-IDR：5’-ACTGAAGGGCAATAGTGAAGAATGT-3’为引物进行PCR扩增，得到PCR扩增产物。将PCR扩增产物进行琼脂糖凝胶电泳,能得到约500bp的目的条带的T 0代拟转基因植株即为T 0代阳性转基因植株。
按照上述方法，将T 0代拟转基因植株叶片的基因组DNA分别替换为水、重组质粒pBSE901-ALS和未转基因西瓜植株叶片的基因组DNA，其它步骤均相同。
结果表明，以水和未转基因西瓜植株叶片的基因组DNA为模板进行PCR扩增后，无目的条带；而以重组质粒pBSE901-ALS为模板进行PCR扩增有约500bp的目的条带。
2、Bar免疫检测试纸鉴定
(1)检测样品处理
取步骤三获得的T 0代拟转基因植株叶片0.1g，放入2mL离心管中，加蒸馏水研磨，得到样品液。
(2)样本检测
完成步骤(1)后，将Bar免疫检测试纸(北京奥创金标生物技术有限公司的产品)垂直插入所述离心管，试纸端淹没入样品液的深度约为0.5cm，1min后取出放平阅读检测结果。
(3)结果判断
检测线及对照线一般可在1-2min内出现，检测标准为：在检测条上仅出现一条紫红色质控线为阴性结果；在检测条上出现两条紫红色条带，一条为紫红色检测线，一条为紫红色质控线，此为阳性结果。
凡能得到两条紫红色条带的T 0代拟转基因植株叶片即为T 0代阳性转基因植株。
经鉴定，共获得199棵T 0代阳性转基因植株。
五、突变类型的分子检测
1、分别以T 0代阳性转基因植株叶片的基因组DNA为模板，以ALS-190-IDF：5’-CGTCACCAATGTCTTCGCTTA-3’和ALS-190-IDR：5’-CAGGCTTCTTAGATTCAGATACCA-3’为引物进行PCR扩增，得到PCR扩增产物并测序。
测序结果表明，199棵T 0代阳性转基因植株中，154株的基因型与野生型(即未转基因西瓜)的基因型完全一致、靶点区并未发生编辑，45株靶点区域的全部或部分C突变成T且均为杂合突变，突变率为22.61％；杂合突变株中CLALS基因有两种突变形式：一种是突变基因1(序列表中序列3所示)，为序列表中序列1(即CLALS基因)自5’末端起第568位的C突变为T得到；另一种是突变基因2(序列表中序列5所示)，为序列表中序列1(即CLALS基因)自5’末端起第568位和第569位的C突变为T得到。突变基因1编码序列表中序列4所示的突变蛋白1，突变基因2编码序列表中序列6所示的突变蛋白2。与CLALS蛋白相比，突变蛋白1是将第190位的脯氨酸突变为丝氨酸，突变蛋白2是将第190位的脯氨酸突变为亮氨酸。
将具有突变基因1的杂合突变命名为P190L突变杂合。将具有突变基因2的杂合突变株命名为P190S突变杂合。
六、P190L纯合突变株、P190S纯合突变株、P190L杂合突变株和P190S杂合突变株的获得
1、将含有P190L杂和突变的植株(作为父本)和未转基因西瓜植株(作为母本)进行杂交，收获杂交种。
2、完成步骤1后，将杂交种种植得到植株。
鉴定植株是否转基因和含有P190L突变。未转基因且含有P190L突变的植株约占25％。
3、完成步骤2后，将未转基因且含有P190L突变的植株自交，收获种子。将该种子种植得到植株，分析植株的基因型。
未转基因且含有P190L纯合突变的植株(即P190L纯合突变株)约占25％。
4、完成步骤3后，将P190L纯合突变株自交，即可繁育大量具有P190L纯合突变的后代。
5、完成步骤4后，将P190L纯合突变株和未转基因西瓜植株进行杂交，杂交种即为P190L杂合突变株。
按照上述步骤，将“含有P190L杂和突变的植株”替换为“含有P190S杂和突变的植株”，其它步骤均不变，得到P190S纯合突变株和P190S杂合突变株。
七、除草剂苯磺隆的抗性鉴定
待测西瓜种子为未转基因西瓜种子、P190L纯合突变株的种子、P190S纯合突变株的种子、P190L杂合突变株的种子或P190S杂合突变株的种子。
实验重复三次，每次重复的步骤如下：
1、在大田中种植20粒待测西瓜种子，常规培养，得到处于两叶一心时期的待测西瓜幼苗。
2、完成步骤1后，取待测西瓜幼苗，使用苯磺隆喷施叶片(喷施剂量为17g ai/ha；g为克，ai为有效成分，ha为公顷)，然后常规培养7天，观察待测西瓜幼苗的生长状态。
部分实验结果见图1(WT为未转基因西瓜种子，P190L为P190L纯合突变株的种子，P190S为P190S纯合突变株的种子)。结果表明，苯磺隆喷施后，未转基因西瓜种子的幼苗很快死亡(苯磺隆喷施后3-7天)，P190L杂合突变株的种子的幼苗、P190S杂合突变株的种子的幼苗、P190L纯合突变株的种子的幼苗和P190S纯合突变株的种子的幼苗均正常生长，且P190L纯合突变株的种子的幼苗和P190S纯合突变株的种子的幼苗的生长状态优于P190L杂合突变株的种子的幼苗和P190S杂合突变株的种子的幼苗。
上述结果表明，含有突变基因1和/或突变基因2的西瓜对苯磺隆具有明显的抗性。
八、除草剂抗性谱鉴定
ALS抑制剂类除草剂按照化学结构可分为五大类：1)磺酰脲类除草剂，如甲基二磺隆、苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、烟嘧磺隆；2)咪唑啉酮类，如甲咪唑烟酸；3)三唑嘧啶类除草剂，如五氟磺草胺、啶磺草胺、双氟磺草胺、唑嘧磺草胺等；4)嘧啶水杨酸类除草剂，如双草醚；5)三唑啉酮类除草剂，如氟唑磺隆。
待测西瓜种子为未转基因西瓜种子、P190L纯合突变株的种子、P190S纯合突变株的种子、P190L杂合突变株的种子或P190S杂合突变株的种子。
实验重复三次，每次重复的步骤如下：
1、在大田中种植5粒待测西瓜种子，常规培养10天至子叶展平，得到待测西瓜幼苗。
2、完成步骤1后，取待测西瓜幼苗，分别使用苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、唑嘧磺草胺和氟唑磺隆喷施叶片(苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、唑嘧磺草胺和氟唑磺的喷施剂量分别为15、33.75、22.5、24、48和31.5g ai/ha；g为克，ai为有效成分，ha为公顷)，然后常规培养7天，观察待测西瓜幼苗的生长状态。
3、完成步骤1后，取待测西瓜幼苗，使用与步骤2中除草剂等体积的水喷施叶片，然后常规培养7天，观察待测西瓜幼苗的生长状态，作为对照。
实验结果见图2(野生型为未转基因西瓜，A为对照，B为喷施苯磺隆的西瓜幼苗，C为喷施氯吡嘧磺隆的西瓜幼苗，D为喷施苄嘧磺隆的西瓜幼苗，E为喷 施吡嘧磺隆的西瓜幼苗，F为喷施氟唑磺隆的西瓜幼苗，G为喷施唑嘧磺草胺的西瓜幼苗)。结果表明，与未转基因西瓜相比，P190L纯合突变株、P190S纯合突变株、P190L杂合突变株和P190S杂合突变株均对苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、氟唑磺隆和唑嘧磺草胺具有明显的抗性。
由此可见，P190L纯合突变株、P190S纯合突变株、P190L杂合突变株和P190S杂合突变株对ALS抑制剂类除草剂存在广谱抗性。
工业应用
本发明通过植物单碱基编辑系统nCas9-PBE获得了P190L突变杂合和P190S突变杂合，进一步获得了P190L纯合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为亮氨酸残基)、P190S纯合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为丝氨酸残基)、P190L杂合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类为脯氨酸残基和亮氨酸残基)和P190S杂合突变株(CLALS蛋白自N末端起第190位的氨基酸残基种类为脯氨酸残基和丝氨酸残基)。用苯磺隆喷施上述突变株的幼苗和未转基因西瓜(CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基)的幼苗，结果表明，未转基因西瓜的幼苗很快死亡(苯磺隆喷施后3-7天)，P190L杂合突变株的幼苗、P190S杂合突变株的幼苗、P190L纯合突变株的幼苗和P190S纯合突变株的幼苗均正常生长，且P190L纯合突变株的幼苗和P190S纯合突变株的幼苗的生长状态优于P190L杂合突变株的幼苗和P190S杂合突变株的幼苗。由此可见，CLALS蛋白自N末端起第190位的氨基酸残基种类可以作为检测对象，预测待测西瓜除草剂抗性。本发明具有重大的应用价值。

Claims (17)

  1. CLALS蛋白，为如下W1)或W2)：
    W1)自N端至C端依次包括区段Ⅰ、区段Ⅱ和区段Ⅲ；
    所述区段Ⅱ为一个氨基酸残基；
    所述区段Ⅰ为如下a1)或a2)或a3)：
    a1)氨基酸序列是序列表中序列2自N末端起第1至189位所示的多肽；
    a2)将a1)所示的多肽经过一个或几个氨基酸残基的替换得到的与除草剂抗性相关的多肽；
    a3)与a1)或a2)所示的多肽具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的多肽；
    所述区段Ⅲ为如下b1)或b2)或b3)：
    b1)氨基酸序列是序列表中序列2自N末端起第191至662位所示的多肽；
    b2)将b1)所示的多肽经过一个或几个氨基酸残基的替换得到的与除草剂抗性相关的多肽；
    b3)与b1)或b2)所示的多肽具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的多肽；
    W2)在W1)的N端或/和C端连接标签得到的融合蛋白质。
  2. 如权利要求1所述的CLALS蛋白，其特征在于：所述区段Ⅱ为脯氨酸残基或非脯氨酸残基；所述非脯氨酸残基为丝氨酸残基或亮氨酸残基。
  3. 如权利要求1或2所述的CLALS蛋白，其特征在于：所述CLALS蛋白为如下c1)或c2)或c3)或c4)或c5)：
    c1)氨基酸序列是序列表中序列2所示的蛋白质；
    c2)氨基酸序列是序列表中序列4所示的蛋白质；
    c3)氨基酸序列是序列表中序列6所示的蛋白质；
    c4)将c1)或c2)或c3)所示的蛋白质的区段Ⅰ和/或区段Ⅲ经过一个或几个氨基酸残基的替换和/或缺失和/或添加得到的与除草剂抗性相关的蛋白质；
    c5)与c1)或c2)或c3)或c4)所示的蛋白质具有80％或80％以上同一性，来源于西瓜且与除草剂抗性相关的蛋白质。
  4. 编码权利要求1至3任一所述CLALS蛋白的核酸分子。
  5. 如权利要求4所述的核酸分子，其特征在于：所述核酸分子为如下d1)或d2)或d3)或d4)或d5)所示的DNA分子：
    d1)核苷酸序列是序列表中序列1所示的DNA分子；
    d2)核苷酸序列是序列表中序列3所示的DNA分子；
    d3)核苷酸序列是序列表中序列5所示的DNA分子；
    d4)与d1)或d2)或d3)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求1至3任一所述CLALS蛋白的DNA分子；
    d5)在严格条件下与d1)或d2)或d3)限定的核苷酸序列杂交，且编码权利要求1至3任一所述CLALS蛋白的DNA分子。
  6. Z1)或Z2)：
    Z1)权利要求1至3任一所述CLALS蛋白、或、权利要求4或5所述核酸分子，在调控西瓜除草剂抗性中的应用；
    Z2)权利要求1至3任一所述CLALS蛋白、或、权利要求4或5所述核酸分子，在培育除草剂抗性改变的西瓜中的应用。
  7. 一种预测待测西瓜除草剂抗性的方法，为S1)或S2)或S3)：
    S1)检测待测西瓜的权利要求1至3任一所述CLALS蛋白自N末端起第190位的氨基酸残基种类；
    “CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”的待测西瓜或“CLALS蛋白自N末端起第190位的氨基酸残基种类为非脯氨酸残基和脯氨酸残基”的待测西瓜的除草剂抗性强于“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基”的待测西瓜；
    S2)检测待测西瓜总RNA的特定转录本中第190个密码子的核苷酸序列；所述特定转录本为权利要求1至3任一所述CLALS蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子；
    “特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”的待测西瓜或“特定转录本中第190个密码子的核苷酸序列编码非脯氨酸和脯氨酸”的待测西瓜的除草剂抗性强于“特定转录本中第190个密码子的核苷酸序列仅编码脯氨酸”的待测西瓜；
    S3)检测待测西瓜总DNA中权利要求1至3任一所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类；
    “CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类仅为c”的待测西瓜的除草剂抗性弱于F1或F2或F3；F1为CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类均不含有c的待测西瓜；F2为CLALS蛋白的编码基因自5’末端起第569位的核苷酸种类含有c且第568位的核苷酸种类不含有c的待测西瓜；F3为CLALS蛋白的编码基因自5’末端起第568位的核苷酸种类含有c且第569位的核苷酸种类不含有c的待测西瓜。
  8. 一种预测待测西瓜除草剂抗性的方法，包括如下步骤：检测待测西瓜总DNA中是否具有序列表的序列1所示的DNA分子、序列表的序列3所示的DNA分子和序列表的序列5所示的DNA分子；
    “待测西瓜总DNA中具有序列表的序列3所示的DNA分子和/或具有序列表的序列5所示的DNA分子”的待测西瓜的除草剂抗性强于“待测西瓜总DNA中仅具有序列表的序列1所示的DNA分子”的待测西瓜。
  9. 物质甲、物质乙或物质丙在预测待测西瓜除草剂抗性中的应用；
    所述物质甲为用于检测权利要求1至3任一所述CLALS蛋白自N末端起第 190位的氨基酸残基种类的物质；
    所述物质乙为用于检测特定转录本中第190个密码子的核苷酸序列的物质；所述特定转录本为权利要求1至3任一所述CLALS蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子；
    所述物质丙为用于检测权利要求1至3任一所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类的物质。
  10. 成套产品甲、成套产品乙或成套产品丙在预测待测西瓜除草剂抗性中的应用；
    所述成套产品甲为权利要求9中所述物质甲和记载有方法甲的载体；
    所述方法甲为：“权利要求1至3任一所述CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”的待测西瓜或“CLALS蛋白自N末端起第190位的氨基酸残基种类为非脯氨酸残基和脯氨酸残基”的待测西瓜的除草剂抗性强于“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为脯氨酸残基”的待测西瓜；
    所述成套产品乙为权利要求9中所述物质乙和记载有方法乙的载体；
    所述方法乙为：“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”的待测西瓜或“特定转录本中第190个密码子的核苷酸序列编码非脯氨酸和脯氨酸”的待测西瓜的除草剂抗性强于“特定转录本中第190个密码子的核苷酸序列仅编码脯氨酸”的待测西瓜；
    所述成套产品丙为权利要求9中所述物质丙和记载有方法丙的载体；
    所述方法丙为：“权利要求1至3任一所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类仅为c”的待测西瓜的除草剂抗性弱于F1或F2或F3；F1为CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类均不含有c的待测西瓜；F2为CLALS蛋白的编码基因自5’末端起第569位的核苷酸种类含有c且第568位的核苷酸种类不含有c的待测西瓜；F3为CLALS蛋白的编码基因自5’末端起第568位的核苷酸种类含有c且第569位的核苷酸种类不含有c的待测西瓜。
  11. 如权利要求7所述的方法或权利要求10所述的应用，其特征在于：
    所述“CLALS蛋白自N末端起第190位的氨基酸残基种类仅为非脯氨酸残基”中非脯氨酸残基的氨基酸种类可以为一种，也可以为两种；
    所述“特定转录本中第190个密码子的核苷酸序列仅编码非脯氨酸”中非脯氨酸的氨基酸种类可以为一种，也可以为两种。
  12. B1)或B2)或B3)：
    B1)权利要求1至3任一所述CLALS蛋白自N末端起第190位的氨基酸残基种类作为检测对象在预测待测西瓜除草剂抗性中的应用；
    B2)特定转录本中第190个密码子的核苷酸序列作为检测对象在预测待测西瓜除草剂抗性中的应用；所述特定转录本为权利要求1至3任一所述CLALS 蛋白的编码基因转录得到的RNA，其第1个密码子为起始密码子；
    B3)权利要求1至3任一所述CLALS蛋白的编码基因自5’末端起第568位和第569位的核苷酸种类作为检测对象在预测待测西瓜除草剂抗性中的应用。
  13. 如权利要求1至3任一所述CLALS蛋白，或，权利要求6、9、10、11或12所述的应用，或，权利要求7、8或11所述的方法，其特征在于：所述除草剂为以所述CLALS蛋白作为靶标的除草剂。
  14. 如权利要求13所述CLALS蛋白、权利要求13所述的应用或权利要求13所述的方法，其特征在于：所述CLALS蛋白作为靶标的除草剂为Y1)或Y2)或Y3)或Y4)或Y5)：Y1)磺酰脲类除草剂；Y2)三唑嘧啶类除草剂；Y3)三唑啉酮类除草剂；Y4)嘧啶水杨酸类除草剂；Y5)咪唑啉酮类。
  15. 如权利要求14所述CLALS蛋白、权利要求14所述的应用或权利要求14所述的方法，其特征在于：
    所述磺酰脲类除草剂为苯磺隆、氯吡嘧磺隆、苄嘧磺隆、吡嘧磺隆、烟嘧磺隆、甲基二磺隆、噻吩磺隆或砜嘧磺隆；
    所述三唑嘧啶类除草剂为唑嘧磺草胺、五氟磺草胺、啶磺草胺或双氟磺草胺；
    所述三唑啉酮类除草剂为氟唑磺隆；
    所述嘧啶水杨酸类除草剂为双草醚；
    所述咪唑啉酮类为甲咪唑烟酸。
  16. CLALS蛋白自N末端起第190位的氨基酸残基的突变与CLALS蛋白中其它氨基酸残基的突变形成的双位点或多位点突变基因。
  17. CLALS蛋白自N末端起第190位的氨基酸残基的突变与CLALS蛋白中其它氨基酸残基的突变形成的双位点或多位点突变基因在调控西瓜除草剂抗性中的应用。
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