WO2023207669A1 - Procédé pour obtenir une protéine résistante au glufosinate et mutante de la glutamine synthétase - Google Patents

Procédé pour obtenir une protéine résistante au glufosinate et mutante de la glutamine synthétase Download PDF

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WO2023207669A1
WO2023207669A1 PCT/CN2023/088915 CN2023088915W WO2023207669A1 WO 2023207669 A1 WO2023207669 A1 WO 2023207669A1 CN 2023088915 W CN2023088915 W CN 2023088915W WO 2023207669 A1 WO2023207669 A1 WO 2023207669A1
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glutamine synthetase
mutant
wild
amino acid
glufosinate
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PCT/CN2023/088915
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Chinese (zh)
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陈容
邓龙群
卢远根
付颖钊
张震
侯青江
胥南飞
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四川天豫兴禾生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N15/8277—Phosphinotricin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)

Definitions

  • the present disclosure relates to the technical field of genetic engineering, and specifically, to a method for obtaining a protein with glufosinate resistance and a glutamine synthetase mutant.
  • Glutamine synthetase is a key enzyme in plant nitrogen metabolism. It catalyzes the condensation of glutamate (Glu) and NH 3 to form glutamine (Gln) in the glutamate synthetase cycle, and is involved in plant nitrogen metabolism. Metabolism of Nitrogen Compounds. According to distribution and subcellular localization, higher plant GS (GSII class) isoenzymes can be divided into two types: one is located in the cytoplasm, called cytoplasmic GS (GS1), with a molecular weight of 38-40kDa; the other is located in the cytoplasm. The chloroplast (or plastid) is called plastid type GS (GS2), with a molecular weight of 44-45kDa.
  • Glufosinate (glufosinate ammonium, trade name Basta) is a glutamine synthetase (GS1) inhibitor developed by Aventis (now Bayer). Its active ingredient is phosphinothricin (PPT for short), and its chemical name is It is (RS)-2-amino-4-(hydroxymethylphosphinyl)ammonium butyrate. The product was launched in 1986, and sales have increased year by year. The target enzyme of glufosinate is GS. Under normal circumstances, GS can form ⁇ -glutamyl phosphate from ATP and glutamate.
  • PPT first binds to ATP, and the phosphorylated PPT occupies 8 reaction centers of the GS molecule, causing the spatial configuration of GS to change, thereby inhibiting the activity of GS. PPT inhibits all known forms of GS.
  • Glufosinate-ammonium's inhibition of GS can lead to nitrogen metabolism disorders in plants, excessive accumulation of ammonium, and disintegration of chloroplasts, thereby inhibiting photosynthesis and ultimately leading to plant death.
  • the main method of cultivating glufosinate-resistant varieties is to use genetic engineering methods to introduce glufosinate-resistant genes from bacteria into crops, thereby cultivating new transgenic glufosinate-resistant crop varieties.
  • glufosinate-resistant genes in agriculture are the bar gene derived from the strain Streptomyces hygroscopicus and the pat gene derived from the strain S. viridochromogenes.
  • the bar gene and the pat gene have 80% homology, and both encode glufosinate acetylase, which can acetylate and inactivate glufosinate.
  • Glufosinate-resistant varieties are of great use value, and resistant rapeseed, corn, etc. have been commercially grown in large areas.
  • genetically modified crops are mainly limited to a few crops such as corn, soybeans, and cotton.
  • the bar gene and pat gene are derived from microorganisms, rather than from the crops themselves, which are more likely to cause consumer resistance.
  • the glufosinate acetylase encoded by the bar gene and the pat gene can acetylate and inactivate glufosinate.
  • overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate, its degree of tolerance is not sufficient for commercial application.
  • the present disclosure provides a method for obtaining a protein with glufosinate resistance and a glutamine synthetase mutant to solve the above technical problems.
  • the present disclosure provides a method for obtaining a protein with glufosinate resistance, including the following steps:
  • Glufosinate resistance-enhancing protein Glufosinate resistance-enhancing protein.
  • the protein is resistant to glufosinate and can maintain its own biological enzyme catalytic activity, thereby meeting the normal nitrogen metabolism of plants and maintaining normal growth and development of plants.
  • the plants or recombinant bacteria transformed into the glufosinate-resistant protein provided by the present disclosure can grow and develop normally in the presence of glufosinate.
  • the glufosinate-resistant protein mutant is not only used for the cultivation of transgenic crops. , can also be used to cultivate glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybeans, corn, wheat, rape, cotton and sorghum, etc., and has broad application prospects.
  • the above reference sequence is wild-type glutamine synthetase derived from rice.
  • the sequence comparison method can use the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for Protein Blast comparison; the same sequence comparison method or tool can also be obtained using other sequence comparison methods or tools well known in the art. result.
  • target proteins include, but are not limited to: proteins with sequences similar to those of natural plant proteins (such as artificially designed and synthesized), and wild-type glutamine synthetase derived from plants. By mutating the above amino acid residue positions, a protein with enhanced glufosinate resistance can be obtained.
  • Glufosinate in this disclosure, also known as glufosinate, refers to ammonium 2-amino-4-[hydroxy(methyl)phosphono]butyrate.
  • the present disclosure also provides a glutamine synthetase mutant with glufosinate resistance, which has at least one of the following amino acid sequences:
  • the amino acid sequence of the glutamine synthetase mutant is obtained by mutating the n-th position of the wild-type glutamine synthetase; the position of the n-th position is determined by the following method: comparing the wild-type glutamine synthetase with the reference sequence Yes, position n of wild-type glutamine synthetase corresponds to position 55 of the reference sequence;
  • the amino acid sequence of the glutamine synthetase mutant is at least 85% identical to the glutamine synthetase mutant shown in (1), and is identical to the glutamine synthetase mutant shown in (1)
  • the amino acid at the n-th position of the body is the same, and it is resistant to glufosinate ammonium;
  • the amino acid sequence of the glutamine synthetase mutant is obtained by mutating the m-th position of wild-type glutamine synthetase; the position of m-th position is determined by the following method: comparing the wild-type glutamine synthetase with the reference sequence Yes, position m of wild-type glutamine synthetase corresponds to position 64 of the reference sequence;
  • the amino acid sequence of the glutamine synthetase mutant is at least 85% identical to the glutamine synthetase mutant shown in (3), and is identical to the glutamine synthetase mutant shown in (3) They have the same amino acid at position m and are resistant to glufosinate ammonium;
  • the reference sequence is shown in SEQ ID NO.1.
  • the research disclosed in this disclosure found that the plant-derived wild-type glutamine synthetase was compared with the reference sequence, and the amino acid position corresponding to the 55th position of the reference sequence, that is, the nth position, was mutated to D, L or T; or mutate the amino acid position corresponding to position 64 of the reference sequence, i.e. position m, to Q, R, S, T, V, W, A, D, F, M, Y Or X, the obtained glutamine synthetase mutant has glufosinate resistance while maintaining its own biological enzyme catalytic activity.
  • the plants or recombinant bacteria transformed into the plant glutamine synthetase mutant provided by the present disclosure can grow and develop normally in the presence of glufosinate.
  • the plant glutamine synthetase mutant is not only used for the cultivation of transgenic crops, but also It can be used to cultivate glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybeans, corn, cotton and sorghum, etc., and has broad application prospects.
  • the above reference sequence is wild-type glutamine synthetase derived from rice.
  • the sequence comparison method can use the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for Protein Blast comparison; the same sequence comparison method or tool can also be obtained using other sequence comparison methods or tools well known in the art. result.
  • the n-th position of wild-type glutamine synthetase may also be the 55th position in its own sequence (such as corn, wheat, soybean, rape, etc.), but it may not be the 55th position.
  • the n-th position The position is determined based on the aforementioned sequence alignment. As long as it is aligned with the reference sequence, the site corresponding to position 55 of the reference sequence is the n-th position described in the present disclosure, that is, the mutation site.
  • the m-th position of wild-type glutamine synthetase may also be the 64th position in its own sequence (such as corn, wheat, soybean, rape, etc.), but it may not be the 64th position.
  • the m-th position The position is determined based on the aforementioned sequence alignment. As long as it is aligned with the reference sequence, the site corresponding to position 64 of the reference sequence is the m-th position described in the present disclosure, which is the mutation site.
  • the wild-type glutamine synthetases of all plants are homologous and have essentially the same functions and structural domains in plants. Therefore, the glutamine synthetase mutant obtained by making the above-mentioned mutation at position 55 or 64 of any plant-derived wild-type glutamine synthetase has glufosinate-ammonium resistance. Therefore, glutamine synthetase mutants obtained by carrying out the above-mentioned mutations on wild-type glutamine synthetase derived from any plant belong to the protection scope of the present disclosure.
  • glutamine synthetase mutant shown in (1) or (3) while maintaining the nth position as
  • the above-mentioned mutated amino acids, and the glutamine synthetase mutant obtained by further mutation has at least 85% (for example, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) or above, and its function Including that the enzyme catalytic activity and glufosinate-ammonium resistance are equivalent to, slightly decreased, slightly increased, or significantly increased compared to the glutamine synthetase mutant shown in (1) or (3). Therefore, such glutamine synthetases should also fall within the scope of the present disclosure.
  • X2 A, D, F, M, Q, W, Y or X, and X is deleted.
  • the rice wild-type glutamine synthetase is SEQ ID NO. 1:
  • the corn wild-type glutamine synthetase is SEQ ID NO. 2:
  • the soybean wild-type glutamine synthetase is SEQ ID NO. 3:
  • the oilseed rape wild-type glutamine synthetase is SEQ ID NO. 4:
  • Figure 1 shows the amino acid sequence comparison results of wild-type glutamine synthetases from different plants; in the figure: OsGS1-WT: rice wild-type glutamine synthetase; ZmGS1-WT: maize wild-type glutamine synthetase; GmGS1-WT: soybean wild-type glutamine synthetase; BnGS1-WT: rapeseed wild-type glutamine synthetase.
  • OsGS1-WT rice wild-type glutamine synthetase
  • ZmGS1-WT maize wild-type glutamine synthetase
  • GmGS1-WT soybean wild-type glutamine synthetase
  • BnGS1-WT rapeseed wild-type glutamine synthetase.
  • the arrows indicate the 55th and 64th amino acids respectively.
  • the comparison method of the above similarity (Similarity) and identity (Identity) is: input the amino acid sequence of a species into the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to perform Protein Blast Compare and find the similarity (Similarity) and identity (Identity) between this species and other species that need to be compared from the comparison results.
  • the present disclosure also provides a nucleic acid molecule encoding the glutamine synthetase mutant described above.
  • nucleic acid sequence encoding the above-mentioned glutamine synthetase mutant based on the degeneracy of codons.
  • corresponding nucleotide mutations can be made on the nucleic acid sequence encoding wild-type glutamine synthetase to obtain the nucleic acid sequence encoding the glutamine synthetase mutant. This is easily accomplished by those skilled in the art.
  • the coding nucleic acid sequence of rice wild-type glutamine synthetase is SEQ ID NO.5:
  • nucleotide mutations are carried out in the codon corresponding to the 55th or 64th position of the encoded amino acid sequence, and the rice glutamine synthetase mutant encoding the above can be obtained.
  • the coding nucleic acid sequence of corn wild-type glutamine synthetase is SEQ ID NO.6:
  • the coding nucleic acid sequence of soybean wild-type glutamine synthetase is SEQ ID NO.7:
  • the coding nucleic acid sequence of rapeseed wild-type glutamine synthetase is SEQ ID NO.8:
  • the present disclosure also provides an expression cassette or vector containing the above-mentioned nucleic acid molecule.
  • the above-mentioned expression cassette is connected with a regulatory sequence for regulating the expression of the above-mentioned nucleic acid molecule.
  • the present disclosure also provides a recombinant bacterium or recombinant cell containing the above-mentioned nucleic acid molecule, or expression cassette or vector.
  • the recombinant bacteria can be selected from Agrobacterium; the recombinant cells can be competent cells.
  • the present disclosure also provides a method for producing a plant tolerant to glufosinate ammonium herbicide, comprising introducing into the genome of the plant a gene encoding the above-mentioned glutamine synthetase mutant having glufosinate resistance.
  • the introduced method is selected from genetic transformation methods, genome editing methods or gene mutation methods.
  • the above genetic transformation methods include, but are not limited to: producing individuals with glufosinate resistance by self-crossing parent plants with genes for glutamine synthetase mutants that are resistant to glufosinate or by crossing with other plant individuals.
  • the above-mentioned transformation methods include but are not limited to Agrobacterium-mediated gene transformation method, gene gun transformation method, and pollen tube channel method.
  • Genome editing method or gene mutation method refers to those skilled in the art who can easily imagine that through conventional transgenic technology, gene editing technology (such as through zinc finger endonuclease (ZFN, zinc-finger nucleases) technology, transcription activator-like effect Target plants can be transformed using transcription activator-like effector nucleases (TALEN, transcription activator-like effector nucleases) technology or CRISPR/Cas9), mutation breeding technology (such as chemical, radiation mutagenesis, etc.) to encode glutamine synthetase as above Mutant genes, thereby obtaining new plant varieties that are resistant to glufosinate and can grow and develop normally. Therefore, no matter what technology is used, as long as it utilizes the glutamine synthetase mutant provided by the present disclosure to confer glufosinate resistance to plants, it falls within the scope of protection of the present disclosure.
  • ZFN zinc finger endonuclease
  • ZFN zinc finger endonuclease
  • ZFN zinc finger endonu
  • plants include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, sweet potato, potato, cotton, rape, sesame, peanut, sunflower, radish, carrot, cauliflower, tomato, eggplant , peppers, leeks, scallions, onions, leeks, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grapes, strawberries, sugar cane, tobacco, brassica vegetables, cucurbits, leguminous plants, pastures, tea or Cassava.
  • the pasture includes, but is not limited to, grasses of the grass family or leguminous grasses.
  • Gramine grasses include but are not limited to: lawn grass.
  • Brassica vegetables include, but are not limited to, turnips, cabbage, mustard greens, cabbage, collard greens, rapeseed, bitter mustard, blueberries, brassicas, greens or beets.
  • cucurbits include, but are not limited to, cucumbers, zucchini, pumpkins, winter melons, bitter melons, luffa, rapeseed melons, watermelons or melons.
  • leguminous plants include, but are not limited to, mung beans, broad beans, peas, lentils, soybeans, beans, cowpeas or edamame.
  • the present disclosure also provides the use of glutamine synthetase mutants, nucleic acid molecules, expression cassettes or vectors, or recombinant bacteria or recombinant cells in cultivating plant varieties with glufosinate resistance.
  • the above-mentioned applications include: mutagenesis and screening of plant cells, tissues, individuals or populations to encode glutamine synthetase mutants.
  • the mutagenesis is performed on plants using a non-lethal dose of physicochemical mutagenesis to obtain plant materials.
  • the above-mentioned non-lethal dose refers to controlling the dose within a range of 20% above or below the semi-lethal dose.
  • Physical and chemical mutagenesis methods include one or a combination of the following physical mutagenesis and chemical mutagenesis methods: physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, ⁇ -ray mutagenesis, ⁇ -ray mutagenesis, and ⁇ -ray mutagenesis.
  • mutagenesis high-energy particle mutagenesis, cosmic ray mutagenesis, microgravity mutagenesis
  • chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, Intercalated dye mutagenesis
  • alkylating agent mutagenesis includes ethyl methyl cyclonate mutagenesis, diethyl sulfate mutagenesis, and ethyleneimine mutagenesis.
  • the above application also includes: modifying the endogenous glutamine synthetase gene of the target plant to encode a glutamine synthetase mutant.
  • Modifying the endogenous glutamine synthetase gene of the target plant means that those skilled in the art can easily think of using conventional transgenic technology and gene editing technology in the field (such as zinc finger endonuclease (ZFN, zinc-finger nucleases) technology, Transcription activator-like effector nucleases (TALEN, transcription activator-like effector nucleases) technology or CRISPR/Cas9), mutation breeding technology (such as chemical, radiation mutagenesis, etc.) are used to transform the target plants so that they have the coding characteristics as above Glutamine synthetase mutant genes can be used to obtain new plant varieties that are resistant to glufosinate and can grow and develop normally. Therefore, no matter what technology is used, as long as it utilizes the glutamine synthetase mutant provided by the present disclosure to confer glufosinate resistance to plants, it falls within the scope of protection of the present disclosure.
  • ZFN zinc finger endonuclease
  • TALEN Tran
  • the above application includes at least one of the following application methods:
  • the isolated nucleic acid molecule is delivered into the target plant cell, and the isolated nucleic acid molecule contains a coding gene encoding a glutamine synthetase mutant;
  • the recombinant bacteria or recombinant cells are introduced into the target plant, and the recombinant bacteria or recombinant cells contain coding genes encoding glutamine synthetase mutants.
  • the isolated nucleic acid molecule can be a plasmid or a DNA fragment.
  • the isolated nucleic acid molecule can be delivered into the target plant cell through a gene bombardment method.
  • the glutamine synthetase mutants and glufosinate-resistant proteins provided by the present disclosure have application potential for constructing expression vectors for transformed plants and cultivating glufosinate-resistant crops.
  • the glutamine synthetase mutant provided by the present disclosure is originally derived from plants and is more easily accepted by consumers. After mutation, glufosinate-ammonium resistance is acquired. Plants transformed into the glutamine synthetase mutant not only have glufosinate-ammonium resistance suitable for commercial application, but can also maintain the normal enzymatic catalytic activity of glutamine synthetase. Can meet the normal growth and development of plants.
  • Figure 1 shows the amino acid sequence comparison results of wild-type glutamine synthetases from different plants; in the figure: OsGS1-WT: rice wild-type glutamine synthetase; ZmGS1-WT: maize wild-type glutamine synthetase; GmGS1-WT: soybean wild-type glutamine synthetase; BnGS1-WT: rapeseed wild-type glutamine synthetase;
  • Figure 2 is a partial alignment result of the amino acid sequences of rice GS1 mutants R55D, R55T, R55L and wild-type rice GS1OsGS1-WT provided in Example 1 of the present disclosure;
  • Figure 3 is a partial alignment result of the amino acid sequences of soybean GS1 mutant G55D and wild-type soybean GS1GmGS1-WT provided in Example 2 of the present disclosure;
  • Figure 4 is a partial alignment result of the amino acid sequences of the maize GS1 mutant Z55D and wild-type maize GS1ZmGS1-WT provided in Example 3 of the present disclosure;
  • Figure 5 is a partial alignment result of the amino acid sequences of rice GS1 mutants R64Q, R64R, R64S, R64T, R64V, R64W, R64X and wild-type rice GS1OsGS1-WT provided in Example 4 of the present disclosure;
  • Figure 6 is a partial alignment result of the amino acid sequences of soybean GS1 mutant G64X and wild-type soybean GS1GmGS1-WT provided in Example 5 of the present disclosure;
  • Figure 7 is a partial alignment result of the amino acid sequences of the maize GS1 mutant Z64X and wild-type maize GS1ZmGS1-WT provided in Example 6 of the present disclosure;
  • Figure 8 is a partial alignment result of the amino acid sequences of the rapeseed GS1 mutants B64A, B64D, B64F, B64M, B64Q, B64Y, B64W, B64X and wild-type rapeseed GS1BnGS1-WT provided in Example 7 of the present disclosure;
  • Figure 9 is a schematic structural diagram of the pADV7 vector provided in Experimental Example 1 of the present disclosure.
  • Figure 10 shows the growth results of Escherichia coli of the rice GS1 mutants R55D, R55T, R55L and wild-type rice GS1OsGS1-WT provided in Transformation Example 1 of the present disclosure on media containing different concentrations of glufosinate ammonium;
  • Figure 11 shows the growth results of E. coli of the soybean GS1 mutant G55D and wild-type soybean GS1GmGS1-WT provided in Transformation Example 2 of the present disclosure on media containing different concentrations of glufosinate ammonium;
  • Figure 12 shows the growth results of E. coli of the corn GS1 mutant Z55D and wild-type corn GS1ZmGS1-WT provided in Transformation Example 3 of the present disclosure on media containing different concentrations of glufosinate ammonium;
  • Figure 13 shows the enzymes of rice GS1 mutant R55D, soybean GS1 mutant G55D, corn GS1 mutant Z55D, wild-type rice GS1OsGS1-WT, wild-type soybean GS1GmGS1-WT and wild-type corn GS1ZmGS1-WT provided in Experimental Example 4 of the present disclosure.
  • Figure 14 shows the rice GS1 mutants R64Q, R64R, R64S, and R64T provided in Transformation Example 4 provided in Experimental Example 5 of the present disclosure. Growth results of R64V, R64W, R64X and wild-type rice GS1OsGS1-WT E. coli on media containing different concentrations of glufosinate;
  • Figure 15 shows the growth results of E. coli of the soybean GS1 mutant G64X and wild-type soybean GS1GmGS1-WT provided in Transformation Example 5 of Experimental Example 6 of the present disclosure on media containing different concentrations of glufosinate ammonium;
  • Figure 16 shows the growth results of the E. coli of the corn GS1 mutant Z64X and wild-type corn GS1ZmGS1-WT provided in Transformation Example 6 provided in Experimental Example 7 of the present disclosure on media containing different concentrations of glufosinate ammonium;
  • Figure 17 shows the transformation of the rapeseed GS1 mutants B64A, B64D, B64F, B64M, B64Q, B64Y, B64W, B64X and wild-type rapeseed GS1BnGS1-WT provided in Transformation Example 7 of the present disclosure in E. coli containing different concentrations of grass. Growth results on ammonium phosphine-based media;
  • Figure 18 shows the rice GS1 mutant R64X, the soybean GS1 mutant G64X, the corn GS1 mutant Z64X, the rape GS1 mutant B64X, the wild type rice GS1OsGS1-WT, the wild type soybean GS1GmGS1-WT, and the wild type provided in Experimental Example 9 of the present disclosure.
  • the rice (Oryza sativa) glutamine synthetase (GS1) mutant provided in this example is composed of wild-type rice glutamine synthetase itself (named OsGS1-WT, and the amino acid sequence is as shown in SEQ ID NO.1, The 55th amino acid residue S of the coding nucleotide sequence (SEQ ID NO. 5) was mutated to D, T, and L.
  • the obtained rice GS1 mutants were named R55D, R55T, and R55L respectively.
  • each rice GS1 mutant is at the position encoding the 55th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • the rice GS1 mutants R55D, R55T and R55L provided in this example and the nucleic acid molecules encoding them can be obtained through chemical synthesis methods.
  • the soybean (Glycine max) GS1 mutant provided in this example is composed of wild-type soybean GS1 itself ((named GmGS1-WT, the amino acid sequence is shown in SEQ ID NO. 3, and the encoding nucleotide sequence is SEQ ID NO. 7) Position 55 (corresponding to position 55 of the reference sequence (SEQ ID NO. 1)) was obtained by mutating the amino acid residue S to D.
  • the obtained soybean GS1 mutants were named G55D.
  • the coding sequence of each soybean GS1 mutant is at the position encoding the 55th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • soybean GS1 mutant G55D and the nucleic acid molecules encoding them provided in this example can be obtained through chemical synthesis methods.
  • the maize (Zea mays) GS1 mutant provided in this example is composed of wild-type maize GS1 itself (named ZmGS1-WT, the amino acid sequence is shown in SEQ ID NO.2, and the encoding nucleotide sequence is SEQ ID NO.6 ) (corresponding to position 55 of the reference sequence (SEQ ID NO. 1)) is obtained by mutating the amino acid residue S to D.
  • the obtained maize GS1 mutants were named Z55D.
  • the coding sequence of each maize GS1 mutant is at the position encoding the 55th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • the maize GS1 mutant Z55D and the nucleic acid molecules encoding them provided in this example can be obtained through chemical synthesis methods.
  • the rice (Oryza sativa) glutamine synthetase (GS1) mutant provided in this example is composed of wild-type rice glutamine synthetase itself (named OsGS1-WT, and the amino acid sequence is as shown in SEQ ID NO.1, The 64th amino acid residue P of the coding nucleotide sequence (SEQ ID NO.5) was mutated to Q, R, S, T, V, W or deleted (X). The resulting rice GS1 mutants were named R64Q. , R64R, R64S, R64T, R64V, R64W, R64X.
  • each rice GS1 mutant is at the position encoding the 64th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • the rice GS1 mutants R64Q, R64R, R64S, R64T, R64V, R64W, and R64X provided in this example and the nucleic acid molecules encoding them can be obtained through chemical synthesis methods.
  • the soybean (Glycine max) GS1 mutant provided in this example is composed of wild-type soybean GS1 itself ((named GmGS1-WT, the amino acid sequence is shown in SEQ ID NO. 3, and the encoding nucleotide sequence is SEQ ID NO. 7) (corresponding to position 64 of the reference sequence (SEQ ID NO.1)) is obtained by mutating the amino acid residue P to X.
  • the obtained soybean GS1 mutants were named G64X and wild-type soybean GS1GmGS1-WT .
  • the coding sequence of each soybean GS1 mutant is at the position encoding the 64th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • soybean GS1 mutant G64X and the nucleic acid molecules encoding them provided in this example can be obtained through chemical synthesis methods.
  • the maize (Zea mays) GS1 mutant provided in this example is composed of wild-type maize GS1 itself (named ZmGS1-WT, the amino acid sequence is shown in SEQ ID NO.2, and the encoding nucleotide sequence is SEQ ID NO.6 ) (corresponding to position 64 of the reference sequence (SEQ ID NO. 1)) is obtained by mutating the amino acid residue P to X.
  • the obtained maize GS1 mutant was named Z64X.
  • the coding sequence of the maize GS1 mutant is at the position encoding the 64th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • the maize GS1 mutant Z64X and the nucleic acid molecules encoding them provided in this example can be obtained by chemical synthesis.
  • the rapeseed (Brassica napus) GS1 mutant provided in this embodiment is composed of wild-type rapeseed GS1 itself (named BnGS1-WT, the amino acid sequence is shown in SEQ ID NO.4, and the encoding nucleotide sequence is SEQ ID NO.8 ) (corresponding to position 64 of the reference sequence (SEQ ID NO. 1)) is obtained by mutating the amino acid residue P to A, D, F, M, Q, Y, W, and X.
  • the obtained rapeseed GS1 mutants were named B64A, B64D, B64F, B64M, B64Q, B64Y, B64W, and B64X respectively.
  • each rapeseed GS1 mutant is at the position encoding the 64th amino acid.
  • the codons used for the corresponding amino acids are as shown in the table below.
  • the nucleotides at the remaining positions are the same as the corresponding wild-type coding sequence.
  • the rape GS1 mutants B64A, B64D, B64F, B64M, B64Q, B64Y, B64W and B64X provided in this example and the nucleic acid molecules encoding them can be obtained through chemical synthesis methods.
  • the glufosinate ammonium resistance of the rice GS1 mutants R55D, R55T and R55L provided in Example 1 was detected as follows:
  • the coding genes encoding the rice GS1 mutants R55D, R55T and R55L were synthesized using chemical synthesis methods. Enzyme cleavage sites (Pac1 and Sbf1) were introduced at both ends. After enzyme cleavage, the genes were connected. Under the action of enzyme, it is connected to an expression vector (such as pADV7 vector, whose structure is shown in Figure 9) that has been digested by the same enzyme, and then transformed into glutamine synthetase-deficient E. coli respectively. After verification, the positive ones are picked Clone, inoculate to M9 medium containing different concentrations of glufosinate, and observe the growth of defective E.
  • an expression vector such as pADV7 vector, whose structure is shown in Figure 9
  • the wild-type rice GS1 mutant was used as a negative control to detect the glufosinate resistance of the GS1 mutants R55D (S55D, the amino acid S at position 55 of rice GS1 was mutated to D), R55T (S55T) and R55L (S55L). The results are shown in Figure 10.
  • the E. coli transformed into wild-type soybean GS1 basically cannot grow, but the growth of E. coli transformed into soybean mutant G55D is significantly better than the negative control, indicating that the single mutant containing G55D The ability to resist glufosinate is significantly better than that of the wild type.
  • the defective strains transformed into genes encoding wild-type maize GS1 ZmGS1-WT
  • the maize GS1 mutant Z55D can grow normally, indicating that GS1 encoded by Z55D is Has normal GS1 enzyme activity;
  • the E. coli transformed into wild-type corn GS1 basically cannot grow, but the growth of E. coli transformed into the corn mutant Z55D is significantly better than the negative control, indicating that the single mutation containing Z55D
  • the ability to resist glufosinate is significantly better than that of the wild type; on the medium with a higher glufosinate concentration (10mM, KP10), the E. coli transformed into the corn GS1 mutant Z55D still grows significantly.
  • the nucleic acid sequence encoding the above mutant was cloned into the prokaryotic expression vector pET32a, and the clone was verified by sequencing.
  • the mutant enzyme protein was purified by 6His and standard methods, and the concentration was determined using a Bradford protein concentration assay kit. The protein was stored in a protein stock solution.
  • the components of the reaction solution for determining glutamine synthetase activity are: 100mM Tris-HCl (pH7.5), 5mM ATP, 10mM sodium L-glutamate, 30mM hydroxylamine, and 20mM MgCl 2 .
  • the Km values of the GS1 mutant are slightly higher, indicating that the GS mutant has a slightly lower sensitivity to glufosinate inhibitors. sensitivity to normal substrates.
  • the V max of the GS1 mutants were all lower than the wild-type control, indicating that the enzyme catalytic ability of these mutants was reduced.
  • the wild-type control is very sensitive to glufosinate, with IC 50s of 0.006mM, 0.005mM and 0.006mM respectively. The IC 50s of the mutants are significantly higher than the wild-type control.
  • the IC 50s of R55D, G55D and Z55D are much higher than the wild-type. control, indicating that the mutant is less sensitive to glufosinate. It can also be seen from the multiple relationship between the IC 50 of the mutant and the IC 50 of the wild type that the IC 50 of R55D, G55D and Z55D are 441.667 times, 2567.8 times and 75066.667 times that of the corresponding wild type GS1 IC 50 respectively. These values also show that the mutants The enzyme activity was much higher than that of the wild-type control. These data illustrate the glufosinate-resistant mechanism of the mutant from the enzyme kinetics.
  • E. coli transformed into wild-type rice GS1 could not grow, but the transformed
  • the growth of E. coli of rice mutants R64Q, R64R, R64S, R64T, R64V, R64W and R64X was significantly better than that of the negative control, indicating that the single mutants containing R64Q, R64R, R64S, R64T, R64V, R64W and R64X are resistant to glufosinate.
  • the ability is significantly better than that of the wild type.
  • the E. coli transformed into wild-type corn GS1 basically cannot grow, but the growth of E. coli transformed into the corn mutant Z64X is significantly better than the negative control, indicating that the single mutation containing Z64X The ability to resist glufosinate is significantly better than that of the wild type; on the medium with high glufosinate concentration (5mM, KP5), the E. coli transformed into the corn GS1 mutant Z64X still grows significantly.
  • E. coli transformed into wild-type rapeseed GS1 basically cannot grow, but the large intestine transformed into rapeseed mutants B64A, B64D, B64F, B64M, B64Q, B64Y, B64W, and B64X
  • the growth of the bacilli was significantly better than that of the negative control, indicating that the single mutants containing B64A, B64D, B64F, B64M, B64Q, B64Y, B64W, and B64X were significantly better than the wild type in resisting glufosinate; at glufosinate concentration (5mM, KP5 ) on the culture medium, the E. coli transformed into the rapeseed GS1 mutants B64W and B64X still grew significantly.
  • the Km values of the GS1 mutant are slightly higher, indicating that the GS mutant reduces the sensitivity to glufosinate ammonium inhibitors. At the same time, the sensitivity to normal substrates is slightly reduced.
  • the V max of both rice and soybean GS1 mutants was higher than that of the wild-type control, indicating that the enzyme catalytic ability of these mutants was improved.
  • the wild-type control is very sensitive to glufosinate, with IC 50s of 0.006mM, 0.005mM, 0.006mM and 0.007mM respectively.
  • the IC 50s of the mutants are significantly higher than those of the wild-type control, and the IC 50s of R64X, G64X, Z64X and B64X are much higher. Much higher than the wild-type control, indicating that the mutant is less sensitive to glufosinate. It can also be seen from the multiple relationship between the IC 50 of the mutant and the IC 50 of the wild type that the IC 50 of R64X, G64X, Z64X and B64X are 2.167 times, 4.6 times, 10.167 times and 2.714 times that of the corresponding wild type GS1 IC 50 respectively. These The values also indicate that the enzyme activity of the mutant is higher than that of the wild-type control. These data illustrate the glufosinate-resistant mechanism of the mutant from the enzyme kinetics.

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Abstract

La présente invention se rapporte au domaine technique du génie génétique et, plus particulièrement, à un mutant de glutamine synthétase présentant une résistance au glufosinate. Selon la présente invention, des mutations sont induites aux positions 55 et 64 de la séquence d'acides aminés d'une glutamine synthétase de type sauvage, et les mutants de glutamine synthétase présentant une résistance accrue au glufosinate sont sélectionnés. Les plantes transformées avec le mutant présentent une résistance au glufosinate adaptée aux applications commerciales tout en conservant l'activité enzymatique normale de la glutamine synthétase, ce qui répond à l'exigence d'une croissance et d'un développement normaux des plantes.
PCT/CN2023/088915 2022-04-25 2023-04-18 Procédé pour obtenir une protéine résistante au glufosinate et mutante de la glutamine synthétase WO2023207669A1 (fr)

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