WO2023040564A1 - Mutant de glutamine synthétase et son application dans la sélection de variétés végétales résistantes au glufosinate-ammonium - Google Patents

Mutant de glutamine synthétase et son application dans la sélection de variétés végétales résistantes au glufosinate-ammonium Download PDF

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WO2023040564A1
WO2023040564A1 PCT/CN2022/113146 CN2022113146W WO2023040564A1 WO 2023040564 A1 WO2023040564 A1 WO 2023040564A1 CN 2022113146 W CN2022113146 W CN 2022113146W WO 2023040564 A1 WO2023040564 A1 WO 2023040564A1
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glutamine synthetase
plant
glufosinate
wild
mutant
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Chinese (zh)
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陈容
侯青江
邓龙群
张震
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
    • C12N15/79—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 disclosure relates to the technical field of genetic engineering, in particular to a glutamine synthetase mutant and its application in breeding glufosinate-resistant plant varieties.
  • Glufosinate ammonium (glufosinate ammonium, trade name Basta) is a glutamine synthetase (GS1) inhibitor developed by Bayer. Its active ingredient is phosphinothricin (abbreviated as PPT), and its chemical name is (RS)-2-amino-4 - Ammonium (hydroxymethylphosphinyl)butyrate.
  • PPT glutamine synthetase
  • RS -2-amino-4 - Ammonium (hydroxymethylphosphinyl)butyrate.
  • glufosinate-resistant plants are mainly obtained through transgenic technology to make target plants express glufosinate-ammonium acetylase, which can acetylate glufosinate-ammonium and inactivate it.
  • glufosinate-ammonium acetylase can acetylate glufosinate-ammonium and inactivate it.
  • glutamine synthetase it is difficult for glufosinate-ammonium acetylase to completely inactivate glufosinate-ammonium. Inhibits the activity of glutamine synthetase on the cell membrane, thereby interfering with nitrogen metabolism in plants.
  • glufosinate-ammonium when glufosinate-ammonium is applied to crops with bar gene and pat gene, it will interfere with the nitrogen metabolism of plants to varying degrees, and at the same time affect the normal growth and development of plants.
  • the sensitivity of transgenic plants to glufosinate can be reduced to a certain extent by overexpressing wild-type glutamine synthetase in plants, the degree of tolerance to glufosinate is far from enough for commercial application.
  • the present disclosure provides a glutamine synthetase mutant, which has glufosinate-ammonium resistance, as shown in (1) or (2):
  • (1) It is obtained by mutating the n-th amino acid of the wild-type glutamine synthetase derived from plants; the position of the n-th position is determined by the following method: comparing the wild-type glutamine synthetase with the reference sequence, wild-type glutamine synthetase The nth position of the type glutamine synthetase corresponds to the 69th position of the reference sequence, wherein the amino acid sequence of the reference sequence is as shown in SEQ ID NO.1;
  • the n-th amino acid of the glutamine synthetase mutant is X, and X includes A, G, M, N, Q, S, T, V or deletion;
  • the inventors found that by comparing the plant-derived wild-type glutamine synthetase with the reference sequence, the amino acid site corresponding to the 69th position of the reference sequence, i.e. the nth position, was mutated into A, G, M, N, Q, S, T, V or deleted, the resulting glutamine synthetase mutant has glufosinate-ammonium resistance, and can maintain its own biological enzyme catalytic activity, so as to meet the normal nitrogen metabolism of plants and maintain The normal growth and development of plants.
  • Plants or recombinant bacteria transformed with the plant glutamine synthetase mutant provided by the present disclosure can grow and develop normally under the conditions of the presence of glufosinate-ammonium.
  • the plant glutamine synthetase mutant is not only used for the cultivation of transgenic crops, but also It can be applied to the cultivation of glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybean, corn, wheat, rape, cotton and sorghum, and has broad application prospects.
  • the above reference sequence is the wild-type glutamine synthetase derived from rice.
  • the sequence alignment method can use the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to perform Protein Blast alignment; other sequence alignment methods or tools well known in the art can also be used to obtain the same result.
  • the nth position of the wild-type glutamine synthetase may also be the 69th position in its own sequence (such as corn, wheat, soybean, rapeseed, etc.), but it may not be the 69th position (for example, peanut corresponds to The 70th position), the specific position of the nth position is determined according to the aforementioned sequence comparison, as long as it is compared with the reference sequence, the position corresponding to the 69th position of the reference sequence is the nth position of the present disclosure, that is, mutation site.
  • the wild-type glutamine synthetases of all plants have homology, and have basically the same functions and structural domains in plants. Therefore, any plant-derived wild-type glutamine synthetase mutants obtained by making the above mutation at position 69 all have glufosinate-ammonium resistance. Therefore, the mutants of glutamine synthetase obtained by performing 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) has at least 85% (such as 85%, 86%, 87%, 88%, 89%) of the glutamine synthetase mutant shown in (1) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) or more identity, and its functions include enzyme catalytic activity and glufosinate-ammonium resistance It is equivalent to or slightly decreased or slightly increased or significantly increased with the glutamine synthetase mutant shown in (1). Therefore, such glutamine synthetase should also belong to the protection scope of the present disclosure.
  • the above-mentioned plants include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, sweet potato, potato, cotton, rapeseed, sesame, peanut, sunflower, radish , carrot, cauliflower, tomato, eggplant, pepper, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, sugar cane, tobacco, brassica vegetables, cucurbits, Legumes, pastures, tea or cassava.
  • the above-mentioned pastures include but not limited to grasses or leguminous pastures.
  • the gramineous herbage is selected from Timothy, Dactylis, Junegrass, fine wheat, fescue, palm leaf, foxtail, etc.; the leguminous forage is selected from alfalfa, clover, three-leaf bean, nest vegetable, corngrass, etc.
  • the pasture grasses mentioned above can also be selected from lawn grasses.
  • the Brassica vegetables include, but are not limited to, turnips, cabbage, mustard greens, cabbage, kale, cabbage, bitter mustard, bluegrass, brassica, green vegetables or sugar beets.
  • the above-mentioned Cucurbitaceae plants include, but are not limited to, cucumber, zucchini, pumpkin, wax gourd, bitter gourd, loofah, snake gourd, watermelon or muskmelon.
  • leguminous plants include but are not limited to mung bean, broad bean, pea, lentil, soybean, kidney bean, cowpea or edamame.
  • the inventors also found that for different plant-derived glutamine synthetases, in addition to mutating the nth position to A, G, M, N, Q, S, T, In addition to V or deletion, mutating its n-th position to other amino acids will also make glutamine synthetase resistant to glufosinate-ammonium.
  • X A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S , T, V, Y or delete.
  • X A, G, L, M, N, Q, S, T, V or deletion.
  • X A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y or deletion.
  • X A, C, D, F, G, H, I, M, N, Q, R, S, T, V, Y or deletion.
  • X A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y or deletion.
  • 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 wheat wild-type glutamine synthetase is SEQ ID NO.4:
  • the rapeseed wild-type glutamine synthetase is SEQ ID NO.5:
  • the comparison method of the above similarity (Similarity) and identity (Identity) is: input the amino acid sequence of a species to the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for Protein Blast Compare, 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 an isolated nucleic acid molecule encoding the above-mentioned glutamine synthetase mutant.
  • nucleic acid sequence encoding the above glutamine synthetase mutant According to 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 described above. This is readily accomplished by those skilled in the art.
  • the coding nucleotide sequence of rice wild-type glutamine synthetase is SEQ ID NO.6:
  • the corresponding nucleotide mutation is carried out at the codon corresponding to the 69th 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.7:
  • the coding nucleic acid sequence of soybean wild-type glutamine synthetase is SEQ ID NO.8:
  • the coding nucleic acid sequence of soybean wild-type glutamine synthetase can also refer to NCBI accession number: NM_001255403.3.
  • the coding nucleic acid sequence of wheat wild-type glutamine synthetase is SEQ ID NO.9:
  • the coding nucleic acid sequence of rapeseed wild-type glutamine synthetase is SEQ ID NO.10:
  • the present disclosure also provides a vector containing the above-mentioned nucleic acid molecule.
  • the present disclosure also provides a recombinant bacterium or a recombinant cell, which contains the above-mentioned nucleic acid molecule or the above-mentioned vector.
  • the recombinant bacteria can be selected from Agrobacterium; the recombinant cells can be competent cells.
  • the present disclosure also provides the application of the above glutamine synthetase mutants, nucleic acid molecules, vectors, recombinant bacteria or recombinant cells with glufosinate-ammonium resistance in cultivating plant varieties with glufosinate-ammonium resistance.
  • the above application includes at least one of the following application methods:
  • the isolated nucleic acid molecule contains the coding gene encoding the glutamine synthetase mutant
  • the vector contains the coding gene encoding the glutamine synthetase mutant
  • the recombinant bacterium or the recombinant cell is introduced into the target plant, and the recombinant bacterium or the recombinant cell contains the coding gene encoding the glutamine synthetase mutant.
  • the isolated nucleic acid molecule can be a plasmid or a DNA fragment, and in an alternative embodiment, the isolated nucleic acid molecule can be delivered into the target plant cell by gene gun method.
  • Transformation methods include, but are not limited to, Agrobacterium-mediated gene transformation, biolistic transformation, and pollen tube passage.
  • Recombinant bacteria or recombinant cells can be introduced into the target plant through infection.
  • the above application includes: modifying the endogenous glutamine synthetase gene of the target plant to encode a glutamine synthetase mutant.
  • glutamine synthetase mutants provided in the present disclosure
  • ZFN zinc finger endonuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas9 transcription activator-like effector nucleases
  • mutation breeding technology such as chemical, radiation mutagenesis, etc.
  • the above application includes: performing mutagenesis and screening on plant cells, tissues, individuals or groups to encode glutamine synthetase mutants.
  • the mutagenesis of the plant is carried out in a non-lethal dose of physicochemical mutagenesis to obtain plant material.
  • the above-mentioned non-lethal dose refers to controlling the dose within the range of 20% above and below the half-lethal dose.
  • Physical and chemical mutagenesis methods include one or more of the following physical and chemical mutagenesis methods: Physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, gamma-ray mutagenesis, beta-ray mutagenesis, alpha-ray mutagenesis mutagenesis, energetic particle mutagenesis, cosmic ray mutagenesis, microgravity mutagenesis; chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, Intercalating dye mutagenesis; alkylating agent mutagenesis includes ethyl methylcycloate mutagenesis, diethyl sulfate mutagenesis, and ethyleneimine mutagenesis.
  • Vegetables include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, sweet potato, potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, cauliflower, tomato, eggplant, pepper, leek , scallions, onions, leeks, spinach, celery, amaranth, lettuce, crown chrysanthemum, day lily, grapes, strawberries, sugar cane, tobacco, brassica vegetables, cucurbits, legumes, grasses, tea or cassava.
  • the forage includes but is not limited to gramineous forage or leguminous forage.
  • Brassica vegetables include, but are not limited to, turnips, cabbage, mustard greens, cabbage, kale, cabbage, bitter mustard, bluegrass, Brassica, greens or sugar beets.
  • the Cucurbitaceae plant includes, but is not limited to, cucumber, zucchini, pumpkin, wax gourd, bitter gourd, loofah, snake gourd, watermelon or muskmelon.
  • legumes include, but are not limited to, mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or edamame.
  • the glutamine synthetase mutant provided by the present disclosure has application potential for constructing expression vectors for transformed plants and cultivating glufosinate-resistant crops.
  • the glutamine synthetase mutants provided by the present disclosure are originally derived from plants and are more easily accepted by consumers. After the mutation, it has glufosinate-ammonium resistance, and the plant transformed with the glutamine synthetase mutant not only has glufosinate-ammonium resistance suitable for commercial application, but also can maintain the normal enzymatic activity of glutamine synthetase, Can meet the normal growth and development of plants.
  • Fig. 1 is the rice GS1 mutants OE69A, OE69C, OE69D, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M, OE69N, OE69P, OE69Q, OE69R, OE69S, OE69T, OE69V, OE69Y and Partial alignment of amino acid sequences between OE69X and wild-type rice GS1OWT;
  • Figure 2 is the result of partial alignment of the amino acid sequences of soybean GS1 mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X and wild-type soybean GS1GWT provided in Example 2 of the present disclosure;
  • 3 is the corn GS1 mutants ZE69A, ZE69C, ZE69D, ZE69F, ZE69G, ZE69H, ZE69I, ZE69K, ZE69L, ZE69M, ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, ZE69T, ZE69V, ZE69Y and Partial alignment results of the amino acid sequences of ZE69X and wild-type maize GS1ZWT;
  • Figure 4 shows the wheat GS1 mutants TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R, TE69S, TE69T, TE69V, TE69Y and TE69X and wild-type wheat GS1TWT provided by Example 4 of the present disclosure
  • Figure 5 shows the rapeseed GS1 mutants BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L, BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, BE69V, BE69W, BE69Y and Partial alignment results of amino acid sequences of BE69X and wild-type rapeseed GS1BWT;
  • FIG. 6 is a schematic structural diagram of the pADV7 vector provided in Experimental Example 1 of the present disclosure.
  • Figure 7 shows the rice GS1 mutants OE69A, OE69C, OE69D, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M, OE69N, OE69P, OE69Q, OE69R, OE69S, The growth results of Escherichia coli of OE69T, OE69V, OE69Y and OE69X and wild-type rice GS1OWT on media containing different concentrations of glufosinate-ammonium;
  • Fig. 8 is Escherichia coli containing different soybean GS1 mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X and wild-type soybean GS1GWT provided by Experimental Example 2 of the present disclosure.
  • the growth result on the medium of concentration glufosinate-ammonium;
  • Fig. 9 shows the maize GS1 mutants ZE69A, ZE69C, ZE69D, ZE69F, ZE69G, ZE69H, ZE69I, ZE69K, ZE69L, ZE69M, ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, Growth results of Escherichia coli of ZE69T, ZE69V, ZE69Y and ZE69X and wild-type maize GS1ZWT on media containing different concentrations of glufosinate-ammonium;
  • Fig. 10 is the wheat GS1 mutants TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R, TE69S, TE69T, TE69V, TE69Y and Growth results of Escherichia coli of TE69X and wild-type wheat GS1TWT on media containing different concentrations of glufosinate-ammonium;
  • Fig. 11 shows the rapeseed GS1 mutants BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L, BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, and Growth results of Escherichia coli BE69V, BE69W, BE69Y and BE69X and wild-type rapeseed GS1BWT on media containing different concentrations of glufosinate-ammonium;
  • Figure 12 shows the rice GS1 mutant OE69M, soybean GS1 mutant GE69M, corn GS1 mutant ZE69M, wheat GS1 mutant TE69M, rapeseed GS1 mutant BE69M, wild-type rice GS1OWT, wild-type soybean GS1GWT, The glufosinate-ammonium resistance parameter IC 50 of wild-type maize GS1ZWT, wild-type wheat GS1TWT and wild-type rape GS1BWT;
  • Figure 13 is the amino acid sequence alignment result of wild-type glutamine synthetase in different plants; in the figure: TWT: wheat wild-type glutamine synthetase; OWT: rice wild-type glutamine synthase; ZWT: corn wild GWT: soybean wild-type glutamine synthetase; BWT: rapeseed wild-type glutamine synthase.
  • the rice (Oryza sativa) glutamine synthetase (GS1) mutant provided by the present embodiment is composed of wild-type rice glutamine synthetase itself (named OWT, the amino acid sequence is as shown in SEQ ID NO.1, and the encoding nucleus
  • the nucleotide sequence is that the 69th amino acid residue E of SEQ ID NO.6) is mutated into A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y or deletion
  • the obtained rice GS1 mutants were named OE69A, OE69C, OE69D, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M, OE69N, OE69P, OE69Q, OE69R, OE69S, OE69T, OE69V, OE69Y, and OE69X.
  • each rice GS1 mutant is at the position encoding the 69th amino acid, and the codons used for the corresponding amino acid are shown in the table below, and the nucleotides at other positions are the same as the corresponding wild-type coding sequence.
  • the soybean (Glycine max) GS1 mutant provided by the present embodiment is composed of the wild-type soybean GS1 itself (named GWT, the amino acid sequence is as shown in SEQ ID NO.3, and the encoding nucleotide sequence is SEQ ID NO.8).
  • the 69th position (corresponding to the 69th position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of the amino acid residue E to A, G, L, M, N, Q, S, T, V or deletion.
  • the obtained soybean GS1 mutants were named GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X, respectively.
  • soybean GS1 mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X and wild-type soybean GS1GWT is shown in Figure 2, in the figure: the position indicated by the arrow is the mutation position point.
  • the coding sequence of the soybean GS1 mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X provided in this example corresponds to SEQ ID NO.3.
  • each soybean GS1 mutant is at the position encoding the 69th amino acid, and the codons used for the corresponding amino acid are shown in the table below, and the nucleotides at other positions are the same as the corresponding wild-type coding sequence.
  • soybean GS1 mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X provided in this example and nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the corn (Zea mays) GS1 mutant provided by the present embodiment is composed of the wild-type corn GS1 itself (named ZWT, the amino acid sequence is as shown in SEQ ID NO.2, and the encoding nucleotide sequence is SEQ ID NO.7).
  • the 69th position (corresponding to the 69th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue E to A, C, D, F, G, H, I, K, L, M, N, P , Q, R, S, T, V, Y or deleted.
  • the maize GS1 mutants obtained were named ZE69A, ZE69C, ZE69D, ZE69F, ZE69G, ZE69H, ZE69I, ZE69K, ZE69L, ZE69M, ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, ZE69T, ZE69V, ZE69Y and ZE69X.
  • each maize GS1 mutant is at the position encoding the 69th amino acid, and the codons used for the corresponding amino acid are shown in the table below, and the nucleotides at other positions are the same as the corresponding wild-type coding sequence.
  • Maize GS1 mutants ZE69A, ZE69C, ZE69D, ZE69F, ZE69G, ZE69H, ZE69I, ZE69K, ZE69L, ZE69M, ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, ZE69T, ZE69V, ZE69Y and ZE69X provided in this example and the codes encoding them Nucleic acid molecules can be obtained by chemical synthesis.
  • the wheat (Triticum aestivum) GS1 mutant provided by the present embodiment is composed of the wild-type wheat GS1 itself (named TWT, the amino acid sequence is shown in SEQ ID NO.4, and the encoded nucleotide sequence is SEQ ID NO.9).
  • the 69th position (corresponding to the 69th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue E to A, C, D, F, G, H, I, M, N, Q, R, S , T, V, Y or delete to get.
  • the obtained wheat GS1 mutants were named as TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R, TE69S, TE69T, TE69V, TE69Y and TE69X.
  • each wheat GS1 mutant is at the position encoding the 69th amino acid, and the codons used for the corresponding amino acid are shown in the table below, and the nucleotides at other positions are the same as the corresponding wild-type coding sequence.
  • the wheat GS1 mutants TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R, TE69S, TE69T, TE69V, TE69Y and TE69X and the nucleic acid molecules encoding them can all be chemically Obtained by synthetic method.
  • the rape (Brassica napus) GS1 mutant provided by the present embodiment is composed of the wild-type rape GS1 (named BWT, the amino acid sequence is as shown in SEQ ID NO.5, and the encoding nucleotide sequence is SEQ ID NO.10)
  • the 69th position (corresponding to the 69th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue E to A, C, D, F, G, H, I, K, L, M, N, Q , R, S, T, V, W, Y or deleted.
  • the obtained rapeseed GS1 mutants were named BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L, BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, BE69V, BE69W, BE69Y and BE69X.
  • each rapeseed GS1 mutant is at the position encoding the 69th amino acid, and the codons used for the corresponding amino acid are shown in the table below, and the nucleotides at other positions are the same as the corresponding wild-type coding sequence.
  • Rapeseed GS1 mutants BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L, BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, BE69V, BE69W, BE69Y and BE69X provided in this embodiment and the codes encoding them Nucleic acid molecules can be obtained by chemical synthesis.
  • the rice GS1 mutants OE69A, OE69C, OE69D, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M, OE69N, OE69P, OE69Q, OE69R, OE69Q, OE69R, Encoding genes of OE69S, OE69T, OE69V, OE69Y, and OE69X were introduced with restriction sites (Pac1 and Sbf1) at both ends.
  • Escherichia coli transformed with wild-type rice GS1OWT could not grow, but transformed rice mutants OE69A, OE69C, OE69D, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M , OE69N, OE69P, OE69Q, OE69R, OE69S, OE69T, OE69V, OE69Y and OE69X Escherichia coli grew significantly better than the negative control, indicating that the E.
  • the single mutants of OE69N, OE69P, OE69Q, OE69R, OE69S, OE69T, OE69V, OE69Y and OE69X have significantly better resistance to glufosinate-ammonium than wild type;
  • the Escherichia coli transformed with rice GS1 mutants OE69A, OE69C, OE69F, OE69G, OE69H, OE69I, OE69K, OE69L, OE69M, OE69N, OE69Q, OE69R, OE69S, OE69T, OE69Y and OE69X still had significant growth.
  • the Escherichia coli transformed with wild-type soybean GS1 basically could not grow, but transformed soybean mutants GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X Escherichia coli grew significantly better than the negative control, indicating that the single mutants containing GE69A, GE69G, GE69L, GE69M, GE69N, GE69Q, GE69S, GE69T, GE69V and GE69X had significantly better resistance to glufosinate-ammonium than the wild type; On the medium with higher glufosinate concentration (10 mM, KP10), Escherichia coli transformed with soybean GS1 mutants GE69M and GE69T still grew significantly.
  • E. coli transformed with wild-type maize GS1ZWT could not grow, but transformed with maize mutants ZE69A, ZE69C, ZE69D, ZE69F, ZE69G, ZE69H, ZE69I, ZE69K, ZE69L, ZE69M , ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, ZE69T, ZE69V, ZE69Y and ZE69X grew significantly better than the negative control, indicating that the E.
  • the single mutants of ZE69N, ZE69P, ZE69Q, ZE69R, ZE69S, ZE69T, ZE69V, ZE69Y and ZE69X have significantly better resistance to glufosinate-ammonium than wild type; on the medium with higher glufosinate-ammonium concentration (20mM, KP20), Escherichia coli transformed with maize GS1 mutants ZE69K, ZE69L, ZE69M, ZE69N, ZE69R, ZE69S, and ZE69T still grew significantly.
  • transformation coding wild-type wheat GS1 TWT
  • wheat GS1 mutants TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R
  • the defective strains of the coding genes of TE69S, TE69T, TE69V, TE69Y and TE69X could all grow normally, indicating that TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N, TE69Q, TE69R, TE69S, TE69T, TE69V , GS1 encoded by TE69Y and TE69X all have normal GS1 enzyme activity;
  • Escherichia coli transformed with wild-type wheat GS1TWT basically could not grow, but transformed wheat mutants TE69A, TE69C, TE69D, TE69F, TE69G, TE69H, TE69I, TE69M, TE69N , TE69Q, TE69R, TE69S, TE69T, TE69V, TE69Y and TE69X Escherichia coli grew significantly better than the negative control, indicating that the E.
  • the single mutants of TE69T, TE69V, TE69Y and TE69X have significantly better resistance to glufosinate-ammonium than the wild type; on the medium with higher glufosinate-ammonium concentration (20mM, KP20), the wheat GS1 mutants TE69M, TE69N, TE69Q were transformed , TE69R, TE69S, TE69Y and TE69X Escherichia coli still had obvious growth.
  • Escherichia coli transformed with wild-type rapeseed GS1 basically could not grow, but transformed with rapeseed mutants BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L , BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, BE69V, BE69W, BE69Y, and BE69X grew significantly better than the negative control, indicating that the cells containing BE69A, BE69C, BE69D, BE69F, BE69G, BE69H, BE69I, BE69K, BE69L, The ability of the single mutants of BE69M, BE69N, BE69Q, BE69R, BE69S, BE69T, BE69V, BE69W, BE69Y and BE69X to resist glufosinate-ammonium was significantly better than that of the wild
  • the OE69M provided by Detecting Example 1 the GE69M provided by Example 2, the ZE69M provided by Example 3, the TE69M provided by Example 4 and the BE69M mutant provided by Example 5 have enzyme kinetic parameters when there is glufosinate-ammonium, to Wild-type rice GS1OWT, wild-type soybean GS1GWT, wild-type corn GS1ZWT, wild-type wheat GS1TWT and wild-type rapeseed GS1BWT were used as controls, and the method was as follows:
  • nucleic acid sequences encoding the above mutants were cloned into the prokaryotic expression vector pET32a, and the clones were verified by sequencing.
  • the mutant enzyme protein was purified by 6His and standard method, and the concentration was determined by Bradford method protein concentration assay kit, and the protein was stored in protein storage solution.
  • the components of the reaction solution for the determination of glutamine synthetase activity are: 100mM Tris-HCl (pH7.5), 5mM ATP, 10mM L-sodium glutamate, 30mM hydroxylamine, 20mM MgCl 2 .
  • 100 ⁇ l of the reaction solution and preheating at 35°C for 5 minutes add 1 ⁇ l of the mutant protein solution (protein concentration: 200ug/ml) to start the reaction.
  • the reaction termination solution 55g/L FeCl 3 (2, 20g/L trichloroacetic acid, 2.1% concentrated hydrochloric acid
  • Centrifuge at 5000 ⁇ g for 10 min, and take 200 ⁇ l to measure the light absorbance at 500 nm.
  • the wild-type controls OWT, GWT, ZWT, TWT, and BWT were very sensitive to glufosinate-ammonium, with IC50 of 7.93 ⁇ M, 13.55 ⁇ M, 8.92 ⁇ M, 7.22 ⁇ M and 1.53 ⁇ M, respectively, and the mutants OE69M, GE69M, ZE69M, TE69M, BE69M IC 50 were much higher than the wild-type control, indicating that the mutants were less sensitive to glufosinate-ammonium.

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Abstract

L'invention concerne un mutant de glutamine synthétase et son application dans la sélection de variétés végétales résistantes au glufosinate-ammonium. Le mutant de glutamine synthétase est obtenu par mutation à la position n d'une glutamine synthétase de type sauvage, la position est A, G, M, N, Q, S, T, V ou supprimée après la mutation, et la position correspond à la position 69 d'une séquence d'acides aminés telle que représentée par SEQ ID NO 1.
PCT/CN2022/113146 2021-09-15 2022-08-17 Mutant de glutamine synthétase et son application dans la sélection de variétés végétales résistantes au glufosinate-ammonium WO2023040564A1 (fr)

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