WO2023071438A1 - 一种谷氨酰胺合成酶突变体及应用 - Google Patents

一种谷氨酰胺合成酶突变体及应用 Download PDF

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WO2023071438A1
WO2023071438A1 PCT/CN2022/113149 CN2022113149W WO2023071438A1 WO 2023071438 A1 WO2023071438 A1 WO 2023071438A1 CN 2022113149 W CN2022113149 W CN 2022113149W WO 2023071438 A1 WO2023071438 A1 WO 2023071438A1
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glutamine synthetase
plant
glufosinate
wild
mutant
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French (fr)
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邓龙群
张震
陈容
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
    • C12N15/79—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.
  • Glutamine synthetase (Glutamine synthetase, GS) is a key enzyme of plant nitrogen metabolism, it catalyzes the condensation of glutamic acid (Glu) and NH3 to form glutamine (Gln) in the glutamate synthetase cycle, and participates in plant Metabolism of nitrogen compounds.
  • Glu glutamic acid
  • Gln glutamine
  • Glufosinate ammonium (glufosinate, glufosinate ammoni ⁇ M, trade name Basta) is a glutamine synthetase (GS1) inhibitor developed by Aventis (now Bayer), its active ingredient is phosphinothricin (referred to as PPT), chemical name It is (RS)-2-amino-4-(hydroxymethylphosphinyl)ammonium butyrate.
  • PPT glutamine synthetase
  • PPT phosphinothricin
  • RS phosphinothricin
  • the target enzyme of glufosinate-ammonium is GS. Under normal circumstances, GS can form ⁇ -glutamyl phosphate from ATP and glutamate.
  • glufosinate-resistant genes are widely used in agriculture to obtain glufosinate-resistant varieties.
  • the most widely used glufosinate-resistant genes are known to be Bar gene and pat gene, both of which can encode grass Ammonium phosphine acetylase, which can acetylate glufosinate-ammonium and inactivate it.
  • Bar gene and pat gene both of which can encode grass Ammonium phosphine acetylase, which can acetylate glufosinate-ammonium and inactivate it.
  • the acceptance of genetically modified crops in the world is still low.
  • the root cause is that the bar gene and pat gene are derived from microorganisms, not from the crop itself, which is likely to cause consumers' resistance.
  • the glufosinate-ammonium acetylase encoded by the bar gene and the pat gene can acetylate and inactivate glufosinate-ammonium, it is difficult for glufosinate-ammonium acetylase to deactivate glufosinate-ammonium before it contacts glutamine synthetase. Completely inactivated, since many glutamine synthetases are distributed on the cell membrane, some non-inactivated glufosinate-ammonium can inhibit the activity of glutamine synthetase on the cell membrane, thereby interfering with the nitrogen metabolism of 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 with glufosinate-ammonium resistance, which is shown in (1) or (2) below:
  • (1) It is obtained by mutating the nth position 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, the wild-type glutamine synthetase The nth position of glutamine synthetase corresponds to the 62nd position of the reference sequence, wherein the amino acid sequence of the reference sequence is shown in SEQ ID NO.1;
  • the n-th amino acid of the glutamine synthetase mutant is X, and X includes K or deletion;
  • the inventors have found that the wild-type glutamine synthetase derived from plants is compared with the reference sequence, and the amino acid site corresponding to the 62nd position of the reference sequence, that is, the nth position, is mutated, mutated to K or deleted , the obtained glutamine synthetase mutant has glufosinate-ammonium resistance while maintaining its own biological enzyme catalytic activity.
  • the 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 can not only be used for the cultivation of transgenic crops, It can also 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 (SEQ ID NO.1) 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 62nd position in its own sequence (such as corn, wheat, soybean, rapeseed, etc.), but it may not be the 62nd position (for example, peanut corresponds to No. 63), the specific position of the nth position is determined according to the aforementioned sequence alignment, as long as it is compared with the reference sequence, the position corresponding to the 62nd position of the reference sequence is the nth position in the present disclosure, That is the 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 62 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) perform simple amino acid substitutions or deletions or additions, and maintain the nth position after the above mutation Amino acid, and the glutamine synthetase mutant obtained by further mutation 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 are selected from wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, sesame, sunflower, radish, carrot, pepper, spinach, Celery, amaranth, lettuce, crown chrysanthemum, daylily, grapes, strawberries, sugar cane, Brassica vegetables, cucurbits, legumes, nightshade, alliums, grasses, tea or cassava.
  • the above-mentioned forage is selected from grasses or leguminous forages.
  • 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 above-mentioned Brassica (also known as Brassica) vegetables include but are not limited to turnips, Chinese cabbage, mustard greens, cabbage, kale, cabbage, bitter mustard, canola, brassica, green vegetables , rapeseed, cauliflower or 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, peanut, or edamame.
  • the above-mentioned plants of the genus Allium include but are not limited to leeks, green onions, onions, leeks or garlic.
  • the above-mentioned Solanaceae plants include but are not limited to eggplant, tomato, tobacco, pepper or potato.
  • the research of the present disclosure also found that for different plant-derived glutamine synthetases, in addition to mutating the nth position to K or deleting it, mutating it to other amino acids will also make glutamine synthetase have glutamine synthetase Phosphine resistance.
  • X A, C, F, G, I, K, L, M, N, P, R, S, W, Y or delete ;
  • X F, G, K, L, M, N, P, W, Y or deletion
  • X C, F, G, K, L, M, P, R, 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 any of the above-mentioned glufosinate-resistant glutamine synthetase mutants.
  • 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 62nd 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 corresponding nucleotide mutation is carried out at the codon corresponding to the 62nd position of the encoded amino acid sequence, so that the corn glutamine synthetase mutant as described above can be obtained.
  • 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 corresponding nucleotide mutation is carried out at the codon corresponding to the 62nd position of the encoded amino acid sequence, and the soybean glutamine synthetase mutant encoding the above can be obtained.
  • the coding nucleic acid sequence of wheat wild-type glutamine synthetase is SEQ ID NO.9:
  • the corresponding nucleotide mutation is carried out at the codon corresponding to the 62nd position of its encoded amino acid sequence, and the wheat glutamine synthetase mutant encoding the above can be obtained.
  • the coding nucleic acid sequence of rapeseed wild-type glutamine synthetase is SEQ ID NO.10:
  • the corresponding nucleotide mutation is carried out at the codon corresponding to the 62nd position of the encoded amino acid sequence, so as to obtain the above-mentioned rapeseed glutamine synthetase mutant.
  • the present disclosure also provides a vector containing the above-mentioned nucleic acid molecule.
  • the present disclosure provides a recombinant bacterium or a recombinant cell, which contains a nucleic acid molecule or a vector.
  • the recombinant bacteria can be selected from Agrobacterium; the recombinant cells can be competent cells.
  • the present disclosure also provides the application of glutamine synthetase mutants, nucleic acid molecules, vectors or 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 bacteria or recombinant cells are introduced into the target plant, and the recombinant bacteria or recombinant cells contain the coding gene encoding 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.
  • Plants include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, sesame, sunflower, radish, carrot, pepper, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, Grapes, strawberries, sugar cane, brassica vegetables, cucurbits, legumes, nightshades, alliums, grasses, tea or cassava.
  • the above-mentioned forage is selected from grasses or leguminous forages.
  • 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 above-mentioned Brassica (also known as Brassica) vegetables include but are not limited to turnips, Chinese cabbage, mustard greens, cabbage, kale, cabbage, bitter mustard, canola, brassica, green vegetables , rapeseed, cauliflower or 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, peanut, or edamame.
  • the above-mentioned plants of the genus Allium include but are not limited to leeks, green onions, onions, leeks or garlic.
  • the above-mentioned Solanaceae plants include but are not limited to eggplant, tomato, tobacco, pepper or potato.
  • 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 has good glufosinate-ammonium resistance, 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, It can meet the normal growth and development needs of plants.
  • Fig. 1 is the rice GS1 mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X (X is a deletion) and wild The results of the partial alignment of the amino acid sequences of rice GS1OWT1;
  • Figure 2 is the result of partial alignment of the amino acid sequences of soybean GS1 mutants GQ62F, GQ62K, GQ62R, GQ62W and GQ62X (X is a deletion) and wild-type soybean GS1GWT1 provided in Example 2 of the present disclosure;
  • Figure 3 is the results of partial alignment of the amino acid sequences of the corn GS1 mutants ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y and ZQ62X (X is a deletion) and wild-type corn GS1ZWT1 provided in Example 2 of the present disclosure ;
  • Figure 4 is the results of partial alignment of the amino acid sequences of wheat GS1 mutants TQ62G, TQ62H, TQ62I, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X (X is a deletion) and wild-type wheat GS1TWT1 provided in Example 2 of the present disclosure;
  • Figure 5 is a partial comparison of the amino acid sequences of rapeseed GS1 mutants BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W, BQ62Y and BQ62X (X is a deletion) and wild-type rapeseed GS1BWT1 provided in Example 2 of the present disclosure to the result;
  • FIG. 6 is a schematic structural diagram of the pADV7 vector provided in Experimental Example 1 of the present disclosure.
  • Fig. 7 is the rice GS1 mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X provided in Experimental Example 1 of the present disclosure.
  • Figure 8 shows the growth of Escherichia coli transformed with soybean GS1 mutants GQ62F, GQ62K, GQ62R, GQ62W and GQ62X and wild-type soybean GS1GWT1 provided in Experimental Example 2 of the present disclosure on media containing different concentrations of glufosinate-ammonium result;
  • Fig. 9 is Escherichia coli containing different corn GS1 mutants ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y and ZQ62X and wild-type corn GS1ZWT1 provided in Experimental Example 3 of the present disclosure.
  • the growth result on the medium of concentration glufosinate-ammonium;
  • Fig. 10 is the Escherichia coli of wheat GS1 mutants TQ62G, TQ62H, TQ62I, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X provided in Experimental Example 4 of the present disclosure provided in Example 4 and wild-type wheat GS1TWT1 in different concentrations of glufosinate-ammonium Growth results on the culture medium;
  • Fig. 11 is the Escherichia coli of rapeseed GS1 mutants BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W, BQ62Y and BQ62X and wild-type rapeseed GS1BWT1 provided in Experimental Example 5 of the present invention. Growth results on media containing different concentrations of glufosinate-ammonium;
  • Figure 12 shows the rice GS1 mutant OQ62X, soybean GS1 mutant GQ62X, corn GS1 mutant ZQ62X, wheat GS1 mutant TQ62X, rapeseed GS1 mutant BQ62X, wild-type rice GS1OWT1, wild-type soybean GS1GWT1, Enzyme kinetic parameters and glufosinate-ammonium resistance parameter IC 50 of wild-type maize GS1ZWT1, wild-type wheat GS1TWT1 and wild-type rapeseed GS1BWT1;
  • Figure 13 shows the amino acid sequence alignment results of wild-type glutamine synthetases in different plants; in the figure: TWT1: wheat wild-type glutamine synthetase; OWT1: rice wild-type glutamine synthase; ZWT1: maize wild GWT1: soybean wild-type glutamine synthetase; BWT1: rapeseed wild-type glutamine synthase.
  • the rice (Oryza sativa) glutamine synthetase (GS1) mutant provided by the present embodiment is composed of the wild-type rice glutamine synthetase itself (named OWT1, the amino acid sequence is as shown in SEQ ID NO.1, and the encoding nucleus
  • the nucleotide sequence is obtained by mutation of the 62nd amino acid residue Q of SEQ ID NO.6) to A, C, F, G, I, K, L, M, N, P, R, S, W, Y or deletion
  • the obtained rice GS1 mutants were named OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X, respectively.
  • each rice GS1 mutant is at the position encoding the 62nd 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 rice GS1 mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X provided in this example and the nucleic acid molecules encoding them can all be chemically synthesized method to obtain.
  • the soybean (Glycine max) GS1 mutant provided by the present embodiment is composed of wild-type soybean GS1 itself ((named GWT1, amino acid sequence as shown in SEQ ID NO.3, encoding nucleotide sequence is SEQ ID NO.8)
  • the 62nd position (corresponding to the 62nd position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of the amino acid residue Q to F, K, R, W or deletion.
  • the obtained rice soybean GS1 mutants are named GQ62F respectively , GQ62K, GQ62R, GQ62W, and GQ62X.
  • the coding sequences of the soybean GS1 mutants GQ62F, GQ62K, GQ62R, GQ62W and GQ62X provided in this example correspond to SEQ ID NO.3.
  • each soybean GS1 mutant is at the position encoding the 62nd 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 GQ62F, GQ62K, GQ62R, GQ62W and GQ62X provided in this example and the 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 ZWT1, the amino acid sequence is as shown in SEQ ID NO.2, and the encoding nucleotide sequence is SEQ ID NO.7).
  • the 62nd position (corresponding to the 62nd position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of the amino acid residue Q to F, G, K, L, M, N, P, W, Y or deletion.
  • the maize GS1 mutants obtained were named ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y and ZQ62X, respectively.
  • each maize GS1 mutant is at the position encoding the 62nd 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 maize GS1 mutants ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y and ZQ62X provided in this example and the nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the wheat (Triticum aestivum) GS1 mutant provided by the present embodiment is composed of wild-type wheat GS1 itself (named TWT1, the amino acid sequence is as shown in SEQ ID NO.4, and the encoded nucleotide sequence is SEQ ID NO.9).
  • the 62nd position (corresponding to the 62nd position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of amino acid residue Q to G, H, I, K, L, R, Y or deletion.
  • the obtained wheat GS1 mutants were named TQ62G, TQ62H, TQ62I, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X, respectively.
  • each wheat GS1 mutant is at the position encoding the 62nd 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 TQ62G, TQ62H, TQ62I, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X provided in this example and nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the rape (Brassica napus) GS1 mutant provided by the present embodiment is composed of wild-type rape GS1 (named BWT1, the amino acid sequence is as shown in SEQ ID NO.5, and the encoding nucleotide sequence is SEQ ID NO.10)
  • the 62nd position (corresponding to the 62nd position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of the amino acid residue Q to C, F, G, K, L, M, P, R, W, Y or deletion.
  • the obtained rapeseed GS1 mutants were named BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W, BQ62Y and BQ62X, respectively.
  • each rapeseed GS1 mutant is at the position encoding the 62nd 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 BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W, BQ62Y and BQ62X provided in this example and nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the glufosinate-ammonium resistance of the rice GS1 mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X provided in Example 1 were detected respectively.
  • the detection method of glufosinate-ammonium resistance is as follows:
  • the rice GS1 mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P, OQ62R, OQ62S, OQ62W, OQ62Y and
  • the coding gene of OQ62X is introduced with restriction sites (Pac1 and Sbf1) at both ends. After digestion, it is connected to the expression vector after the same restriction treatment under the action of ligase (for example, pADV7 vector, its structure is shown in Figure 6 shown), and then transform glutamine synthetase-deficient Escherichia coli respectively.
  • ligase for example, pADV7 vector, its structure is shown in Figure 6 shown
  • Escherichia coli transformed with wild-type rice GS1 could not grow on the medium containing 10 mM glufosinate-ammonium (KP10), but transformed rice mutants OQ62A, OQ62C, OQ62F, OQ62G, OQ62I, OQ62K, OQ62L, OQ62M, OQ62N, OQ62P , OQ62R, OQ62S, OQ62W, OQ62Y and OQ62X Escherichia coli grew significantly better than the negative control, indicating that the E. Q62Y and The ability of the single mutant of OQ62X to resist glufosinate was significantly better than that of the wild type.
  • KP10 mM glufosinate-ammonium
  • the defective strains transformed with coding genes encoding wild-type soybean GS1 (GWT1) and soybean GS1 mutants GQ62F, GQ62K, GQ62R, GQ62W and GQ62X could grow normally, indicating that GS1 encoded by GQ62F, GQ62K, GQ62R, GQ62W and GQ62X all have normal GS1 enzyme activity;
  • the Escherichia coli transformed with wild-type soybean GS1 basically could not grow, but the Escherichia coli transformed with soybean mutants GQ62F, GQ62K, GQ62R, GQ62W and GQ62X grew significantly better than negative
  • the control shows that the ability of the single mutant containing GQ62F, GQ62K, GQ62R, GQ62W and GQ62X to resist glufosinate is significantly better than that of the wild type; on the medium with higher glufosinate concentration (20mM, KP20), the transformed soybean GS1 mutation The Escherichia coli of body GQ62X all still has obvious growth.
  • Escherichia coli transformed with wild-type maize GS1 basically could not grow, but transformed maize mutants ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y
  • the growth of Escherichia coli and ZQ62X was significantly better than that of the negative control, indicating that the ability of the single mutants containing ZQ62F, ZQ62G, ZQ62K, ZQ62L, ZQ62M, ZQ62N, ZQ62P, ZQ62W, ZQ62Y and ZQ62X to resist glufosinate-ammonium was significantly better than that of the wild type;
  • Escherichia coli transformed with maize GS1 mutants ZQ62K and ZQ62X still
  • the Escherichia coli transformed with wild-type wheat GS1 basically could not grow, but the E. Bacterial growth is obviously better than negative control, shows that the single mutant containing TQ62G, TQ62H, TQ62I, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X is obviously better than wild type in resistance to glufosinate-ammonium; , KP20) culture medium, the Escherichia coli transformed with wheat GS1 mutants TQ62G, TQ62H, TQ62K, TQ62L, TQ62R, TQ62Y and TQ62X also had obvious growth.
  • Escherichia coli transformed with wild-type rapeseed GS1 basically could not grow, but transformed rapeseed mutants BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W , BQ62Y and BQ62X Escherichia coli grew significantly better than the negative control, indicating that the single mutants containing BQ62C, BQ62F, BQ62G, BQ62K, BQ62L, BQ62M, BQ62P, BQ62R, BQ62W, BQ62Y and BQ62X had significantly better resistance to glufosinate-ammonium Wild type; Escherichia coli transformed with rapeseed GS1 mutants BQ62P, BQ62R and BQ62X still grow significantly on the medium with higher glufo
  • 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 mutant protein solution (protein concentration: 200ug/ml) to start the reaction.
  • the Km values of the GS1 mutant were higher, indicating that the GS mutant not only reduced the sensitivity to glufosinate-ammonium inhibitors, but also reduced the sensitivity to normal bottom. object sensitivity.
  • the Vmax of 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-ammonium, with IC 50 of 7.93 ⁇ M, 13.55 ⁇ M, 8.92 ⁇ M, 7.22 ⁇ M and 1.53 ⁇ M, and the IC 50 of the mutants were significantly higher than that of the wild-type control. IC50 is much higher than the wild-type control, indicating that the mutant is less sensitive to glufosinate-ammonium.
  • mutant IC 50 OQ62X, GQ62X, ZQ62X, TQ62X and BQ62X is 3.70 times, 20.88 times, 22.05 times, 28.38 times and 110.56 times, these values also show that the enzyme activity of the mutant is much higher than that of the wild type control. These data illustrate the mechanism of the mutant's resistance to glufosinate-ammonium from the enzyme kinetics.

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Abstract

本公开提供了一种谷氨酰胺合成酶突变体及其在培育抗草铵膦的植物品种中的应用。该谷氨酰胺合成酶突变体是在野生型谷氨酰胺合成酶第n位进行突变获得的，该位点对应于如SEQ ID NO.1所示的氨基酸序列的第62位。

Description

一种谷氨酰胺合成酶突变体及应用
相关申请的交叉引用
本公开要求于2021年10月26日提交中国专利局的申请号为CN202111244191.2、名称为“一种谷氨酰胺合成酶突变体及应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及基因工程技术领域，具体而言，涉及一种谷氨酰胺合成酶突变体及应用。
背景技术
谷氨酰胺合成酶(Glutamine synthetase,GS)是植物氮代谢的关键酶，它在谷氨酸合成酶循环中催化谷氨酸(Glu)与NH 3缩合形成谷氨酰胺(Gln)，参与植物含氮化合物的新陈代谢。
草铵膦(glufosinate，glufosinate ammoniμM，商品名称Basta)是由安万特公司(现为拜耳公司)开发的谷氨酰胺合成酶(GS1)抑制剂，其有效成分为phosphinothricin(简称PPT)，化学名称为(RS)-2-氨基-4-(羟基甲基氧膦基)丁酸铵。草铵膦的靶标酶是GS，在正常情况下，GS可以由ATP及谷氨酸(glutamate)形成λ-磷酸谷氨酰(λ-glutamyl phosphate)。但在PPT处理后，PPT先与ATP结合，磷酸化的PPT占据GS分子的8个反应中心，使GS的空间构型发生变化，从而GS的活性受到抑制。PPT能抑制GS所有已知的形式。
草铵膦抑制GS的结果，可以导致植物体内氮代谢紊乱，铵的过量积累，叶绿体解体，从而抑制植物的光合作用，最终可导致植物死亡。
目前，农业上广泛应用细菌的抗草铵膦基因导入农作物的方法得到抗草铵膦品种，已知应用最广的抗草铵膦基因是Bar基因和pat基因，这两种基因都可以编码草铵膦乙酰化酶，而该酶可以使草铵膦乙酰化而失活。然而转基因作物在全世界的接受程度仍然较低，根源在于bar基因和pat基因来源于微生物，而不是来源于农作物本身，容易造成消费者的抵触心理。
bar基因和pat基因编码的草铵膦乙酰化酶虽然可以使草铵膦乙酰化而失活，但是在草铵膦接触谷氨酰胺合成酶之前，草铵膦乙酰化酶很难使草铵膦彻底失活，由于很多谷氨酰胺合成酶分布在细胞膜上，部分未失活的草铵膦可以抑制细胞膜上谷氨酰胺合成酶的活性，从而干扰植物的氮代谢。因此草铵膦在转bar基因和pat基因农作物上应用时，会不同程度的干扰植物的氮代谢，同时影响植物正常的生长和发育。通过在植物中过量表达野生型谷氨酰胺合成酶虽然可以一定程度上降低转基因植物对草铵膦的敏感程度，但其对草铵膦的耐性程度远不足以商业化应用。
发明内容
本公开提供了一种具有草铵膦抗性的谷氨酰胺合成酶突变体，其如下(1)或(2)所示：
(1)：其由来源于植物的野生型谷氨酰胺合成酶的第n位发生突变得到；第n位的位置通过如下方式确定：野生型谷氨酰胺合成酶与参考序列比对，野生型谷氨酰胺合成酶的第n位对应于参考序列的第62位，其中，参考序列的氨基酸序列如SEQ ID NO.1所示；
谷氨酰胺合成酶突变体的第n位的氨基酸为X，X包括K或删除；
(2)：其与(1)所示的谷氨酰胺合成酶突变体至少具有85％以上的同一性、且与(1)所示的谷氨酰胺合成酶突变体在第n位的氨基酸相同、以及具有草铵膦抗性。
发明人研究发现，将植物来源的野生型谷氨酰胺合成酶与参考序列进行比对，将其序列上对应于参考序列第62位的氨基酸位点即第n位进行突变，突变为K或删除，所得到的谷氨酰胺合成酶突变体具有草铵膦抗性，同时保持自身的生物酶催化活性。转化本公开提供的植物谷氨酰胺合成酶突变体的植株或重组菌均能够在草铵膦存在的条件下正常生长和发育，该植物谷氨酰胺合成酶突变体不仅可以用于转基因作物培育，也可应用于培育抗草铵膦非转基因植物或转基因植物例如水稻、烟草、大豆、玉米、小麦、油菜、棉花和高粱等，具有广阔的应用前景。
上述参考序列(SEQ ID NO.1)为水稻来源的野生型谷氨酰胺合成酶。
序列比对方法可使用Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行Protein Blast比对；采用本领域熟知的其他序列比对方法或工具也可以得到相同的结果。
需要说明的是，野生型谷氨酰胺合成酶的第n位在其自身序列上可能也是第62位(例如玉米、小麦、大豆、油菜等)，但也可能不是第62位(例如花生对应为第63位)，第n位的具体位置根据前述序列比对后确定，只要其通过与参考序列比对后，对应于参考序列第62位的位点即为本公开所述的第n位，也就是突变位点。
所有植物的野生型谷氨酰胺合成酶都具有同源性，在植物体内具有基本相同的功能和结构域。因此，任意植物来源的野生型谷氨酰胺合成酶在第62位作上述突变后所得到的谷氨酰胺合成酶突变体都具有草铵膦抗性。因此，由任意植物来源的野生型谷氨酰胺合成酶作上述突变后得到的谷氨酰胺合成酶突变体均属于本公开的保护范围。
此外，本领域技术人员知晓并容易实现，在(1)所示的谷氨酰胺合成酶突变体的非保守区域进行简单的氨基酸替换或删除或增加等操作并维持第n位为上述突变后的氨基酸，并使进一步突变得到的谷氨酰胺合成酶突变体与(1)所示的谷氨酰胺合成酶突变体具有至少具有85％(例如85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％等)以上的同一性，且其功能包括酶催化活性和草铵膦抗性与(1)所示的谷氨酰胺合成酶突变体相当或略有下降或略有提高或大幅提高等。因此，此类谷氨酰胺合成酶也应属于本公开的保护范围。
在本公开应用可选的实施方式中，上述植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、棉花、芝麻、向日葵、萝卜、胡萝卜、辣椒、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、芸薹属蔬菜、葫芦科植物、豆科植物、茄科植物、葱属植物、牧草、茶或木薯。
在一种实施方式中，上述牧草选自禾本科牧草或豆科牧草。禾本科牧草选自梯牧草、鸭茅、六月禾、细麦、羊茅、棕叶、狗尾草等；豆科牧草选自苜蓿、三叶草、三叶豆、巢菜、鸡眼草等。此外，在其他实施方式中，上述牧草也可选自草坪草。
在一种可选的实施方式中，上述芸薹(亦称为芸苔)属蔬菜包括不限于芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜、油菜、花椰菜或甜菜。
在一种可选的实施方式中，上述葫芦科植物包括不限于黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜。
在一种可选的实施方式中，上述豆科植物包括不限于绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆、花生、或毛豆。
在一种可选的实施方式中，上述葱属植物包括不限于韭菜、大葱、洋葱、韭葱或大蒜。
在一种可选的实施方式中，上述茄科植物包括不限于茄子、番茄、烟草、辣椒或马铃薯。
本公开的研究还发现，针对不同的植物来源的谷氨酰胺合成酶，将其第n位突变为K或删除之外，将其突变为其他的氨基酸也会使得谷氨酰胺合成酶具有草铵膦抗性。
例如，在本公开应用可选的实施方式中，当植物为水稻时，X＝A、C、F、G、I、K、L、M、N、P、R、S、W、Y或删除；
当植物为大豆时，X＝F、K、R、W或删除；
当植物为玉米时，X＝F、G、K、L、M、N、P、W、Y或删除；
当植物为小麦时，X＝G、H、I、K、L、R、Y或删除；
当植物为油菜时，X＝C、F、G、K、L、M、P、R、W、Y或删除。
需要说明的是，X＝删除，是指野生型谷氨酰胺合成酶第n位氨基酸被删除，即缺失突变。
可选的，在本公开的一些实施方案中，当所述植物为水稻时，水稻野生型谷氨酰胺合成酶为SEQ ID NO.1：
Figure PCTCN2022113149-appb-000001
可选的，在本公开的一些实施方案中，当所述植物为玉米时，玉米野生型谷氨酰胺合成酶为SEQ ID NO.2：
Figure PCTCN2022113149-appb-000002
可选的，在本公开的一些实施方案中，当所述植物为大豆时，大豆野生型谷氨酰胺合成酶为SEQ ID NO.3：
Figure PCTCN2022113149-appb-000003
可选的，在本公开的一些实施方案中，当所述植物为小麦时，小麦野生型谷氨酰胺合成酶为SEQ ID NO.4：
Figure PCTCN2022113149-appb-000004
可选的，在本公开的一些实施方案中，当所述植物为油菜时，油菜野生型谷氨酰胺合成酶为SEQ ID NO.5：
Figure PCTCN2022113149-appb-000005
部分植物来源的野生型谷氨酰胺合成酶相互间的相似性(Similarity)和同一性(Identity)如下表所示，其序列比对的部分结果见图13，箭头所示为第62位氨基酸。
Figure PCTCN2022113149-appb-000006
上述相似性(Similarity)和同一性(Identity)的比对方法为：将一个物种的氨基酸序列输入到Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行Protein Blast比对，从比对结果中查找此物种和其他需要比对的物种的相似性(Similarity)和同一性(Identity)。
本公开还提供了一种分离的核酸分子，其编码上述任一种具有草铵膦抗性的谷氨酰胺合成酶突变体。
在本公开提供了上述氨基酸序列的情况下，本领域技术人员根据密码子的简并性容易获得编码上述谷氨酰胺合成酶突变体的核酸序列。例如，可以在编码野生型谷氨酰胺合成酶的核酸序列上作对应的核苷酸突变得到编码上述谷氨酰胺合成酶突变体的核酸序列。这对本领域技术人员来说是容易实现的。
例如，水稻野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.6：
Figure PCTCN2022113149-appb-000007
据此，在序列基础上，在对应于其编码氨基酸序列第62位的密码子进行对应的核苷酸突变，即可得到编码如上所述的水稻谷氨酰胺合成酶突变体。
玉米野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.7：
Figure PCTCN2022113149-appb-000008
据此，在序列基础上，在对应于其编码氨基酸序列第62位的密码子进行对应的核苷酸突变，即可得到编码如上所述的玉米谷氨酰胺合成酶突变体。
大豆野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.8：
Figure PCTCN2022113149-appb-000009
大豆野生型谷氨酰胺合成酶的编码核酸序列也可以参见NCBI登记号：NM_001255403.3。
据此，在上述序列基础上，在对应于其编码氨基酸序列第62位的密码子进行对应的核苷酸突变，即可得到编码如上所述的大豆谷氨酰胺合成酶突变体。
小麦野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.9：
Figure PCTCN2022113149-appb-000010
据此，在上述序列基础上，在对应于其编码氨基酸序列第62位的密码子进行对应的核苷酸突变，即可得到编码如上所述的小麦谷氨酰胺合成酶突变体。
油菜野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.10：
Figure PCTCN2022113149-appb-000011
据此，在上述序列基础上，在对应于其编码氨基酸序列第62位的密码子进行对应的核苷酸突变，即可得到编码如上所述的油菜谷氨酰胺合成酶突变体。
本公开还提供了一种载体，其含有上述的核酸分子。
本公开提供了一种重组菌或重组细胞，其含有核酸分子或载体。重组菌可以选自农杆菌；重组细胞可以是感受态细胞。
本公开还提供了具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子、载体或重组菌或重组细胞在培育具有草铵膦抗性的植物品种中的应用。
上述应用包括如下至少一种的应用方式：
将分离的核酸分子送入目的植物细胞，分离的核酸分子含有编码谷氨酰胺合成酶突变体的编码基因；
将载体转化目的植物，载体含有编码谷氨酰胺合成酶突变体的编码基因；
或，将重组菌或重组细胞导入目的植物，重组菌或重组细胞含有编码谷氨酰胺合成酶突变体的编码基因。
分离的核酸分子可以是质粒或DNA片段，在可选的实施方式中，可以通过基因枪法将分离的核酸分子送入目的植物细胞。
转化的方法包括不限于农杆菌介导基因转化法，基因枪转化法、花粉管通道法。
重组菌或重组细胞可通过侵染的方式导入目的植物体内。
在本公开应用可选的实施方式中，上述应用包括：修饰目的植物的内源谷氨酰胺合成酶基因，使其编码谷氨酰胺合成酶突变体。
在本公开提供了谷氨酰胺合成酶突变体的基础上，本领域技术人员容易想到通过本领域常规的转基因技术、基因编辑技术(如通过锌指核酸内切酶(ZFN，zinc-finger nucleases)技术、类转录激活因子效应物核酸酶(TALEN，transcription activator-like effector nucleases)技术或CRISPR/Cas9)、诱变育种技术(如化学、辐射诱变等)等对目标植物进行改造，使其具有编码如上谷氨酰胺合成酶突变体的基因，进而获得草铵膦抗性并能够正常生长和发育的植物新品种。因此，无论采用何种技术，只要其利用了本公开提供的谷氨酰胺合成酶突变体赋予植物草铵膦抗性，均属于本公开的保护范围。
在本公开应用可选的实施方式中，上述应用包括：对植物细胞、组织、个体或群体进行诱变和筛选，使其编码谷氨酰胺合成酶突变体。
在一种可选的实施方式中，诱变为非致死剂量的理化诱变方式对植物进行诱变以获得植物材料。
上述非致死剂量是指将剂量控制在半致死剂量上下浮动20％的范围。
理化诱变方式包括以下物理诱变、化学诱变方式中的一种或多种的组合：物理诱变包括紫外线诱变、X射线诱变、γ射线诱变、β射线诱变、α射线诱变、高能粒子诱变、宇宙射线诱变、微重力诱变；化学诱变包括烷化剂诱变、叠氮化物诱变、碱基类似物诱变、氯化锂诱变、抗生素诱变、嵌入染料诱变；烷化剂诱变包括甲基环酸乙酯诱变、硫酸二乙酯诱变、乙烯亚胺诱变。
植物包括不限于小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、棉花、芝麻、向日葵、萝卜、胡萝卜、辣椒、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、芸薹属蔬菜、葫芦科植物、豆科植物、茄科植物、葱属植物、牧草、茶或木薯。
在一种实施方式中，上述牧草选自禾本科牧草或豆科牧草。禾本科牧草选自梯牧草、鸭茅、六月禾、细麦、羊茅、棕叶、狗尾草等；豆科牧草选自苜蓿、三叶草、三叶豆、巢菜、鸡眼草等。此外，在其他实施方式中，上述牧草也可选自草坪草。
在一种可选的实施方式中，上述芸薹(亦称为芸苔)属蔬菜包括不限于芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜、油菜、花椰菜或甜菜。
在一种可选的实施方式中，上述葫芦科植物包括不限于黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜。
在一种可选的实施方式中，上述豆科植物包括不限于绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆、花生、或毛豆。
在一种可选的实施方式中，上述葱属植物包括不限于韭菜、大葱、洋葱、韭葱或大蒜。
在一种可选的实施方式中，上述茄科植物包括不限于茄子、番茄、烟草、辣椒或马铃薯。
本公开提供的谷氨酰胺合成酶突变体，具有用于构建转化植物的表达载体、及培育抗草铵膦作物的应用潜力。本公开提供的谷氨酰胺合成酶突变体原始来源于植物，更容易被消费者接受。突变后具有良好的草铵膦抗性，转化该谷氨酰胺合成酶突变体的植物不仅具有适于商业化应用的草铵膦抗性，也能够保持谷氨酰胺合成酶正常的酶催化活性，可以满足植物正常的生长和发育需求。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例1提供的水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X(X为删除)和野生型水稻GS1OWT1的氨基酸序列部分比对结果；
图2为本公开实施例2提供的大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X(X为删除)和野生型大豆GS1GWT1的氨基酸序列部分比对结果；
图3为本公开实施例2提供的玉米GS1突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X(X为删除)和野生型玉米GS1ZWT1的氨基酸序列部分比对结果；
图4为本公开实施例2提供的小麦GS1突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X(X为删除)和野生型小麦GS1TWT1的氨基酸序列部分比对结果；
图5为本公开实施例2提供的油菜GS1突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X(X为删除)和野生型油菜GS1BWT1的氨基酸序列部分比对结果；
图6为本公开实验例1提供的pADV7载体的结构示意图；
图7为本公开实验例1提供的转化实施例1提供的水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X和野生型水稻GS1OWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果；
图8为本公开实验例2提供的转化实施例2提供的大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X和野生型大豆GS1GWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果；
图9为本公开实验例3提供的转化实施例3提供的玉米GS1突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X和野生型玉米GS1ZWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果；
图10为本公开实验例4提供的转化实施例4提供的小麦GS1突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X和野生型小麦GS1TWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果；
图11为本发明实验例5提供的转化实施例5提供的油菜GS1突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X和野生型油菜GS1BWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果；
图12为本公开实验例6提供的水稻GS1突变体OQ62X、大豆GS1突变体GQ62X、玉米GS1突变体ZQ62X、小麦GS1突变体TQ62X、油菜GS1突变体BQ62X、野生型水稻GS1OWT1、野生型大豆GS1GWT1、野生型玉米GS1ZWT1、野生型小麦GS1TWT1和野生型油菜GS1BWT1的酶动力学参数和草铵膦抗性参数IC 50；
图13为不同植物野生型谷氨酰胺合成酶的氨基酸序列比对结果；图中：TWT1：小麦野生型谷氨酰胺合成酶体；OWT1：水稻野生型谷氨酰胺合成酶体；ZWT1：玉米野生型谷氨酰胺合成酶体；GWT1：大豆野生型谷氨酰胺合成酶体；BWT1：油菜野生型谷氨酰胺合成酶体。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用植物生理学、植物分子遗传学、细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《植物生理学》(苍晶等人，2017)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《植物分子遗传学》(Monica A.Hughes等人著)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)，所述文献中的每个文献均通过引用明确并入本文中。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供的水稻(Oryza sativa)谷氨酰胺合成酶(GS1)突变体，其由野生型水稻谷氨酰胺合成酶自身(命名为OWT1，氨基酸序列如SEQ ID NO.1所示，编码核苷酸序列为SEQ ID NO.6)的第62位氨基酸残基Q突变为A、C、F、G、I、K、L、M、N、P、R、S、W、Y或删除得到，得到的水稻GS1突变体分别命名为OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X。
水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y、OQ62X和野生型水稻GS1的氨基酸序列比对如图1所示，图中：箭头所指示的位置为突变位点。
本实施例中，各水稻GS1突变体的编码序列在编码第62位氨基酸的位置上，对应氨基酸所用的密码子如下表所示，其余位置的核苷酸同相应的野生型编码序列。
氨基酸 A C F G I
密码子 GCC TGC TTC GGC ATC
氨基酸 K L M N P
密码子 AAG CTC ATG AAC CCC
氨基酸 R S W Y 删除
密码子 CGG TCT TGG TAC 无
本实施例提供的水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例2
本实施例提供的大豆(Glycine max)GS1突变体，其由野生型大豆GS1自身((命名为GWT1，氨基酸序列如SEQ ID NO.3所示，编码核苷酸序列为SEQ ID NO.8)的第62位(对应于参考序列(SEQ ID NO.1)的第62位)由氨基酸残基Q突变为F、K、R、W或删除得到。得到的水稻大豆GS1突变体分别命名为GQ62F、GQ62K、GQ62R、GQ62W和GQ62X。
大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W、GQ62X和野生型大豆GS1的氨基酸序列比对如图2所示，图中：箭头所指示的位置为突变位点。
本实施例提供的大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X的编码序列对应于SEQ ID NO.3。
本实施例中，各大豆GS1突变体的编码序列在编码第62位氨基酸的位置上，对应氨基酸所用的密码子如下表所示，其余位置的核苷酸同相应的野生型编码序列。
氨基酸 F K R W 删除
密码子 TTC AAG CGG TGG 无
本实施例提供的大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例3
本实施例提供的玉米(Zea mays)GS1突变体，其由野生型玉米GS1自身(命名为ZWT1，氨基酸序列如SEQ ID NO.2所示，编码核苷酸序列为SEQ ID NO.7)的第62位(对应于参考序列(SEQ ID NO.1)的第62位)由氨基酸残基Q突变为F、G、K、L、M、N、P、W、Y或删除得到。得到的玉米GS1突变体分别命名为ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X。
玉米GS1突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y、ZQ62X和野生型玉米GS1的氨基酸序列比对如图3所示，图中：箭头所指示的位置为突变位点。
本实施例中，各玉米GS1突变体的编码序列在编码第62位氨基酸的位置上，对应氨基酸所用的密码子如下表所示，其余位置的核苷酸同相应的野生型编码序列。
氨基酸 F G K L M
密码子 TTC GGC AAG CTC ATG
氨基酸 N P W Y 删除
密码子 AAC CCC TGG TAC 无
本实施例提供的玉米GS1突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例4
本实施例提供的小麦(Triticum aestivum)GS1突变体，其由野生型小麦GS1自身(命名为TWT1，氨基酸序列如SEQ ID NO.4所示，编码核苷酸序列为SEQ ID NO.9)的第62位(对应于参考序列(SEQ ID NO.1)的第62位)由氨基酸残基Q突变为G、H、I、K、L、R、Y或删除得到。得到的小麦GS1突变体分别命名为TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X。
小麦GS1突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y、TQ62X和野生型小麦GS1的氨基酸序列比对如图4所示，图中：箭头所指示的位置为突变位点。
本实施例中，各小麦GS1突变体的编码序列在编码第62位氨基酸的位置上，对应氨基酸所用的密码子如下表所示，其余位置的核苷酸同相应的野生型编码序列。
Figure PCTCN2022113149-appb-000012
本实施例提供的小麦GS1突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例5
本实施例提供的油菜(Brassica napus)GS1突变体，其由野生型油菜GS1自身(命名为BWT1，氨基酸序列如SEQ ID NO.5所示，编码核苷酸序列为SEQ ID NO.10)的第62位(对应于参考序列(SEQ ID NO.1)的第62位)由氨基酸残基Q突变为C、F、G、K、L、M、P、R、W、Y或删除得到。得到的油菜GS1突变体分别命名为BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X。
油菜GS1突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y、BQ62X和野生型油菜GS1的氨基酸序列比对如图5所示，图中：箭头所指示的位置为突变位点。
本实施例中，各油菜GS1突变体的编码序列在编码第62位氨基酸的位置上，对应氨基酸所用的密码子如下表所示，其余位置的核苷酸同相应的野生型编码序列。
Figure PCTCN2022113149-appb-000013
本实施例提供的油菜GS1突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X和编码它们的核酸分子均可以通过化学合成的方法获得。
实验例1
分别检测实施例1提供的水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的草铵膦抗性。草铵膦抗性检测方法如下：
根据实施例1提供的核酸分子的序列，采用化学合成的方法合成编码水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的编码基因，两端引入酶切位点(Pac1和Sbf1)，酶切后，在连接酶的作用下连接至经相同酶切处理后的表达载体(例如pADV7载体，其结构如图6所示)上，然后分别转化谷氨酰胺合成酶缺陷型大肠杆菌，经验证后，挑取阳性克隆，接种至含不同浓度草铵膦的M9培养基上生长，观察缺陷型大肠杆菌生长情况。以野生型水稻GS1突变体作为负对照，检测含有GS1突变体OQ62A(Q62A，水稻GS1的第62位的氨基酸Q突变为A)、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X(Q62X，水稻GS1的第62位的氨基酸Q删除)的草铵膦抗性。结果如图7所示。
在含0mM草铵膦(KP0)的培养基上，转化编码野生型水稻GS1(OWT1)及水稻GS1突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的编码基因的缺陷型菌株均能正常生长，表明由OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X编码的GS1都具有正常GS1酶活力。
在含10mM草铵膦(KP10)的培养基上，转化野生型水稻GS1的大肠杆菌不能生长，但转化了水稻突变体OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的大肠杆菌生长明显优于负对照，说明含OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的单突变体抗草铵膦的能力明显优于野生型。
在更好草铵膦浓度(20mM，KP20)的培养基上，转化水稻GS1突变体OQ62A、OQ62F、OQ62G、OQ62I、OQ62K、OQ62N、OQ62P、OQ62R、OQ62W、OQ62Y和OQ62X的大肠杆菌都还有明显生长。
这些结果说明OQ62A、OQ62C、OQ62F、OQ62G、OQ62I、OQ62K、OQ62L、OQ62M、OQ62N、OQ62P、OQ62R、OQ62S、OQ62W、OQ62Y和OQ62X的单突变体都具有抗草铵膦的能力。
实验例2
参考实验例1的检测方法，验证实施例2提供的大豆GS1突变体GQ62F(Q62F，大豆GS1的第62位的氨基酸Q突变为F)、GQ62K、GQ62R、GQ62W和GQ62X(Q62X，大豆GS1的第62位的氨基酸Q删除)的草铵膦抗性。结果如图8所示。
根据图8的结果可看出：
在含0mM草铵膦(KP0)的培养基上，转化编码野生型大豆GS1(GWT1)及大豆GS1突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X的编码基因的缺陷型菌株均能正常生长，表明由GQ62F、GQ62K、GQ62R、GQ62W和GQ62X编码的GS1都具有正常GS1酶活力；
在含1mM草铵膦(KP1)的培养基上，转化野生型大豆GS1的大肠杆菌基本上不能生长，但转化了大豆突变体GQ62F、GQ62K、GQ62R、GQ62W和GQ62X的大肠杆菌生长明显优于负对照，说明含GQ62F、GQ62K、GQ62R、GQ62W和GQ62X的单突变体抗草铵膦的能力明显优于野生型；在更高草铵膦浓度(20mM，KP20)的培养基上，转化大豆GS1突变体GQ62X的大肠杆菌都还有明显生长。
这些结果说明GQ62F、GQ62K、GQ62R、GQ62W和GQ62X的单突变体都具有抗草铵膦的能力，且大豆GS1突变体GQ62X的抗草铵膦能力更强。
实验例3
参考实验例1的检测方法，验证实施例3提供的玉米GS1突变体ZQ62F(Q62F，玉米GS1的第62位的氨基酸Q突变为F)、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X(Q62X，玉米GS1的第62位的氨基酸Q删除)的草铵膦抗性。结果如图9所示。
根据图9的结果可看出：
在含0mM草铵膦(KP0)的培养基上，转化编码野生型玉米GS1(ZWT1)及玉米GS1突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X的编码基因的缺陷型菌株均能正常生长，表明由ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X编码的GS1都具有正常GS1酶活力；
在含2mM草铵膦(KP2)的培养基上，转化野生型玉米GS1的大肠杆菌基本上不能生长，但转化了玉米突变体ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X的大肠杆菌生长明显优于负对照，说明含ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X的单突变体抗草铵膦的能力明显优于野生型；在更高草铵膦浓度(20mM，KP20)的培养基上，转化玉米GS1突变体ZQ62K和ZQ62X的大肠杆菌都还有明显生长。
这些结果说明ZQ62F、ZQ62G、ZQ62K、ZQ62L、ZQ62M、ZQ62N、ZQ62P、ZQ62W、ZQ62Y和ZQ62X的单突变体都具有抗草铵膦的能力。
实验例4
参考实验例1的检测方法，验证实施例4提供的小麦GS1突变体TQ62G(Q62G，小麦GS1的第62位的氨基酸Q突变为G)、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X(Q62X，小麦GS1的第62位的氨基酸Q进行删除)的草铵膦抗性。结果如图10所示。
根据图10的结果可看出：
在含0mM草铵膦(KP0)的培养基上，转化编码野生型小麦GS1(TWT1)及小麦GS1突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的编码基因的缺陷型菌株均能正常生长，表明由TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X编码的GS1都具有正常GS1酶活力；
在含2mM草铵膦(KP2)的培养基上，转化野生型小麦GS1的大肠杆菌基本上不能生长，但转化了小麦突变体TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的大肠杆菌生长明显优于负对照，说明含TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的单突变体抗草铵膦的能力明显优于野生型；在更高草铵膦浓度(20mM，KP20)的培养基上，转化小麦GS1突变体TQ62G、TQ62H、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的大肠杆菌都还有明显生长。
这些结果说明TQ62G、TQ62H、TQ62I、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的单突变体都具有抗草铵膦的能力，且小麦GS1突变体TQ62G、TQ62H、TQ62K、TQ62L、TQ62R、TQ62Y和TQ62X的抗草铵膦能力更强。
实验例5
参考实验例1的检测方法，验证实施例5提供的油菜GS1突变体BQ62C(Q62C，油菜GS1的第62位的氨基酸Q突变为C)、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X(Q62X，油菜GS1的第62位的氨基酸Q进行删除)的草铵膦抗性。结果如图11所示。
根据图11的结果可看出：
在含0mM草铵膦(KP0)的培养基上，转化编码野生型油菜GS1(BWT1)及油菜GS1突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X的编码基因的缺陷型菌株均能正常生长，表明由BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X编码的GS1都具有正常GS1酶活力；
在含1mM草铵膦(KP1)的培养基上，转化野生型油菜GS1的大肠杆菌基本上不能生长，但转化了油菜突变体BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X的大肠杆菌生长明显优于负对照，说明含BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X的单突变体抗草铵膦的能力明显优于野生型；在更高草铵膦浓度(20mM，KP20)的培养基上，转化油菜GS1突变体BQ62P、BQ62R和BQ62X的大肠杆菌都还有明显生长。
这些结果说明BQ62C、BQ62F、BQ62G、BQ62K、BQ62L、BQ62M、BQ62P、BQ62R、BQ62W、BQ62Y和BQ62X的单突变体都具有抗草铵膦的能力，且油菜GS1突变体BQ62P、BQ62R和BQ62X的抗草铵膦能力更强。
实验例6
分别检测实施例1提供的OQ62X、实施例2提供的GQ62X、实施例3提供的ZQ62X、实施例4提供的TQ62X和实施例5提供的BQ62X突变体的酶动力学参数和在有草铵膦时的酶动力学参数，分别以野生型水稻GS1OWT1、野生型大豆GS1GWT1、野生型玉米GS1ZWT1、野生型小麦GS1TWT1和野生型油菜GS1BWT1为对照，方法如下：
载体构建：
将编码上述突变体的核酸序列克隆到原核表达载体pET32a中，测序验证克隆。
6His蛋白纯化：
通过6His和用标准方法纯化突变体酶蛋白，用Bradford法蛋白浓度测定试剂盒测定浓度，蛋白保存在蛋白贮存液中。
酶活测定：
1.仪器和试剂：酶标仪(德铁：HBS-1096A)，草铵膦(利尔化学股份有限公司)，底物L-谷氨酸钠(CAS：6106-04-3)。
2.操作步骤：
谷氨酰胺合成酶酶活测定反应液组分为：100mM Tris-HCl(pH7.5)，5mM ATP，10mM L-谷氨酸钠，30mM hydroxylamine，20mM MgCl 2。100μl反应液混匀后35℃预热5min后，加入1μl突变体蛋白液(蛋白浓度为200ug/ml)开始反应，35℃反应60min后，加入110μl反应终止液(55g/L FeCl 3·6H 2O，20g/L三氯乙酸，2.1％浓盐酸)终止反应，静置10min。5000×g离心10min，取200μl在500nm处测定光吸收值。
结果如图12所示。
根据图12的结果可以看出：
相对于野生型对照OWT1、GWT1、ZWT1、TWT1和BWT1，GS1突变体的Km值都较之偏高，说明GS突变体在降低对草铵膦抑制剂的敏感度的同时，降低了对正常底物的敏感度。GS1突变体的Vmax均高于野生型对照，说明这些突变体的酶催化能力有所提高。野生型对照对草铵膦很敏感，IC 50分别为7.93μM、13.55μM、8.92μM、7.22μM和1.53μM，突变体的IC 50均明显高于野生型对照，GQ62X、ZQ62X、TQ62X和BQ62X的IC 50远远高于野生型对照，表明突变体对草铵膦更不敏感。
从突变体IC 50和野生型IC 50的倍数关系上也可以看出，OQ62X、GQ62X、ZQ62X、TQ62X和BQ62X的IC 50分别是对应野生型GS1IC50的3.70倍、20.88倍、22.05倍、28.38倍和110.56倍，这些数值也说明突变体的酶活性远远高于野生型对照。这些数据从酶动力学上说明了突变体的抗草铵膦机制。
以上所述仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (10)

  1. 一种具有草铵膦抗性的谷氨酰胺合成酶突变体，其特征在于，其如下(1)或(2)所示：
    (1)：其由来源于植物的野生型谷氨酰胺合成酶的第n位发生突变得到；所述第n位的位置通过如下方式确定：所述野生型谷氨酰胺合成酶与参考序列比对，所述野生型谷氨酰胺合成酶的所述第n位对应于所述参考序列的第62位，其中，所述参考序列的氨基酸序列如SEQ ID NO.1所示；
    所述谷氨酰胺合成酶突变体的所述第n位的氨基酸为X，X包括K或删除；
    (2)：其与(1)所示的谷氨酰胺合成酶突变体至少具有85％以上的同一性、且与(1)所示的谷氨酰胺合成酶突变体在第n位的氨基酸相同、以及具有草铵膦抗性。
  2. 根据权利要求1所述的具有草铵膦抗性的谷氨酰胺合成酶突变体，其特征在于，所述植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、棉花、芝麻、向日葵、萝卜、胡萝卜、辣椒、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、芸薹属蔬菜、葫芦科植物、豆科植物、茄科植物、葱属植物、牧草、茶或木薯；
    优选地，所述牧草选自禾本科牧草或豆科牧草；
    优选地，所述芸薹属蔬菜选自芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜、油菜、花椰菜或甜菜；
    优选地，所述葫芦科植物选自黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜；
    优选地，所述豆科植物选自绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆、花生、或毛豆；
    葱属植物选自韭菜、大葱、洋葱、韭葱或大蒜；
    茄科植物选自茄子、番茄、烟草、辣椒或马铃薯。
  3. 根据权利要求1或2所述的具有草铵膦抗性的谷氨酰胺合成酶突变体，其特征在于，当所述植物为水稻时，X＝A、C、F、G、I、K、L、M、N、P、R、S、W、Y或删除；
    当所述植物为大豆时，X＝F、K、R、W或删除；
    当所述植物为玉米时，X＝F、G、K、L、M、N、P、W、Y或删除；
    当所述植物为小麦时，X＝G、H、I、K、L、R、Y或删除；
    当所述植物为油菜时，X＝C、F、G、K、L、M、P、R、W、Y或删除。
  4. 一种分离的核酸分子，其特征在于，其编码权利要求1-3任一项所述的具有草铵膦抗性的谷氨酰胺合成酶突变体。
  5. 一种载体，其特征在于，其含有权利要求4所述的核酸分子。
  6. 一种重组菌或重组细胞，其特征在于，其含有权利要求4所述的核酸分子或权利要求5所述的载体。
  7. 权利要求1-3任一项所述的具有草铵膦抗性的谷氨酰胺合成酶突变体、权利要求4所述的核酸分子、权利要求5所述的载体或权利要求6所述的重组菌或重组细胞在培育具有草铵膦抗性的植物品种中的应用。
  8. 根据权利要求7所述的应用，其特征在于，其包括如下至少一种的应用方式：
    将分离的核酸分子送入目的植物细胞，所述分离的核酸分子含有编码所述谷氨酰胺合成酶突变体的编码基因；
    将所述载体转化目的植物，所述载体含有编码所述谷氨酰胺合成酶突变体的编码基因；
    或，将所述重组菌或重组细胞导入目的植物，所述重组菌或重组细胞含有编码所述谷氨酰胺合成酶突变体的编码基因。
  9. 根据权利要求7所述的应用，其特征在于，其包括：修饰目的植物的内源谷氨酰胺合成酶基因，使其编码所述谷氨酰胺合成酶突变体。
  10. 根据权利要求8或9所述的应用，其特征在于，其包括：对植物细胞、组织、个体或群体进行诱变和筛选，使其编码所述谷氨酰胺合成酶突变体；
    所述植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、棉花、芝麻、向日葵、萝卜、胡萝卜、辣椒、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、芸薹属蔬菜、葫芦科植物、豆科植物、茄科植物、葱属植物、牧草、茶或木薯；
    优选地，所述牧草选自禾本科牧草或豆科牧草；
    优选地，所述芸薹属蔬菜选自芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜、油菜、花椰菜或甜菜；
    优选地，所述葫芦科植物选自黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜；
    优选地，所述豆科植物选自绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆、花生、或毛豆；
    葱属植物选自韭菜、大葱、洋葱、韭葱或大蒜；
    茄科植物选自茄子、番茄、烟草、辣椒或马铃薯。
PCT/CN2022/113149 2021-10-26 2022-08-17 一种谷氨酰胺合成酶突变体及应用 WO2023071438A1 (zh)

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