WO2023040565A1 - 一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用 - Google Patents

一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用 Download PDF

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WO2023040565A1
WO2023040565A1 PCT/CN2022/113148 CN2022113148W WO2023040565A1 WO 2023040565 A1 WO2023040565 A1 WO 2023040565A1 CN 2022113148 W CN2022113148 W CN 2022113148W WO 2023040565 A1 WO2023040565 A1 WO 2023040565A1
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glutamine synthetase
glufosinate
plant
wild
ammonium
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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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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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 with glufosinate-ammonium resistance, nucleic acid molecules and applications.
  • Glufosinate-ammonium also known as glufosinate, the trade name is basta, baston, and the chemical name is 4-[hydroxy(methyl)phosphono]-DL-homoalanine or 2-amino- Ammonium 4-[hydroxy(methyl)phosphono]butyrate.
  • Broad-spectrum contact herbicide developed by Bayer. By inhibiting the activity of glutamine synthetase (Glutamine synthetase, GS), the synthesis of glutamine in plants is blocked, and then the nitrogen metabolism in plants is disordered, the synthesis of proteins and nucleotides and other substances is reduced, and photosynthesis is blocked. Chlorophyll synthesis decreased. At the same time, the content of ammonium ions in the cells increases, causing the cell membranes to be damaged, the chloroplasts to disintegrate, and eventually the plants to die.
  • glufosinate-ammonium has a wide herbicidal spectrum, rapid inactivation and degradation in soil, and low toxicity to non-target organisms, crops can be made resistant to glufosinate-ammonium through transgenic technology, thereby selectively killing glufosinate-ammonium. Kills weeds without harming crops.
  • glufosinate-resistant genes in agriculture are the bar gene from the strain Streptomyces hygroscopicus and the pat gene from the strain S. viridochromogenes.
  • the bar gene and the pat gene have 80% homology, both can encode glufosinate-ammonium acetylase, and the enzyme can acetylate glufosinate-ammonium and inactivate it.
  • Glufosinate-ammonium resistance genes have been introduced into more than 20 crops including rice, wheat, corn, sugar beet, tobacco, soybean, cotton, potato, tomato, rape, sugarcane, etc. Commercial cultivation.
  • glufosinate-ammonium acetylase encoded by bar gene and pat gene can inactivate glufosinate-ammonium, but before glufosinate-ammonium contacts GS, it is difficult for glufosinate-ammonium acetylase to completely inactivate glufosinate-ammonium Since many GS are distributed on the cell membrane, some non-inactivated glufosinate can inhibit the activity of GS on the cell membrane, thereby interfering with the nitrogen metabolism of plants. Therefore, 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. Although the overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate-ammonium to a certain extent, the degree of tolerance to glufosinate-ammonium 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 57th 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 C, E, F, I, M, N, P, S, Y 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 57th position of the reference sequence, that is, the nth position, was mutated into C, E, F, I, M, N, P, S, Y or deletion, the resulting glutamine synthetase mutants are all resistant to glufosinate-ammonium, while maintaining their own glutamine synthetase with normal catalytic activity . Moreover, the plants or recombinant bacteria transformed with the plant glutamine synthetase mutant provided by the present disclosure can grow and develop normally in the presence of glufosinate-ammonium.
  • the plant glutamine synthetase mutant is not only 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 reference sequence shown in the above 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 the result of.
  • the nth position of the wild-type glutamine synthetase may also be the 57th position in its own sequence (such as corn, wheat, soybean, rapeseed, etc.), but it may not be the 57th position (for example, peanut corresponds to No. 58), 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 57th 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 57 all have glufosinate-ammonium resistance. That is to say, glutamine synthetase mutants obtained by performing the above mutations on wild-type glutamine synthetase derived from any plant also 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 target plant is selected from the group consisting of wheat, rice, barley, oat, 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, day lily, grape, strawberry, sugar cane, tobacco, Brassica vegetables, cucurbits, legumes, grasses, tea or cassava.
  • the 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.
  • 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 inventors also found that for glutamine synthetases from different plant sources, in addition to mutating the nth position to C, E, F, I, M, N, P, In addition to S, Y or deletion, mutating its nth position to other amino acids will also make glutamine synthetase resistant to glufosinate-ammonium.
  • X A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, V, W, Y or deletion;
  • X C, E, F, I, M, N, P, S, T, Y or delete.
  • 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 having glufosinate-ammonium resistance.
  • 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 57th 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 recombinant cell, which contains the above-mentioned nucleic acid molecule or 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.
  • the above application includes: performing mutagenesis and screening on plant cells, tissues, individuals or groups to encode glutamine synthetase mutants.
  • glutamine synthetase mutants provided in the present disclosure
  • ZFN zinc finger endonuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas9 CRISPR/Cas9
  • mutation breeding technology such as chemical, radiation mutagenesis, etc.
  • the target plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, potato, cotton, rapeseed, sesame, peanut, sunflower, radish, carrot , cauliflower, tomato, eggplant, peppers, leeks, green onions, onions, leeks, spinach, celery, amaranth, lettuce, crown chrysanthemum, day lily, grapes, strawberries, sugar cane, tobacco, brassica vegetables, cucurbits, legumes Plants, grass, tea or cassava.
  • the forage is selected from grasses or leguminous forages.
  • the Brassica vegetables are selected from turnips, Chinese cabbage, mustard greens, cabbage, kale, cabbage, bitter mustard, bluegrass, brassica, green vegetables or sugar beets.
  • the Cucurbitaceae plant is selected from cucumber, zucchini, pumpkin, wax gourd, bitter gourd, loofah, snake melon, watermelon or muskmelon.
  • the leguminous plant is selected from mung bean, broad bean, pea, lentil, soybean, kidney bean, cowpea or edamame.
  • the glutamine synthetase mutant with glufosinate-ammonium resistance 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 OG57A, OG57C, OG57D, OG57E, OG57F, OG57H, OG57I, OG57K, OG57L, OG57M, OG57N, OG57P, OG57Q, OG57R, OG57S, OG57V, OG57W, OG57Y and Partial alignment results of amino acid sequences of OG57X and wild-type rice GS1OWT;
  • Fig. 2 is the soybean GS1 mutants GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M, GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, GG57V, GG57W, GG57Y and Partial alignment results of the amino acid sequences of GG57X and wild-type soybean GS1GWT;
  • 3 shows the corn GS1 mutants ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, ZG57W, ZG57Y and Partial alignment results of the amino acid sequences of ZG57X and wild-type maize GS1ZWT;
  • Figure 4 is the result of partial alignment of the amino acid sequences of wheat GS1 mutants TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T, TG57Y and TG57X and wild-type wheat GS1TWT provided in Example 4 of the present disclosure;
  • Figure 5 shows the rapeseed GS1 mutants BG57C, BG57D, BG57E, BG57F, BG57H, BG57I, BG57K, BG57L, BG57M, BG57N, BG57P, BG57Q, BG57R, BG57S, BG57T, BG57V, BG57W, BG57Y and Partial alignment of amino acid sequences between BG57X 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 OG57A, OG57C, OG57D, OG57E, OG57F, OG57H, OG57I, OG57K, OG57L, OG57M, OG57N, OG57P, OG57Q, OG57R, OG57S, The growth results of Escherichia coli of OG57V, OG57W, OG57Y and OG57X and wild-type rice GS1OWT on media containing different concentrations of glufosinate-ammonium;
  • Figure 8 shows the soybean GS1 mutants GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M, GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, The growth results of Escherichia coli of GG57V, GG57W, GG57Y and GG57X and wild-type soybean GS1GWT on the medium containing different concentrations of glufosinate-ammonium;
  • Fig. 9 shows the maize GS1 mutants ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, Growth results of Escherichia coli of ZG57V, ZG57W, ZG57Y and ZG57X and wild-type maize GS1ZWT on media containing different concentrations of glufosinate-ammonium;
  • Fig. 10 is the Escherichia coli of wheat GS1 mutants TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T, TG57Y and TG57X provided in Experimental Example 4 of the present disclosure provided in Example 4 and wild-type wheat GS1TWT. Growth results on media containing different concentrations of glufosinate-ammonium;
  • Figure 11 shows the rape GS1 mutants BG57C, BG57D, BG57E, BG57F, BG57H, BG57I, BG57K, BG57L, BG57M, BG57N, BG57P, BG57Q, BG57R, BG57S, BG57T, BG57R, BG57S, BG57T, The growth results of Escherichia coli of BG57V, BG57W, BG57Y and BG57X and wild-type rapeseed GS1BWT on the medium containing different concentrations of glufosinate-ammonium;
  • Figure 12 shows the rice GS1 mutant OG57P, soybean GS1 mutant GG57P, corn GS1 mutant ZG57P, wheat GS1 mutant TG57P, rapeseed GS1 mutant BG57P, 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 57th amino acid residue G of SEQ ID NO.6) is mutated into A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, V, W, Y or deletion
  • the obtained rice GS1 mutants were named OG57A, OG57C, OG57D, OG57E, OG57F, OG57H, OG57I, OG57K, OG57L, OG57M, OG57N, OG57P, OG57Q, OG57R, OG57S, OG57V, OG57W, OG57Y, and OG57X.
  • each rice GS1 mutant is at the position encoding the 57th 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.
  • Rice GS1 mutants OG57A, OG57C, OG57D, OG57E, OG57F, OG57H, OG57I, OG57K, OG57L, OG57M, OG57N, OG57P, OG57Q, OG57R, OG57S, OG57V, OG57W, OG57Y and OG57X and their coding Nucleic acid molecules can be obtained by chemical synthesis.
  • 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 57th position (corresponding to the 57th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue G to C, D, E, F, H, I, K, L, M, N, P, Q , R, S, T, V, W, Y or deleted.
  • soybean GS1 mutants were named GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M, GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, GG57V, GG57W, GG57Y and GG57X.
  • soybean GS1 mutants GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M, GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, GG57V, GG57W, GG57Y and GG57X provided in this example correspond to In SEQ ID NO.3.
  • each soybean GS1 mutant is at the position encoding the 57th 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 GS1 mutants GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M, GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, GG57V, GG57W, GG57Y and GG57X provided by the present embodiment and their coding Nucleic acid molecules 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 57th position (corresponding to the 57th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue G to C, D, E, F, H, I, K, L, M, N, P, Q , R, S, T, V, W, Y or deleted.
  • the maize GS1 mutants obtained were named ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, ZG57W, ZG57Y and ZG57X.
  • each maize GS1 mutant is at the position encoding the 57th 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 ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, ZG57W, ZG57Y and ZG57X 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 57th position (corresponding to the 57th position of the reference sequence (SEQ ID NO.1)) is obtained by mutation of the amino acid residue G to C, E, F, I, M, N, P, S, T, Y or deletion.
  • the obtained wheat GS1 mutants were named TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T, TG57Y and TG57X, respectively.
  • each wheat GS1 mutant is at the position encoding the 57th 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 TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T, TG57Y and TG57X provided in this example and the 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 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 57th position (corresponding to the 57th position of the reference sequence (SEQ ID NO.1)) is mutated from amino acid residue G to C, D, E, F, H, I, K, L, M, N, P, Q , R, S, T, V, W, Y or deleted.
  • the obtained rapeseed GS1 mutants were named BG57C, BG57D, BG57E, BG57F, BG57H, BG57I, BG57K, BG57L, BG57M, BG57N, BG57P, BG57Q, BG57R, BG57S, BG57T, BG57V, BG57W, BG57Y and BG57X.
  • each rapeseed GS1 mutant is at the position encoding the 57th 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 BG57C, BG57D, BG57E, BG57F, BG57H, BG57I, BG57K, BG57L, BG57M, BG57N, BG57P, BG57Q, BG57R, BG57S, BG57T, BG57V, BG57W, BG57Y and BG57X and their encodings provided in this example Nucleic acid molecules can be obtained by chemical synthesis.
  • Escherichia coli transformed with wild-type rice GS1OWT could not grow on medium containing 5 mM glufosinate-ammonium (KP5), but transformed rice mutants OG57A, OG57C, OG57D, OG57E, OG57F, OG57H, OG57I, OG57K, OG57L, OG57M , OG57N, OG57P, OG57Q, OG57R, OG57S, OG57V, OG57W, OG57Y and OG57X Escherichia coli grew significantly better than the negative control, indicating that the E.
  • KP5 mM glufosinate-ammonium
  • the single mutants of OG57N, OG57P, OG57Q, OG57R, OG57S, OG57V, OG57W, OG57Y and OG57X have significantly better resistance to glufosinate-ammonium than the wild type; on the medium with better glufosinate concentration (20mM, KP20),
  • the Escherichia coli transformed with rice GS1 mutants OG57D, OG57E, OG57F, OG57H, OG57I, OG57L, OG57M, OG57P, OG57Q, OG57S, OG57V, OG57W, OG57Y and OG57X still grew significantly.
  • the Escherichia coli transformed with wild-type soybean GS1 basically could not grow, but transformed soybean mutants GG57C, GG57D, GG57E, GG57F, GG57H, GG57I, GG57K, GG57L, GG57M , GG57N, GG57P, GG57Q, GG57R, GG57S, GG57T, GG57V, GG57W, GG57Y and GG57X Escherichia coli grew significantly better than the negative control, indicating that the E.
  • ZWT transformation coding wild-type maize GS1
  • maize GS1 mutants ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P
  • the defective strains of ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, and ZG57Y could grow normally, indicating that ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R , ZG57S, ZG57T, ZG57V, and ZG57Y encoded GS1 all have normal GS1 enzyme activity;
  • E. coli transformed with wild-type maize GS1 could not grow, but transformed maize mutants ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N , ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, ZG57W, ZG57Y and ZG57X Escherichia coli grew significantly better than the negative control, indicating that the E.
  • the single mutants of ZG57P, ZG57Q, ZG57R, ZG57S, ZG57T, ZG57V, ZG57W, ZG57Y and ZG57X have significantly better resistance to glufosinate-ammonium than wild type; on the medium with higher glufosinate-ammonium concentration (20mM, KP20),
  • the Escherichia coli transformed with maize GS1 mutants ZG57C, ZG57D, ZG57E, ZG57F, ZG57H, ZG57I, ZG57K, ZG57L, ZG57M, ZG57N, ZG57P, ZG57Q, ZG57R, ZG57T, ZG57V, ZG57W, ZG57Y and ZG57X still had significant growth.
  • the Escherichia coli transformed with wild-type wheat GS1 basically could not grow, but the transformed wheat mutants TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T , TG57Y and TG57X Escherichia coli grew significantly better than the negative control, indicating that the ability of single mutants containing TG57C, TG57E, TG57F, TG57I, TG57M, TG57N, TG57P, TG57S, TG57T, TG57Y and TG57X to resist glufosinate-ammonium was significantly better than Wild type; Escherichia coli transformed with wheat GS1 mutants TG57C, TG57E, TG57F, TG57M, TG57N, TG57P, TG57T, TG57Y and TG57X to resist glufosinate-ammonium
  • Escherichia coli transformed with wild-type rapeseed GS1 basically could not grow, but transformed rapeseed mutants BG57C, BG57D, BG57E, BG57F, BG57H, BG57I, BG57K, BG57L, BG57M , BG57N, BG57P, BG57Q, BG57R, BG57S, BG57T, BG57V, BG57W, BG57Y and BG57X grew significantly better than the negative control, indicating that the E.
  • rapeseed GS1 mutants BG57C, BG57D, BG57E, BG57F, BG57K, BG57L, BG57M, BG57N, BG57P, BG57Q, BG57R, BG57W and BG57X had stronger resistance to glufosinate-ammonium.
  • the OG57P provided by Detecting Example 1 the GG57P provided by Example 2, the ZG57P provided by Example 3, the TG57P provided by Example 4 and the BG57P 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 OG57P, GG57P, ZG57P, TG57P and BG57P IC50 is much higher than the wild-type control, indicating that the mutant is less sensitive to glufosinate-ammonium.

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Abstract

提供了一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用，涉及基因工程技术领域。在野生型谷氨酰胺合成酶第n位进行突变可以获得谷氨酰胺合成酶突变体，突变后为C、E、F、I、M、N、P、S、Y或删除，该突变能赋予谷氨酰胺合成酶适于商业化应用的草铵膦抗性。该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。

Description

一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用
相关申请的交叉引用
本公开要求于2021年09月15日提交中国专利局的申请号为CN202111083393.3、名称为“一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用”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，涉及一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用。
背景技术
草铵膦，又称草丁膦，商品名为保试达(basta)、百速顿，化学名为4-[羟基(甲基)膦酰基]-DL-高丙氨酸或2-氨基-4-[羟基(甲基)膦酰基]丁酸铵。由拜耳公司开发的广谱触杀型灭生性除草剂。其通过抑制谷氨酰胺合成酶(Glutamine synthetase,GS)的活性，使得植物体内谷氨酰胺的合成受阻，继而植物体内氮代谢发生紊乱，蛋白质和核苷酸等物质的合成减少，光合作用受阻，叶绿素合成减少。同时细胞内的铵离子的含量增加，使得细胞膜遭到破坏，叶绿体解体，最终导致植物死亡。
由于草铵膦具有杀草谱广、在土壤中迅速失活降解、对非靶标生物低毒的特性，可以通过转基因技术使作物对草铵膦产生抗性，从而对草铵膦能够选择地杀死杂草而不危害作物。目前农业上应用最广的抗草铵膦基因是来源于菌株Streptomyces hygroscopicus的bar基因和菌株S.viridochromogenes的pat基因。bar基因和pat基因具有80％的同源性，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。草铵膦的抗性基因已经被导入了包括水稻、小麦、玉米、甜菜、烟草、大豆、棉花、马铃薯、番茄、油菜、甘蔗等20多种作物中，其中抗性油菜、玉米等已大面积商业化种植。
研究表明，bar基因和pat基因编码的草铵膦乙酰化酶可以使草铵膦乙酰化而失活，但是在草铵膦接触GS之前，草铵膦乙酰化酶很难使草铵膦完全失活，由于很多GS分布在细胞膜上，部分未失活的草铵膦可以抑制细胞膜上GS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GS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57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C、E、F、I、M、N、P、S、Y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57位的氨基酸位点即第n位进行突变，突变为C、E、F、I、M、N、P、S、Y或删除，所得到的谷氨酰胺合成酶突变体均具有草铵膦抗性，同时还能保持自身的谷氨酰胺合成酶具有正常的催化活性。而且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SEQ ID NO.1所示参考序列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均可得到相同的结果。
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57位(例如玉米、小麦、大豆、油菜等)，但也可能不是第57位(例如花生对应为第58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57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57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。也即，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也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，在一种可选的实施方式中，目的植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯。
在一种可选的实施方式中，所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，芸薹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、青菜或甜菜。
在一种可选的实施方式中，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，豆科植物包括不限于绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
在本公开应用可选的实施方式中，发明人还发现，针对不同的植物来源的谷氨酰胺合成酶，除了将其第n位突变为C、E、F、I、M、N、P、S、Y或删除之外，将其第n位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。
当目的植物为水稻时，X＝A、C、D、E、F、H、I、K、L、M、N、P、Q、R、S、V、W、Y或删除；
当植物为大豆、玉米、油菜时，X＝C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y或删除；
当植物为小麦时，X＝C、E、F、I、M、N、P、S、T、Y或删除。
可选的，在本公开的一些实施方案中，当植物为水稻时，水稻野生型谷氨酰胺合成酶为SEQ ID NO.1：
Figure PCTCN2022113148-appb-000001
可选的，在本公开的一些实施方案中，当植物为玉米时，玉米野生型谷氨酰胺合成酶为SEQ ID NO.2：
Figure PCTCN2022113148-appb-000002
可选的，在本公开的一些实施方案中，当植物为大豆时，大豆野生型谷氨酰胺合成酶为SEQ ID NO.3：
Figure PCTCN2022113148-appb-000003
可选的，在本公开的一些实施方案中，当植物为小麦时，小麦野生型谷氨酰胺合成酶为SEQ ID NO.4：
Figure PCTCN2022113148-appb-000004
可选的，在本公开的一些实施方案中，当植物为油菜时，油菜野生型谷氨酰胺合成酶为SEQ ID NO.5：
Figure PCTCN2022113148-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57位氨基酸。
Figure PCTCN2022113148-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，其编码上述的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 PCTCN2022113148-appb-000007
据此，在序列基础上，在对应于其编码氨基酸序列第57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 PCTCN2022113148-appb-000008
Figure PCTCN2022113148-appb-000009
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 PCTCN2022113148-appb-000010
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。
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 PCTCN2022113148-appb-000011
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 PCTCN2022113148-appb-000012
本公开还提供了一种载体，其含有上述的核酸分子。
本公开还提供了一种重组菌或重组细胞，其含有上述的核酸分子或载体。
重组菌可以选自农杆菌；重组细胞可以是感受态细胞。
本公开还提供了上述具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子、载体、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。
在本公开应用可选的实施方式中，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；
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。
在一种可选的实施方式中，目的植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯。
在一种可选的实施方式中，所述牧草选自禾本科牧草或豆科牧草。
在一种可选的实施方式中，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、青菜或甜菜。
在一种可选的实施方式中，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。
在一种可选的实施方式中，所述豆科植物选自绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
本公开提供的具有草铵膦抗性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。通过突变后具有了草铵膦抗性，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。
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OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X和野生型水稻GS1OWT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GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X和野生型大豆GS1GWT的氨基酸序列部分比对结果；
图3为本公开实施例3提供的玉米GS1突变体ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X和野生型玉米GS1ZWT的氨基酸序列部分比对结果；
图4为本公开实施例4提供的小麦GS1突变体TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X和野生型小麦GS1TWT的氨基酸序列部分比对结果；
图5为本公开实施例5提供的油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X和野生型油菜GS1BWT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OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X和野生型水稻GS1OWT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GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X和野生型大豆GS1GWT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ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X和野生型玉米GS1ZWT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TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X和野生型小麦GS1TWT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为本公开实验例5提供的转化实施例5提供的油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X和野生型油菜GS1BWT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OG57P、大豆GS1突变体GG57P、玉米GS1突变体ZG57P、小麦GS1突变体TG57P、油菜GS1突变体BG57P、野生型水稻GS1OWT、野生型大豆GS1GWT、野生型玉米GS1ZWT、野生型小麦GS1TWT和野生型油菜GS1BWT的草铵膦抗性参数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：TWT：小麦野生型谷氨酰胺合成酶体；OWT：水稻野生型谷氨酰胺合成酶体；ZWT：玉米野生型谷氨酰胺合成酶体；GWT：大豆野生型谷氨酰胺合成酶体；BWT：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。
本实施例及实验例中，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。
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OWT，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57位氨基酸残基G突变为A、C、D、E、F、H、I、K、L、M、N、P、Q、R、S、V、W、Y或删除得到，得到的水稻GS1突变体分别命名为OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X。
水稻GS1突变体OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X和野生型水稻GS1OWT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57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 PCTCN2022113148-appb-000013
本实施例提供的水稻GS1突变体OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X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GWT，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57位(对应于参考序列(SEQ ID NO.1)的第57位)由氨基酸残基G突变为C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y或删除得到。得到的大豆GS1突变体分别命名为GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X。
大豆GS1突变体GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X和野生型大豆GS1GWT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GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X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57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 PCTCN2022113148-appb-000014
本实施例提供的大豆GS1突变体GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X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ZWT，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57位(对应于参考序列(SEQ ID NO.1)的第57位)由氨基酸残基G突变为C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y或删除得到。得到的玉米GS1突变体分别命名为ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X。
玉米GS1突变体ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X和野生型玉米GS1ZWT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57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 PCTCN2022113148-appb-000015
本实施例提供的玉米GS1突变体ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X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TWT，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57位(对应于参考序列(SEQ ID NO.1)的第57位)由氨基酸残基G突变为C、E、F、I、M、N、P、S、T、Y或删除得到。得到的小麦GS1突变体分别命名为TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X。
小麦GS1突变体TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X和野生型小麦GS1TWT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57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 PCTCN2022113148-appb-000016
本实施例提供的小麦GS1突变体TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X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BWT，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57位(对应于参考序列(SEQ ID NO.1)的第57位)由氨基酸残基G突变为C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y或删除得到。得到的油菜GS1突变体分别命名为BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X。
油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X和野生型油菜GS1BWT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57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 PCTCN2022113148-appb-000017
本实施例提供的油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X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
检测实施例1提供的水稻GS1突变体OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X的草铵膦抗性，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OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X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OG57A(G57A，水稻GS1的第57位的氨基酸G突变为A)、OG57C(G57C)、OG57D(G57D)、OG57E(G57E)、OG57F(G57F)、OG57H(G57H)、OG57I(G57I)、OG57K(G57K)、OG57L(G57L)、OG57M(G57M)、OG57N(G57N)、OG57P(G57P)、OG57Q(G57Q)、OG57R(G57R)、OG57S(G57S)、OG57V(G57V)、OG57W(G57W)、OG57Y(G57Y)和OG57X(G57Δ，水稻GS1的第57位的氨基酸G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(OWT)及水稻GS1突变体OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X的编码基因的缺陷型菌株均能正常生长，表明由OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X编码的GS1都具有正常GS1酶活力；
在含5mM草铵膦(KP5)的培养基上，转化野生型水稻GS1OWT的大肠杆菌不能生长，但转化了水稻突变体OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X的大肠杆菌生长明显优于负对照，说明含OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X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OG57D、OG57E、OG57F、OG57H、OG57I、OG57L、OG57M、OG57P、OG57Q、OG57S、OG57V、OG57W、OG57Y和OG57X的大肠杆菌都还有明显生长。
这些结果说明OG57A、OG57C、OG57D、OG57E、OG57F、OG57H、OG57I、OG57K、OG57L、OG57M、OG57N、OG57P、OG57Q、OG57R、OG57S、OG57V、OG57W、OG57Y和OG57X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GG57C(G57C，大豆GS1的第57位的氨基酸G突变为C)、GG57D(G57D)、GG57E(G57E)、GG57F(G57F)、GG57H(G57H)、GG57I(G57I)、GG57K(G57K)、GG57L(G57L)、GG57M(G57M)、GG57N(G57N)、GG57P(G57P)、GG57Q(G57Q)、GG57R(G57R)、GG57S(G57S)、GG57T(G57T)、GG57V(G57V)、GG57W(G57W)、GG57Y(G57Y)和GG57X(G57Δ，大豆GS1的第57位的氨基酸G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(GWT)及大豆GS1突变体GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y的编码基因的缺陷型菌株均能正常生长，表明由GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y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GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X的大肠杆菌生长明显优于负对照，说明含GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X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GG57P和GG57T的大肠杆菌都还有明显生长。
这些结果说明GG57C、GG57D、GG57E、GG57F、GG57H、GG57I、GG57K、GG57L、GG57M、GG57N、GG57P、GG57Q、GG57R、GG57S、GG57T、GG57V、GG57W、GG57Y和GG57X的单突变体都具有抗草铵膦的能力，且大豆GS1突变体GG57P和GG57T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ZG57C(G57C，玉米GS1的第57位的氨基酸G突变为C)、ZG57D(G57D)、ZG57E(G57E)、ZG57F(G57F)、ZG57H(G57H)、ZG57I(G57I)、ZG57K(G57K)、ZG57L(G57L)、ZG57M(G57M)、ZG57N(G57N)、ZG57P(G57P)、ZG57Q(G57Q)、ZG57R(G57R)、ZG57S(G57S)、ZG57T(G57T)、ZG57V(G57V)、ZG57W(G57W)、ZG57Y(G57Y)和ZG57X(G57Δ，玉米GS1的第57位的氨基酸G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(ZWT)及玉米GS1突变体ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57Y的编码基因的缺陷型菌株均能正常生长，表明由ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57Y编码的GS1都具有正常GS1酶活力；
在含5mM草铵膦(KP5)的培养基上，转化野生型玉米GS1的大肠杆菌不能生长，但转化了玉米突变体ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X的大肠杆菌生长明显优于负对照，说明含ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X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ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X的大肠杆菌都还有明显生长。
这些结果说明ZG57C、ZG57D、ZG57E、ZG57F、ZG57H、ZG57I、ZG57K、ZG57L、ZG57M、ZG57N、ZG57P、ZG57Q、ZG57R、ZG57S、ZG57T、ZG57V、ZG57W、ZG57Y和ZG57X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TG57C(G57C，小麦GS1的第57位的氨基酸G突变为C)、TG57E(G57E)、TG57F(G57F)、TG57I(G57I)、TG57M(G57M)、TG57N(G57N)、TG57P(G57P)、TG57S(G57S)、TG57T(G57T)、TG57Y(G57Y)和TG57X(G57Δ，小麦GS1的第57位的氨基酸G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(TWT)及小麦GS1突变体TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X的编码基因的缺陷型菌株均能正常生长，表明由TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X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，转化野生型小麦GS1的大肠杆菌基本上不能生长，但转化了小麦突变体TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X的大肠杆菌生长明显优于负对照，说明含TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、 TG57S、TG57T、TG57Y和TG57X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TG57C、TG57E、TG57F、TG57M、TG57N、TG57P、TG57S、TG57Y和TG57X的大肠杆菌都还有明显生长。
这些结果说明TG57C、TG57E、TG57F、TG57I、TG57M、TG57N、TG57P、TG57S、TG57T、TG57Y和TG57X的单突变体都具有抗草铵膦的能力。
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BG57C(G57C，油菜GS1的第57位的氨基酸G突变为C)、BG57D(G57D)、BG57E(G57E)、BG57F(G57F)、BG57H(G57H)、BG57I(G57I)、BG57K(G57K)、BG57L(G57L)、BG57M(G57M)、BG57N(G57N)、BG57P(G57P)、BG57Q(G57Q)、BG57R(G57R)、BG57S(G57S)、BG57T(G57T)、BG57V(G57V)、BG57W(G57W)、BG57Y(G57Y)和BG57X(G57Δ，油菜GS1的第57位的氨基酸G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(BWT)及油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y的编码基因的缺陷型菌株均能正常生长，表明由BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y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BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X的大肠杆菌生长明显优于负对照，说明含BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X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BG57C、BG57D、BG57E、BG57F、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57W和BG57X的大肠杆菌都还有明显生长。
这些结果说明BG57C、BG57D、BG57E、BG57F、BG57H、BG57I、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57S、BG57T、BG57V、BG57W、BG57Y和BG57X的单突变体都具有抗草铵膦的能力，且油菜GS1突变体BG57C、BG57D、BG57E、BG57F、BG57K、BG57L、BG57M、BG57N、BG57P、BG57Q、BG57R、BG57W和BG57X的抗草铵膦能力更强。
实验例6
检测实施例1提供的OG57P、实施例2提供的GG57P、实施例3提供的ZG57P、实施例4提供的TG57P和实施例5提供的BG57P突变体在有草铵膦时的酶动力学参数，以野生型水稻GS1OWT、野生型大豆GS1GWT、野生型玉米GS1ZWT、野生型小麦GS1TWT和野生型油菜GS1BWT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：
野生型对照OWT、GWT、ZWT、TWT、BWT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OG57P、GG57P、ZG57P、TG57P和BG57P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，OG57P、GG57P、ZG57P、TG57P和BG57P的IC 50分别是对应野生型GS1IC 50的74.36倍、61.20倍、44.24倍、20.60倍和481.95倍，这些数据从酶动力学上说明了突变体的谷氨酰胺合成酶酶活力保持了较高的水平，也说明了抗草铵膦机制。
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57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C、E、F、I、M、N、P、S、Y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、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、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、豇豆或毛豆。
  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、D、E、F、H、I、K、L、M、N、P、Q、R、S、V、W、Y或删除；
    当所述植物为大豆、玉米、油菜时，X＝C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y或删除；
    当所述植物为小麦时，X＝C、E、F、I、M、N、P、S、T、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：
    将所述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；
    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、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、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、豇豆或毛豆。
PCT/CN2022/113148 2021-09-15 2022-08-17 一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用 WO2023040565A1 (zh)

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