WO2022142936A1 - Mutant de glutamine synthase résistant au glufosinate-ammonium dérivé de plante, molécule d'acide nucléique et applications - Google Patents

Mutant de glutamine synthase résistant au glufosinate-ammonium dérivé de plante, molécule d'acide nucléique et applications Download PDF

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WO2022142936A1
WO2022142936A1 PCT/CN2021/133918 CN2021133918W WO2022142936A1 WO 2022142936 A1 WO2022142936 A1 WO 2022142936A1 CN 2021133918 W CN2021133918 W CN 2021133918W WO 2022142936 A1 WO2022142936 A1 WO 2022142936A1
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glufosinate
plant
glutamine synthase
wild
mutant
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Chinese (zh)
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邓龙群
陈容
张震
候青江
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
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    • 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)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present disclosure relates to the technical field of genetic engineering, in particular, to a plant-derived glutamine synthase mutant with glufosinate-ammonium resistance, a nucleic acid molecule and applications.
  • Glutamine synthetase is a key enzyme in plant nitrogen metabolism. It catalyzes the condensation of glutamate (Gln) and NH3 to form glutamine (Glu) in the glutamate synthetase cycle, and is involved in plant nitrogen-containing Metabolism of compounds.
  • the isoenzymes of higher plant GS (which belong to GSII) can be divided into two types: one is located in the cytoplasm called cytoplasmic GS (GS1) with a molecular weight of 38-40kDa; the other is located in the cytoplasm.
  • the chloroplast (or plastid) is called plastid-type GS (GS2), with a molecular weight of 44-45kDa.
  • Glufosinate (glufosinate, glufosinate ammoni ⁇ M, trade name Basta) is a glutamine synthase (GS1) inhibitor developed by Aventis (now Bayer), its active ingredient is phosphinothricin (referred to as PPT), chemical name It is (RS)-2-amino-4-(hydroxymethyl phosphinyl) ammonium butyrate.
  • PPT glutamine synthase
  • PPT phosphinothricin
  • RS phosphinothricin
  • the product was launched in 1986, and sales have increased year by year.
  • the target enzyme of glufosinate-ammonium is GS. Under normal circumstances, GS can form ⁇ -glutamyl phosphate from ATP and glutamate.
  • PPT is first combined with ATP, and the phosphorylated PPT occupies the eight reaction centers of GS molecule, which changes the spatial configuration of GS and inhibits the activity of GS. PPT inhibits all known forms of GS.
  • glufosinate-ammonium inhibition of GS can lead to disorder of nitrogen metabolism in plants, excessive accumulation of ammonium, disintegration of chloroplasts, inhibition of photosynthesis, and ultimately plant death.
  • the main method of cultivating glufosinate-resistant varieties is to use genetic engineering methods to introduce the glufosinate-resistant gene from bacteria into crops, so as to cultivate new varieties of transgenic glufosinate-resistant crops.
  • the most widely used glufosinate-resistant genes in agriculture are the bar gene from the strain Streptomyces hygroscopicus and the pat gene from the strain S. viridochromogenes. Bar gene and pat gene share 80% homology, and both can encode glufosinate acetylase, which can inactivate glufosinate acetylation.
  • Glufosinate-resistant varieties have great use value, among which resistant rape and corn have been commercialized in large areas.
  • GM due to the anti-GM wave, the acceptance of GM crops in the world is still low. Even in the Americas, where the GM crops are planted with the largest area, GM is mainly limited to a few crops such as corn, soybean, and cotton. In particular, the bar gene and pat gene are derived from microorganisms, rather than from the crops themselves, which are more likely to cause consumer resistance.
  • the glufosinate acetylase encoded by Bar and pat genes can acetylate and inactivate glufosinate, but it is difficult for the enzyme to completely inactivate glufosinate before the glufosinate is exposed to GS, because many GS are distributed in the Therefore, when glufosinate-ammonium is applied to bar gene and pat gene crops, 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. Overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate, but the degree of tolerance is insufficient for commercial application.
  • the present disclosure provides a glutamine synthase mutant with glufosinate resistance, which is shown in the following (1) or (2):
  • (1) it is obtained by mutating the n-th position of a plant-derived wild-type glutamine synthetase; the position of the n-th position is determined by comparing the wild-type glutamine synthase with the reference sequence. Right, the nth position of the wild-type glutamine synthetase corresponds to the 68th position of the reference sequence, wherein the amino acid sequence of the reference sequence is as shown in SEQ ID NO.1;
  • the research of the present disclosure found that the wild-type glutamine synthetase derived from plants was compared with the reference sequence, and the amino acid position corresponding to the 68th position of the reference sequence, that is, the n-th position, was mutated to mutate to D, E, G, H, N, P, Q, V or deletion, the obtained glutamine synthase mutant has glufosinate resistance, while maintaining its own biological enzyme catalytic activity.
  • Plants or recombinant bacteria transformed with the plant glutamine synthase mutant provided by the present disclosure can grow and develop normally in the presence of glufosinate-ammonium, and the plant glutamine synthase mutant is not only used for transgenic crop cultivation, but also It can be used to cultivate glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybean, corn, wheat, rapeseed, cotton and sorghum, etc., and has broad application prospects.
  • the above reference sequence is a wild-type glutamine synthase 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.
  • position n of wild-type glutamine synthase may also be position 68 on its own sequence (eg, corn, wheat, soybean, canola, etc.), but may not be position 68,
  • the position of the nth position is determined according to the aforementioned sequence alignment. As long as it is aligned with the reference sequence, the position corresponding to the 68th position of the reference sequence is the nth position described in the present disclosure, that is, the mutation site.
  • the plants include, but are not limited to, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato , cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon , tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, grass, lawn Any of grass, tea and tapioca.
  • any plant-derived wild-type glutamine synthase mutant obtained by mutating the above-mentioned 68th position has glufosinate resistance. Therefore, the glutamine synthetase mutant obtained by the above-mentioned mutation of any plant-derived wild-type glutamine synthase belongs to the protection scope of the present disclosure.
  • the glutamine synthetase mutant obtained by further mutation has at least 85% (for example, 85%, 86%, 87%, 88%, 89% of the glutamine synthetase mutant shown in (1)) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) or more identity, and its functions include enzymatic activity and glufosinate resistance Compared with the glutamine synthase mutant shown in (1), it is equivalent to or slightly decreased, or slightly increased or greatly increased, etc. Therefore, such glutamine synthase should also fall within the scope of the present disclosure.
  • the nth position is mutated to D, E, G, H, N, P, Q, V or deleted, and it is mutated to other
  • the amino acid also makes glutamine synthase resistant to glufosinate.
  • X A, C, D, E, F, G, H, I, K, L, M, N , P, Q, R, T, V, W, Y or delete;
  • X A, C, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y or deletion.
  • mutating the nth position to D, E, G, H, N, P, Q, V and other amino acids other than deletions will also make Glutamine synthase is glufosinate-resistant.
  • the rice wild-type glutamine synthase is SEQ ID NO.1:
  • the maize 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 rape wild-type glutamine synthase is SEQ ID NO.5:
  • the alignment method of the above similarity (Similarity) and identity (Identity) is: input the amino acid sequence of a species into the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for Protein Blast Align, find the similarity (Similarity) and identity (Identity) of this species and other species that need to be aligned from the alignment results.
  • the present disclosure provides an isolated nucleic acid molecule encoding the glufosinate-resistant glutamine synthase mutant of any of the above.
  • nucleic acid sequences encoding the above-mentioned glutamine synthase mutants can easily obtain nucleic acid sequences encoding the above-mentioned glutamine synthase mutants according to the degeneracy of codons.
  • the nucleic acid sequence encoding the above-mentioned glutamine synthase mutant can be obtained by making corresponding nucleotide mutations in the nucleic acid sequence encoding wild-type glutamine synthase. This is easily accomplished by those skilled in the art.
  • the encoding nucleic acid sequence of rice wild-type glutamine synthase is SEQ ID NO.6:
  • the corresponding nucleotide mutation is performed at the codon corresponding to the 68th position of the encoded amino acid sequence to obtain the rice glutamine synthase mutant encoding the above.
  • 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 wheat wild-type glutamine synthetase is SEQ ID NO.9:
  • the coding nucleic acid sequence of rape wild-type glutamine synthetase is SEQ ID NO.10:
  • the present disclosure provides a vector containing the nucleic acid molecule as described above.
  • the present disclosure provides a recombinant bacteria or a recombinant cell containing the above-mentioned nucleic acid molecule or the above-mentioned vector.
  • the present disclosure provides the glufosinate-resistant glutamine synthetase mutant as described in any one of the above, the nucleic acid molecule as described above, the vector as described above, or the recombinant bacteria or recombinant cells as described above when cultivating Application of glufosinate-ammonium-resistant plant varieties.
  • it comprises: transforming a plant of interest with a vector containing a gene encoding the glutamine synthase mutant.
  • it includes: modifying the endogenous glutamine synthase gene of the target plant to encode the glutamine synthase mutant.
  • it includes mutagenizing and screening plant cells, tissues, individuals or populations to encode the glutamine synthase mutant.
  • the present disclosure provides glutamine synthetase mutants
  • gene editing technology such as zinc finger endonucleases (ZFN, zinc-finger nucleases) in the art technology, transcription activator-like effector nucleases (TALEN, transcription activator-like effector nucleases) technology or CRISPR/Cas9), mutagenesis breeding technology (such as chemical, radiation mutagenesis, etc.), etc.
  • ZFN zinc finger endonucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR/Cas9 CRISPR/Cas9
  • mutagenesis breeding technology such as chemical, radiation mutagenesis, etc.
  • the plants of interest include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, Potato, cotton, soybean, canola, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter squash, bitter gourd, loofah, vegetable melon, watermelon, Melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, pasture, Any of lawn grass, tea and cassava.
  • Figure 1 provides rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, Results of partial alignment of amino acid sequences of OsY and OsX and wild-type rice GS1 OsGS1_WT.
  • Fig. 2 The partial ratio of amino acid sequences of soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX and wild-type soybean GS1 GmGS1_WT provided in Example 2 of the present disclosure to the results.
  • Figure 3 provides the maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, Results of partial alignment of amino acid sequences of ZmY and ZmX with wild-type maize GS1 ZmGS1_WT.
  • Figure 4 shows the partial alignment results of the amino acid sequences of wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX and wild-type wheat GS1 TaGS1_WT provided in Example 2 of the present disclosure.
  • Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and Partial alignment of amino acid sequences of BnX and wild-type rape GS1 BnGS1_WT.
  • FIG. 6 is a schematic structural diagram of the pADV7 vector provided in Experimental Example 1 of the present disclosure.
  • FIG. 7 is provided in Experimental Example 1 of the present disclosure.
  • Figure 8 provides the transformation example 2 of the present disclosure provided the soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX and wild-type soybean GS1 Growth results of GmGS1_WT Escherichia coli on media containing different concentrations of glufosinate.
  • Fig. 9 is the maize GS1 mutant ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, Growth results of E. coli ZmT, ZmV, ZmW, ZmY and ZmX and wild-type maize GS1 ZmGS1_WT on media containing different concentrations of glufosinate.
  • Figure 10 is provided in Experimental Example 4 of the present disclosure for the transformation of wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX and wild-type wheat GS1 TaGS1_WT provided in Example 4 in E. coli containing different concentrations Growth results on glufosinate-ammonium medium.
  • Fig. 11 The Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, Growth results of BnV, BnW, BnY and BnX and wild-type canola GS1 BnGS1_WT E. coli on media containing different concentrations of glufosinate.
  • FIG. 12 Rice GS1 mutant OsP, soybean GS1 mutant GmQ, corn GS1 mutant ZmV, wheat GS1 mutant TaG, rape GS1 mutant BnE, wild-type rice GS1 OsGS1_WT, wild-type soybean GS1 provided in Experimental Example 6 of the present disclosure Enzyme kinetic parameters and glufosinate resistance parameters IC50 of GmGS1_WT, wild-type maize GS1 ZmGS1_WT, wild-type wheat GS1 TaGS1_WT and wild-type rape GS1 BnGS1_WT.
  • Figure 13 is the amino acid sequence alignment results of different plant wild-type glutamine synthases; in the figure: TaGS1_WT: wheat wild-type glutamine synthase; OsGS1_WT: rice wild-type glutamine synthase; ZmGS1_WT: maize wild-type type glutamine synthase; GmGS1_WT: soybean wild-type glutamine synthase; BnGS1_WT: rape wild-type glutamine synthase.
  • Some embodiments of the present disclosure provide a plant-derived glufosinate-resistant glutamine synthase mutant, a nucleic acid molecule, and uses.
  • the glutamine synthase mutant provided by the present disclosure is originally derived from plants, and has glufosinate resistance after mutation. Plants transformed with the glutamine synthase mutant not only have glufosinate resistance, but also can grow normally. and development.
  • the rice (Oryza sativa) glutamine synthase (GS1) mutant provided in this example is composed of the wild-type rice glutamine synthase itself (named OsGS1_WT, the amino acid sequence is shown in SEQ ID NO.
  • the amino acid residue S at the 68th position of the nucleotide sequence of SEQ ID NO.6) is mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y or deletion
  • the obtained rice GS1 mutants were named OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX.
  • each rice GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.
  • the rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX provided in this example and coding Their nucleic acid molecules can be obtained by chemical synthesis.
  • the soybean (Glycine max) GS1 mutant provided in this example is derived from the wild-type soybean GS1 itself ((named GmGS1_WT, the amino acid sequence is shown in SEQ ID NO.3, and the encoding nucleotide sequence is SEQ ID NO.8)
  • the 68th position of (corresponding to the 68th position of the reference sequence SEQ ID NO.
  • soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY, GmX and wild-type soybean GS1 is shown in Figure 2, in the figure: the arrow The indicated positions are mutation sites.
  • soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX correspond to SEQ ID NO.3.
  • each soybean GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.
  • soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX provided in this example and the nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the maize (Zea mays) GS1 mutant provided in this example is composed of wild-type maize GS1 itself (named ZmGS1_WT, the amino acid sequence is shown in SEQ ID NO.2, and the coding nucleotide sequence is SEQ ID NO.7).
  • Position 68 (corresponding to position 68 of the reference sequence (SEQ ID NO. 1)) was mutated from amino acid residue S to A, C, D, E, F, G, H, I, K, L, M, N , P, Q, R, T, V, W, Y or delete.
  • the resulting maize GS1 mutants were named ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX, respectively.
  • Amino acids of maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY, ZmX and wild-type maize GS1
  • the sequence alignment is shown in Figure 3, in the figure: the position indicated by the arrow is the mutation site.
  • each maize GS1 mutant is at the position encoding the 68th amino acid, and the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.
  • Their nucleic acid molecules can be obtained by chemical synthesis.
  • the wheat (Triticum aestivum) GS1 mutant provided in this example is derived from wild-type wheat GS1 itself (named TaGS1_WT, the amino acid sequence is shown in SEQ ID NO.4, and the coding nucleotide sequence is SEQ ID NO.9).
  • Position 68 results from mutation of amino acid residue S to D, E, G, H, N, P, Q, V or deletion.
  • the obtained wheat GS1 mutants were named TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX, respectively.
  • each wheat GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.
  • the wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX provided in this example and the nucleic acid molecules encoding them can be obtained by chemical synthesis.
  • the Brassica napus GS1 mutant provided in this example is derived from the wild-type Brassica napus GS1 itself (named BnGS1_WT, the amino acid sequence is shown in SEQ ID NO.5, and the coding nucleotide sequence is SEQ ID NO.10).
  • Position 68 (corresponding to position 68 of the reference sequence (SEQ ID NO. 1)) was mutated from amino acid residue S to A, C, D, E, F, G, H, I, K, L, M, N , P, Q, T, V, W, Y or delete.
  • the resulting rapeseed GS1 mutants were named BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX, respectively.
  • Amino acid sequence ratio of rapeseed GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY, BnX and wild-type rapeseed GS1 As shown in Figure 5, in the figure: the position indicated by the arrow is the mutation site.
  • each rapeseed GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.
  • Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX and their encoding Nucleic acid molecules can be obtained by chemical synthesis.
  • the method of chemical synthesis was used to synthesize the rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ,
  • the coding genes of OsR, OsT, OsV, OsW, OsY and OsX were introduced with restriction enzyme sites (Pac1 and Sbf1) at both ends.
  • the GS1 mutants containing OsA (S68A, the amino acid S at position 68 of rice GS1 was mutated to A), OsC (S68C), OsD (S68D), OsE (S68E), and OsF were detected.
  • transform coding wild-type rice GS1 (OsGS1_WT) and rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX coding genes of the deficient strains can grow normally, indicating that OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL , OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX encoded GS1 all have normal GS1 enzymatic activity;
  • E. coli transformed with wild-type rice GS1 could not grow on medium containing 10 mM glufosinate-ammonium (KP10), but transformed rice mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL , OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX E.
  • KP10 mM glufosinate-ammonium
  • E. coli transformed with wild-type soybean GS1 could not grow substantially, but soybean mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP were transformed , GmQ, GmV, GmY and GmX E.
  • KP2 glufosinate-ammonium
  • ZmGS1_WT transform coding wild-type maize GS1
  • maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM
  • the defective strains of ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX can grow normally, indicating that ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL , ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX encoded GS1 all have normal GS1 enzymatic activity;
  • E. coli transformed with wild-type maize GS1 were essentially incapable of growing, but maize mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK were transformed , ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX E.
  • Transformation of genes encoding wild-type wheat GS1 (TaGS1_WT) and wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX on medium containing 0 mM glufosinate-ammonium (KP0) All strains can grow normally, indicating that GS1 encoded by TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX all have normal GS1 enzyme activity;
  • E. coli transformed with wild-type wheat GS1 were essentially unable to grow on medium containing 1 mM glufosinate-ammonium (KP1), but transformed wheat mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX
  • the growth of Escherichia coli was significantly better than that of the negative control, indicating that the single mutants containing TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX had significantly better glufosinate resistance than the wild type; E. coli transformed with wheat GS1 mutant TaN still grew significantly on the medium with phosphine concentration (10 mM, KP10).
  • E. coli transformed with wild-type rape GS1 could not grow substantially, but the rape mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK were transformed , BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX E.
  • coli growth was significantly better than the negative control, indicating that BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, The single mutants of BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY, and BnX were significantly more resistant to glufosinate-ammonium than wild-type; in medium with higher glufosinate concentration (20mM, KP20) E.
  • the nucleic acid sequence encoding the above mutant was cloned into the prokaryotic expression vector pET32a, and the clone was verified by sequencing.
  • the mutant enzyme protein was purified by 6His and standard methods, the concentration was determined with the Bradford method protein concentration assay kit, and the protein was stored in protein stock solution.
  • Instruments and reagents microplate reader (Deutsche Bronze: HBS-1096A), glufosinate-ammonium, substrate L-sodium glutamate (CAS: 6106-04-3).
  • the components of the glutamine synthase enzyme activity assay reaction solution were: 100 mM Tris-HCl (pH7.5), 5 mM ATP, 10 mM sodium L-glutamate, 30 mM hydroxylamine, 20 mM MgCl 2 . After mixing 100 ⁇ l of the reaction solution, it was preheated at 35°C for 5 minutes, and then 1 ⁇ l of mutant protein solution (protein concentration of 200ug/ml) was added to start the reaction. After 60 minutes of reaction at 35°C, 110 ⁇ l of reaction stop solution (55g/L FeCl 3 ⁇ 6H 2 was added) O, 20g/L trichloroacetic acid, 2.1% concentrated hydrochloric acid) to terminate the reaction and let stand for 10min. Centrifuge at 5000Xg for 10min, take 200 ⁇ l and measure the light absorption value at 500nm.
  • the Km values of the GS1 mutants were slightly higher, indicating that the GS mutants reduced the sensitivity to glufosinate-ammonium inhibitors while slightly reducing the sensitivity to glufosinate-ammonium inhibitors. normal substrate sensitivity.
  • the V max of the GS1 mutants was higher than that of the wild-type control, indicating that the enzymatic catalytic ability of these mutants was improved.
  • the wild-type control was sensitive to glufosinate-ammonium with IC50s of 7.93 ⁇ M, 13.55 ⁇ M, 8.92 ⁇ M, 7.22 ⁇ M and 1.5 ⁇ M, respectively.
  • the IC50s of the mutants were significantly higher than those of the wild-type control.
  • the IC50 was much higher than the wild-type control, indicating that the mutant was less sensitive to glufosinate.
  • the IC 50 of OsP, GmQ, ZmV, TaG and BnE are 63.05 times, 32.34 times, 36.69 times and 23.83 times that of the corresponding wild type GS1 IC 50 , respectively. and 15.83 times, these values also indicate that the enzyme activity of the mutant is much higher than that of the wild-type control.

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Abstract

L'invention concerne un mutant de glutamine synthase de glufosinate-ammonium dérivé d'une plante, une molécule d'acide nucléique et des applications. Par comparaison avec les glutamine synthases de type sauvage, le mutant a une mutation en position 68, qui change en D, E, G, H, N, P, Q, V ou une délétion après la mutation, et une telle mutation donne à la glutamine synthase une résistance au glufosinate-ammonium. Le mutant de glutamine synthase peut être utilisé pour la sélection de nouvelles variétés de plantes ayant une résistance au glufosinate-ammonium.
PCT/CN2021/133918 2020-12-31 2021-11-29 Mutant de glutamine synthase résistant au glufosinate-ammonium dérivé de plante, molécule d'acide nucléique et applications WO2022142936A1 (fr)

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