GS mutant protein as well as mutant gene and application thereof

Abstract

The invention discloses a glutamine synthetase (GS) mutant protein as well as a mutant gene and application thereof. According to the rice GS A2N mutant disclosed by the invention, the glufosinate-ammonium tolerance concentration of rice wild type GS can be improved by about 25 times while the enzyme activity is improved; according to the combined mutant rice GS A2N Y158C mutant, the glufosinate-ammonium tolerance concentration of rice wild type GS can be improved by about 150 times while the enzyme activity is improved by about 2 times, and transgenic rice can tolerate glufosinate-ammonium treatment with 4 times of field recommended dosage. After corresponding homologous sites are mutated, GS mutant transgenic plants of wheat, corn, soybean and rape can also tolerate treatment of glufosinate-ammonium with 4 times of recommended dosage in the field.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of protein engineering and plant herbicide resistance, and particularly relates to a GS mutant protein, a mutant gene thereof and application thereof. BACKGROUND

[0002] In recent years, due to the rapid development of resistant weed populations, the lack of new herbicide targets, the frequent occurrence of rice phytotoxicity and the shortening of the service life of existing herbicides, the weed control in rice fields in China is facing severe challenges. It is of great significance to use biological technologies such as enzyme engineering directed evolution and gene editing to accelerate the breeding of new varieties of herbicide-resistant rice, so as to effectively control the outbreak of resistant weed populations, reduce herbicide phytotoxicity and expand the application range of existing herbicides.

[0003] Glufosinate-ammonium, also known as phosphinothricin, is a contact-type herbicide developed by Bayer Company, which has a broad application prospect due to its broad spectrum, high efficiency and slow resistance development. At present, the number of glufosinate-ammonium-resistant weeds reported is very small, so glufosinate-ammonium is an ideal target herbicide for breeding of herbicide-resistant crops. The target of glufosinate-ammonium is glutamine synthetase, and the inhibition of glutamine synthetase activity will lead to glutamine deficiency and ammonium accumulation in plants. The accumulation of ammonium will inhibit the light reaction in photosynthesis, block the electron transport chain in photosynthesis, cause the rupture of the thylakoid membrane, and ultimately lead to the death of plants.

[0004] At present, the main method for breeding glufosinate-ammonium-resistant crop varieties is to introduce glufosinate-ammonium-degrading genes to breed new transgenic glufosinate-ammonium-resistant crop varieties. At present, the bar and pat The most widely used gene in agriculture encodes glufosinate acetylase, which can acetylate glufosinate to lose herbicidal activity. However, in order to introduce exogenous resistance genes, it is almost inevitable to use transgenic technology to construct resistant crops. At present, the safety of transgenic crop varieties is still controversial in the world, and different countries have different attitudes towards the commercialization of transgenic crops. At present, affected by the policy and the cost of registration of transgenic crops, the application and promotion of transgenic herbicide-resistant crops have great resistance. In recent years, the gene editing technology represented by CRISPR system makes it possible to edit the herbicide target gene. Therefore, it is of great significance to use enzyme engineering means such as directed evolution to modify the herbicide target to obtain mutants with herbicide resistance for herbicide-resistant crop breeding. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a GS mutant protein capable of improving the glufosinate resistance of plants.

[0006] The present application also aims to provide an expression vector for constructing a transformed plant to cultivate a glufosinate-resistant transgenic crop or a gene-edited crop GS a mutant gene.

[0007] The present application also aims to provide an expression cassette, a recombinant vector or a recombinant bacterium.

[0008] The present application also aims to provide a use of the GS mutant protein, the GS mutant gene, the expression cassette, the recombinant vector or the recombinant bacterium in cultivating a glufosinate-resistant plant.

[0009] Technical solution: In order to solve the above problems, the present application provides a GS mutant protein, which comprises the following (a), (b) or (c): (a) a protein in which the 2nd and / or 158th amino acid of a wild-type GS protein of rice or a homologous site of a GS protein of other plants is mutated to have higher glutamine synthetase activity and / or glufosinate resistance; the amino acid sequence of the wild-type GS protein of rice is shown in SEQ ID No. 1; (b) a protein derived from (a) in which the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and has glutamine synthetase activity and / or glufosinate resistance; (c) a protein having at least 85% or more amino acid sequence identity with any one of the GS mutant proteins in (a) or (b) and having higher glutamine synthetase activity and / or glufosinate resistance than the wild-type GS protein of plants.

[0010] The mutant protein of the present application has higher glutamine synthetase activity and glufosinate tolerance concentration than the wild-type GS protein of rice. As a preferred embodiment, when the 2nd amino acid of the wild-type GS protein of rice or the homologous site of other plants is mutated to T, M, Q, L, K, Y, N, G, H, I, P, F or S, the glutamine synthetase activity and / or the glufosinate tolerance concentration are both improved. As a preferred embodiment, the 2nd amino acid of the wild-type GS protein of rice or the homologous site of other plants is mutated to H or N.

[0011] The application also provides a GS mutant protein, wherein the amino acid sequence of the GS mutant protein is mutated to N at the 2nd amino acid of the wild-type GS protein of rice or the homologous site of other plants, and the glutamine synthetase activity is higher than that of the wild-type GS protein of rice when the 158th amino acid of the wild-type GS protein of rice or the homologous site of other plant GS proteins is mutated to S, G, M, N, A, V, C, W, T, F, Q, L or H; the glufosinate tolerance concentration is higher than that of the wild-type GS protein of rice when the 158th amino acid of the wild-type GS protein of rice or the homologous site of other plant GS proteins is mutated to C, D, A, F, N, I, L, V, E, G, W, H, Q, S, M or T; preferably, the enzyme activity and glufosinate tolerance concentration of the rice mutant GS2 A2N Y158C, rice mutant GS2 A2N Y158A, rice mutant GS2 A2N Y158T and rice mutant GS2 A2N Y158M are all higher than those of the rice GS A2N mutant protein, and are about 150 times higher than those of the wild-type GS protein of rice.

[0012] wherein, because the wild-type GS sequences of all plants are highly homologous and conservative, and have basically the same function and structure in the plant body. Therefore, the wild-type GS from any plant source can have glufosinate resistance after mutation at the corresponding homologous sites. Therefore, the other plants include but are not limited to wheat, corn, soybean or rape, and other plants are also included. The GS mutant obtained by mutation of the wild-type GS from any plant source belongs to the protection scope of the application.

[0013] when the plant is wheat, corn, soybean or rape, the homologous sites of the 2nd and 158th amino acid sites of the wild-type GS protein of rice are located at the 2nd and 158th amino acids of the wild-type GS protein of wheat, the wild-type GS protein of corn, the wild-type GS protein of soybean and the wild-type GS protein of rape, respectively; the amino acid sequence of the wild-type GS protein of wheat is shown in SEQ ID No. 2, the amino acid sequence of the wild-type GS protein of corn is shown in SEQ ID No. 3, the amino acid sequence of the wild-type GS protein of soybean is shown in SEQ ID No. 4, and the amino acid sequence of the wild-type GS protein of rape is shown in SEQ ID No. 5.

[0014] The application also provides a method for preparing a GS mutant protein, comprising the following steps: GS The application also provides a mutant gene encoding the GS mutant protein.

[0015] wherein, the mutant gene is a wild-type GS gene of rice GS The mutant gene is a wild-type GS gene of rice GS The 4th, 5th and 6th nucleotides of the wild-type GS gene of rice are mutated, and the nucleotides encoding the homologous amino acid sites in other plant GS genes are also mutated; and the mutant gene is a wild-type GS gene of riceGS the 4th, 5th, 6th, 472nd, 473rd and / or 474th nucleotide of the gene is mutated, and other plants GS the nucleotide in the gene encoding the corresponding homologous amino acid site is mutated.

[0016] wherein the GS the base corresponding to the mutation of the 2nd amino acid of the wild-type GS protein of rice, wild-type GS protein of wheat, wild-type GS protein of corn, wild-type GS protein of soybean or wild-type GS protein of rape to K is aaa or aag, to S is tct, tca, tcc, tcg, agt or agc, to G is ggt, ggc, gga or ggg, to M is atg, to Y is tat or tac, to N is aat or aac, to T is act, acc, acg or aca, to F is ttt or ttc, to I is att, atc or ata, to P is cct, cca, ccg or ccc, to Q is caa or cag, to L is ctt, ctc, cta, ctg, tta or ttg, to H is cat or cac; the base corresponding to the mutation of the 158th amino acid Y of the wild-type GS protein of rice, wild-type GS protein of wheat, wild-type GS protein of corn, wild-type GS protein of soybean or wild-type GS protein of rape to A is gct, gcc, gca or gcg; to V is gtt, gtg, gta or gtg; to C is tgt or tgc; to W is tgg; to S is tct, tca, tcc, tcg, agt or agc; to G is ggt, ggc, gga or ggg; to M is atg; to N is aat or aac; to T is act, acc, acg or aca; to F is ttt or ttc; to Q is caa or cag; to L is ctt, ctc, cta, ctg, tta or ttg; to H is cat or cac; to I is att, atc or ata; to D is gat or gac; to E is gaa or gag.

[0017] As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10. GS As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10. GS As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10.

[0018] As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10.

[0019] As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10.

[0020] As preferred, the corresponding base sequence of the wild type GS protein mutated to different amino acids is shown in Table 1, the wild type GS gene sequence of rice is shown in SEQ ID No. 6, the wild type GS gene sequence of wheat is shown in SEQ ID No. 7, the wild type GS gene sequence of corn is shown in SEQ ID No. 8, the wild type GS gene sequence of soybean is shown in SEQ ID No. 9, and the wild type GS gene sequence of rape is shown in SEQ ID No. 10.

[0021] The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene. GS The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene.

[0022] The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene. GS The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene.

[0023] The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene.

[0024] The application also includes an expression cassette, a recombinant vector or a recombinant bacteria containing the mutant gene.

[0025] Beneficial effects: Compared with the prior art, the present application has the following advantages: by comparing the growth curves of mutant and wild type glutamine synthetase of rice complementing auxotrophic E. coli, the present application improves the glutamine synthetase activity of rice at the 2nd amino acid site. Among them, the A2N mutation can greatly improve the enzyme activity while increasing the glufosinate tolerance concentration of wild type GS of rice by about 25 times; on the basis of A2N mutation, the combination mutation Y158C can further improve the enzyme activity and increase the glufosinate tolerance concentration to about 150 times of wild type GS of rice. The experimental results of spraying glufosinate in the field show that the transgenic rice and wheat containing the GS mutant protein gene of the present application can tolerate 4 times the recommended dose of glufosinate treatment in the field. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Rice GS A2 site mutant complementing auxotrophic E. coli growth curve under different glufosinate concentrations; Figure 2 Wild type rice GS complementing auxotrophic E. coli growth curve under different glufosinate concentrations; Figure 3 Rice GS A2N Y158 site mutant complementing auxotrophic E. coli growth curve under different glufosinate concentrations; the different colored curves in the figure represent wild type and mutant, respectively, wherein WT represents wild type rice GS complementing auxotrophic E. coli, Y represents rice GS A2N mutant bacteria (Y158 does not mutate), and the rest C, D, A, F, N, I, K, L, V, E, G, R, W, P, H, Q, S, M or T represent mutant bacteria with the above-mentioned site mutation at the 158th amino acid of the rice GS A2N mutant protein.

[0027] Figure 4 Effect of glufosinate treatment on transgenic plants of rice GS mutant; Figure 5 Effect of glufosinate treatment on transgenic plants of wheat GS mutant; Figure 6 Effect of glufosinate treatment on transgenic plants of corn GS mutant; Figure 7 Effect of glufosinate treatment on transgenic plants of soybean GS mutant; Figure 8 Effect of glufosinate treatment on transgenic plants of rapeseed GS mutant. DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] Example 1: Screening of mutants of rice GS

[0030] Rice GS mutants resistant to glufosinate were screened using spontaneously mutant strains of *E. coli* XL-1 Red. The specific steps are as follows: Wild-type rice G mutants were synthesized using... S Gene fragment (GenScript, sequence shown in SEQ ID No. 6), pSC-19 plasmid (the pUC19 vector replication origin site was replaced with pSC101, and deletion was performed) lacO Using the pSC-19 plasmid (sequence shown in SEQ ID No. 11) as a template to relieve repression, wild-type rice was amplified using a high-fidelity enzyme (PhantaMax Super-Fidelity DNA Polymerase, Novizan). GS Gene fragments and pSC-19 linearized vector.

[0031] The primer sequences for amplifying the GS gene fragment are as follows: F: 5'- ACACAGGAAACAGCT ATGGCTTCTCTCACCGATCTCG -3'; R: 5'- TCGGGGCTGGCTTAA TCAGGGCTTCCAGATGATGGTG -3'.

[0032] The primer sequences for amplifying the linearized pSC-19 vector are as follows: F: 5'- TTAAGCCAGCCCCGACACC -3'; R: 5'-AGCTGTTTCCTGTGTGAAAGAAACCG-3'.

[0033] The PCR annealing temperature was 55℃, and the system and other reaction conditions were performed in accordance with the Novizan high-fidelity enzyme instructions.

[0034] The amplified wild-type rice GS The PCR products of the gene fragment and the linearized pSC-19 vector product were purified and then ligated (ClonExpress II One Step Cloning Kit, Novizan) to replace the plasmid. lacZ alphaOpen reading frame. The ligation product was transformed into *E. coli* DH5α. After the clones grew, transformants were selected for sequencing verification. Transformants with correct sequencing results were transferred, and the plasmid was extracted to obtain the rice wild-type GS complement plasmid (FastPure Plasmid Mini Kit, Novizan). This plasmid was transformed into *E. coli* XL-1 Red competent cells (Agilent Technologies), and transformants were selected and passaged in M9 basal medium plates containing 5 μM glufosinate. Colonies that grew were selected, cultured, and the plasmid (FastPure Plasmid Mini Kit, Novizan) was extracted and sequenced to identify the rice GS mutation. This invention preliminarily screened a rice GS mutant protein with glufosinate resistance using this screening method, namely rice GS A2T, which is a mutation of A to T at the second amino acid position of the GS protein.

[0035] Example 2: Enzyme activity and resistance assessment of GS and related mutants

[0036] After initial screening of GS mutants in Example 1, their enzyme activity and resistance were subsequently verified a second time.

[0037] 1. Site-directed mutagenesis of GS: Using the wild-type GS complement plasmid from Example 1 as a template, a saturated mutant of rice GS A2 was constructed by site-directed mutagenesis using PCR. Primer F: 5'- CAGGAAACAGCTAT GnnnTCTCTCACCGATCTCGTCAACCTC -3'; R: 5'- CATAGCTGTTTCCTGTGTGAAAGAAACCG -3' (uppercase letters represent template-specific sequences, lowercase letters nnn represent sequences identical to the mutated amino acid codons; specific base sequences are shown in Table 1, underlined parts are recombinant homologous arm sequences) was amplified by PCR using Phanta Max Super-Fidelity DNA Polymerase (Novizan). The PCR annealing temperature was 55℃, and the PCR system and other reaction conditions were performed according to the manufacturer's instructions. The PCR product was then digested with the restriction fast cutase QuickCut. ™ DpnI (Takara, item number 1609) were digested, and then circularized using a homologous recombination kit (ClonExpress II One Step Cloning Kit, Novagen). The ligation products were transformed into E. coli DH5a, and after the clones grew, the transformants were picked for sequencing to verify that the sequencing of the correct transformants was correct. The rice GS site-directed mutant A2N plasmid, A2G plasmid, A2E plasmid, A2V plasmid, A2L plasmid, A2I plasmid, A2P plasmid, A2F plasmid, A2Y plasmid, A2W plasmid, A2S plasmid, A2T plasmid, A2C plasmid, A2M plasmid, A2Q plasmid, A2D plasmid, A2K plasmid, A2R plasmid, and A2H plasmid (FastPure Plasmid Mini Kit, Novagen) were extracted for standby use. The above mutant plasmids and the rice wild-type GS complementation plasmid were transformed into the competent cells of the BL21 (DE3) -glnA knockout strain (purchased from Chongqing Yingmaoshengye Biotechnology Co., Ltd., BL21 (DE3) -glnA knockout strain, item number EC1070) for use in evaluating GS enzyme activity and resistance using growth curves.

[0038] Table 1. Corresponding base sequence of mutant amino acid codons in site-directed mutation primers

[0039] 2. The E. coli BL21 (DE3) containing the rice wild-type GS complementation plasmid constructed in Example 1, the rice GS site-directed mutant A2N plasmid, A2G plasmid, A2E plasmid, A2V plasmid, A2L plasmid, A2I plasmid, A2P plasmid, A2F plasmid, A2Y plasmid, A2W plasmid, A2S plasmid, A2T plasmid, A2C plasmid, A2M plasmid, A2Q plasmid, A2D plasmid, A2K plasmid, A2R plasmid, and A2H plasmid were respectively streaked on LB plates containing ampicillin (100 ppm). Delta glnA The normal growth of single colonies was picked and inoculated into 24-well deep well plates for overnight culture, and the deep well plates contained LB liquid medium containing ampicillin (100 ppm). For the bacterial solution transformed with the rice GS A2 site-directed mutant plasmid or the rice wild-type GS complementation plasmid, the bacteria were washed with ultrapure water and the bacterial solution was diluted to OD 600 nm=0.2, 1 μL of the bacterial solution was taken and inoculated on the plate. The plate was M9 solid medium (6.78 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 0.241 g / L MgSO4, 0.011 g / L CaCl2, 4 g / L glucose; 2% agar) containing ampicillin (100 ppm), and different concentrations of glufosinate (0, 10, 20, 25 μM) were added according to the actual situation to evaluate the herbicide resistance of mutant bacteria and wild-type bacteria; the plate was placed at 30°C for culture, and the growth state of the bacterial colony was compared without the addition of glufosinate to determine the activity of different GS enzymes; the growth state of the bacterial colony was compared with the addition of the same concentration of glufosinate to determine the tolerance of wild-type GS protein and different GS mutant proteins to glufosinate, and the results are shown in Figure 1 In addition, since the wild-type GS protein has weak resistance, in order to improve the discrimination, the growth curve was used to further refine the evaluation of the tolerance of the wild-type GS protein to glufosinate. 2 μL of the bacterial solution after washing with ultrapure water was inoculated into a 24-well deep well plate, and the deep well plate was 300 μL of M9 liquid medium (6.78 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 0.241 g / L MgSO4, 0.011 g / L CaCl2, 4 g / L glucose) containing ampicillin (100 ppm), and different concentrations (0, 0.3, 0.5, 1, 3, 5 μM) of glufosinate were added according to the actual situation to evaluate the herbicide resistance of wild-type bacteria; the enzyme marker was used to measure the OD 600 nm value of the deep well plate every 20 min at 30°C with shaking, and the growth curve was drawn. By comparing the growth curve of the wild-type rice GS complementation bacteria with the addition of different concentrations of glufosinate with the growth curve of the wild-type rice GS complementation bacteria without the addition of glufosinate, the tolerance of the wild-type rice GS to glufosinate was determined.

[0040] According to the results Figure 1 , when the 2nd amino acid A of rice GS is mutated to T, M, Q, L, K, Y, N, G, H, I, P, F or S, the enzyme activity is improved; among them, the rice GS A2H mutant protein can tolerate about 10 μM glufosinate concentration, and the rice GS A2N mutant protein can tolerate about 25 μM glufosinate concentration. As a comparison, the wild-type rice GS protein can only tolerate about 1 μM glufosinate Figure 2 . Therefore, compared with the wild-type rice GS, the glufosinate tolerance concentrations of the rice GS A2H and rice GS A2N mutant proteins are increased by about 10 times and 25 times, respectively.

[0041] Example 3: Iterative screening of GS mutants and enzyme activity and resistance evaluation of related mutants

[0042] The rice GS site-directed mutant A2N plasmid constructed in Example 2 was transformed into E. coli XL-1 Red competent cells (Agilent), and the transformants were picked and subcultured on M9 minimal medium plates containing 100 μΜ phosphinothricin. The colonies that could grow were picked and cultured, and the plasmids were extracted (FastPure Plasmid Mini Kit, Omega) for sequencing to identify the rice GS mutation. Through this screening method, the present application iteratively screened a rice GS mutant protein with higher phosphinothricin resistance than rice GS A2N, i.e., rice GS A2N Y158C.

[0043] The rice GS A2N Y158 saturation mutant was constructed using the method in Example 2. Among them, the site-directed mutation template was the rice GS site-directed mutant A2N plasmid constructed in Example 2, and the primers used were F: 5'- nnnTGTGGTATCGGTGCTGACAAGTC -3'; R: 5'- GTAAGGACCCTGAGGACCAGGG -3' (capital letters are template-specific sequences, and lowercase letters nnn represent the same sequence as the mutant amino acid codon, and the specific base sequence is shown in Table 1, and the underlined part is the recombination homologous arm sequence). The remaining experimental steps and methods were the same as in Example 2. CCTCAGGGTCCTTAC

[0044] Since the rice GS A2N mutant protein has a significantly improved resistance compared to the wild-type rice GS, in order to improve the discrimination, the enzyme activity and resistance of the rice GS A2N Y158 saturation mutant were evaluated using the growth curve. E. coli BL21 (DE3) containing the wild-type rice GS complementation plasmid, the rice GS A2N plasmid, the rice GS A2N Y158N plasmid, the rice GS A2N Y158G plasmid, the rice GS A2N Y158E plasmid, the rice GS A2N Y158V plasmid, the rice GS A2N Y158L plasmid, the rice GS A2N Y158I plasmid, the rice GS A2N Y158P plasmid, the rice GS A2N Y158F plasmid, the rice GS A2N Y158A plasmid, the rice GS A2N Y158W plasmid, the rice GS A2N Y158S plasmid, the rice GS A2N Y158T plasmid, the rice GS A2N Y158C plasmid, the rice GS A2N Y158M plasmid, the rice GS A2N Y158Q plasmid, the rice GS A2N Y158D plasmid, the rice GS A2N Y158K plasmid, the rice GS A2N Y158R plasmid, and the rice GS A2N Y158H plasmid, respectively, were used.​Delta glnA Streaked on LB plates containing ampicillin (100 ppm). Positive strains were obtained - rice mutant GSA2N Y158N, GSA2N Y158G, GSA2N Y158E, GSA2N Y158V, GSA2N Y158L, GSA2N Y158I, GSA2N Y158P, GSA2N Y158F, GSA2N Y158A, GSA2N Y158W, GSA2N Y158S, GSA2N Y158T, GSA2N Y158C, GSA2N Y158M, GSA2N Y158Q, GSA2N Y158D, GSA2N Y158K, GSA2N Y158R, GSA2N Y158H.

[0045] Picked normal growth of single colonies, inoculated into 24-well deep well plates, deep well plates containing ampicillin (100 ppm) 300 μL M9 liquid medium (6.78 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 0.241 g / L MgSO4, 0.011 g / L CaCl2, 4 g / L glucose), and according to the actual situation to add different concentrations (0 μM, 150 μM) of glufosinate to evaluate the herbicide resistance of wild type and mutant bacteria; using microplate reader at 30°C, every 20 min to determine the OD 600 nm value of deep well plate, and draw the growth curve. By comparing the trend of the growth curve without adding glufosinate to judge the level of different GS enzyme activities; by comparing the trend of the growth curve when adding the same concentration of glufosinate to judge the tolerance of different GS to glufosinate.

[0046] According to Figure 3 the growth curve results, when the rice GS A2N mutant plasmid is used as a template, the rice GS A2N mutant protein is further mutated from Y to S, G, M, N, A, V, C, W, T, F, Q, L or H at the 158th amino acid relative to the wild type rice GS protein, the enzyme activity is higher than that of the wild type rice GS; when Y is mutated to C, D, A, F, N, I, L, V, E, G, W, H, Q, S, M or T, the tolerance to 150 μM glufosinate treatment is higher than that of the wild type rice GS (WT) in Figure 3 the rice mutant GSA2N Y158C, the rice mutant GSA2N Y158A, the rice mutant GSA2N Y158T, and the rice mutant GSA2N Y158M enzyme activity and glufosinate tolerance concentration are higher than those of the wild type rice GS A2N mutant protein (Figure 3 The levels of Y in the mutants were significantly higher, and the growth curves of these mutants treated with 150 μM glufosinate were still better than those of the wild-type GS supplemented rice strain without glufosinate treatment. In contrast, the wild-type GS protein in rice could only tolerate approximately 1 μM glufosinate (…). Figure 2 Therefore, compared to wild-type GS rice, the glufosinate tolerance concentration of the above mutants is at least 150 times higher.

[0047] Example 4: Agrobacterium-mediated genetic transformation of rice and identification of transgenic positive plants 1. Construction of rice expression vector for mutant genes and Agrobacterium-mediated transformation Using the rice GS site-directed mutant A2N Y158C plasmid constructed in Example 3 as a template, gene-specific primer F: 5'- ACTAGGTACCTGCAGGTCGACGGATCC ATGGCTTCTCTCACCGATCTCG-3', R: 5'- GACTCCTCT TAGAATTCCCGGGGATCC The gene sequence containing the corresponding rice mutant GS A2N Y158C protein (as shown in SEQ ID No. 12) was amplified using TCAGGGCTTCCAGATGATGGTG-3' (the underlined part is the homologous arm). A corresponding mutant vector was constructed using homologous recombination (ClonExpress II One Step Cloning Kit, Novizan). This vector used the pCAMBIA1390 vector as a backbone, Hyg as a selection marker gene, and an expression cassette driven by the ZmUbi promoter-NOS terminator. The complete vector sequence is shown in SEQ ID No. 13, with the insertion site being BamHI. An expression vector for the mutant rice GS A2N Y158C gene with the corresponding mutation site was then constructed. The constructed plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured. The conventional Agrobacterium-mediated transformation of japonica rice Nipponbare was carried out, and the operation procedure was performed in accordance with the method described in the reference (Agrobacterium-mediated transformation of rice using immature embryos orcalli induced from mature seed [J]. Nat Protoc, 2008; 3(5): 824-34.).

[0048] The transgenic rice seedlings obtained in the above steps were transplanted into nutrient soil. When the seedlings reached the 3-leaf stage, leaves were taken from the transgenic seedlings, and DNA samples were extracted using the CTAB method for subsequent testing. The gene-specific primers used in this example were used for amplification, and the PCR products were sequenced to verify the correctness of the amino acids at the mutation sites, thus identifying positive transgenic plants.

[0049] 2. Treatment of transgenic plants expressing the mutant protein GS A2N Y158C in rice with glufosinate. Transgenic rice seedlings selected through molecular screening were transplanted into large pots in a greenhouse and evenly distributed in the same experimental area. Foliar spraying with glufosinate at the recommended concentration of 4X (200 gae / ha is the generally accepted recommended concentration of glufosinate) was applied. A pneumatic sprayer was used to spray the transgenic positive plants, ensuring even distribution of droplets on the leaf surface. The recipient variety was used as a control. The plants were cultured for approximately 10 days, and their growth was observed.

[0050] from Figure 4 The results showed that wild-type rice plants died after being sprayed with the recommended 4X concentration of glufosinate; the mutant rice plants grew well and showed significant resistance to the recommended 4X concentration of glufosinate. In conclusion, the rice GS A2N Y158C mutant protein exhibits significantly better resistance to glufosinate than the wild type.

[0051] Example 5: Agrobacterium-mediated genetic transformation of wheat and identification of transgenic positive plants 1. Construction of wheat expression vector for mutant genes and Agrobacterium-mediated transformation

[0052] A gene fragment of the wheat GS A2N Y158C mutant (GenScript Biotech; mutant gene sequence shown in SEQ ID No. 14) was synthesized, and using this gene fragment as a template, gene-specific primers F: 5'- ACTAGGTACCTGCAGGTCGACGGATCC ATGAACCTCCTCACCGATCTCCT-3', R: 5'- GACTCCTCTTAGAAT TCCCGGGGATCC The gene sequence containing the corresponding wheat mutant GS A2N Y158C protein was amplified using TCAGGGCTTCCACAGGATGG-3' (underlined part is the homologous arm). A corresponding mutant vector was constructed using homologous recombination (ClonExpress II One Step Cloning Kit, Novizan). This vector used pCAMBIA1390 as the backbone, Hyg as the selection marker gene, and a ZmUbi promoter-NOS terminator expression cassette to drive the expression of the target gene. The complete vector sequence is shown in SEQ ID No. 13, with the insertion site being BamHI. An expression vector for the mutant wheat GS A2N Y158C gene with the corresponding mutation site was then constructed. This plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured. Fielder wheat was transformed using the conventional Agrobacterium-mediated transformation method. The transformation procedure is described in the reference (An efficient and reproducible...). Agrobacterium-mediated transformation method for hexaploid wheat ( Triticum aestivum The operation is performed according to the method described in [J]. Plant Methods, 2019; 15(121).

[0053] The transgenic wheat seedlings obtained in the above steps were transplanted into nutrient soil. When the seedlings reached the 3-leaf stage, leaves were taken from the transgenic seedlings, and DNA samples were extracted using the CTAB method for subsequent testing. The gene-specific primers used in this example were used for amplification, and the PCR products were sequenced to verify the correctness of the amino acids at the mutation sites, thus identifying positive transgenic plants.

[0054] 2. Glufosinate treatment of transgenic plants expressing the wheat GS A2N Y158C mutant protein

[0055] Wheat transgenic positive seedlings, selected through molecular screening, were transplanted into large pots in a greenhouse and evenly distributed within the same experimental area. Foliar spraying with glufosinate at the recommended concentration of 4X (200 gae / ha is the generally accepted recommended concentration for glufosinate) was applied. A pneumatic sprayer was used to apply the foliar spray to the transgenic positive plants, ensuring even distribution of droplets on the leaf surface. The recipient variety was used as a control. The plants were cultured for approximately 10 days, and their growth was observed.

[0056] from Figure 5 The results showed that wild-type wheat plants died after being sprayed with the recommended 4X concentration of glufosinate; the mutant wheat plants grew well and showed significant resistance to the recommended 4X concentration of glufosinate. In conclusion, the wheat GS A2N Y158C mutant protein exhibits significantly better resistance to glufosinate than the wild type.

[0057] Example 6: Agrobacterium-mediated genetic transformation of maize and identification of transgenic positive plants 1. Construction of maize expression vector for mutant genes and Agrobacterium-mediated transformation

[0058] A gene fragment of the maize GS A2N Y158C mutant (GenScript Biotech; mutant gene sequence shown in SEQ ID No. 15) was synthesized, and using this gene fragment as a template, gene-specific primers F: 5'- ACTAGGTACCTGCAGG TCGACGGATCC ATGAACTGCCTCACCGACCT-3',R: 5'- GACTCCTCTTAGAATTCCCGGGGATCCTCAGGGCTTCCAGATGATGGTG-3' (the underlined part is the homologous arm) to amplify the gene sequence containing the corresponding maize mutant GS A2N Y158C protein, and construct the corresponding mutant vector (using pCAMBIA1390 vector as the backbone, using Hyg as the screening marker gene, using the expression frame of ZmUbi promoter-NOS terminator to drive the expression of the target gene, the complete sequence of the vector is shown as SEQ ID No. 13, and the insertion site is BamH I) through homologous recombination (ClonExpress II One Step Cloning Kit, Novozyme). The mutant maize mutant GS A2N Y158C gene expression vector corresponding to the mutant site was constructed. The above-mentioned plasmid vector was transformed into Agrobacterium EHA105, and the bacterial body was cultured. The conventional Agrobacterium-mediated method was used to transform KN5585 variety of corn, and the operation process was performed according to the method described in the reference (Research on Transformation of Bar Gene into Maize Inbred Line by Agrobacterium-mediated Hybrid Embryo. Corn Science, 2017, 25(4)).

[0059] The corn transgenic seedlings obtained in the above step were transplanted into nutrient soil, and when the seedlings grew to the 3-leaf stage, the leaves of the transgenic seedlings were taken, and the sample DNA was extracted by the CTAB method for subsequent detection. The PCR product was sequenced to verify the correctness of the amino acid of the mutant site, and the positive transgenic plant was determined.

[0060] 2, Glufosinate treatment of transgenic plants expressing maize GS A2N Y158C mutant protein

[0061] The corn transgenic positive seedlings screened by molecular detection were transplanted into a greenhouse pot, and were uniformly arranged in the same experimental area. Glufosinate was sprayed on the leaf surface at a recommended concentration of 4X (200 g ae / ha is the recommended concentration of glufosinate recognized). An air pressure sprayer was used to spray the transgenic positive plants on the leaf surface, so that the liquid droplets were uniformly distributed on the leaf surface. The recipient variety was used as a control; continue to cultivate for about 10 days, and observe the growth of the corresponding plants.

[0062] From Figure 6 As can be seen from the results, after spraying glufosinate at a recommended concentration of 4X, the wild-type corn plants died; the mutant corn plants grew well and had obvious resistance to glufosinate at a recommended concentration of 4X. In summary, the maize GS A2N Y158C mutant protein has obvious resistance to glufosinate, which is obviously better than the wild type.

[0063] Example 7: Agrobacterium-mediated genetic transformation of soybean and identification of transgenic positive plants 1, Construction of mutant gene soybean expression vector and Agrobacterium transformation

[0064] A gene fragment of the soybean GS A2N Y158C mutant (GenScript Biotech; mutant gene sequence shown in SEQ ID No. 16) was synthesized, and using this gene fragment as a template, gene-specific primers F: 5'- ACTAGGTACCTGCAGG TCGACGGATCC ATGAACTTGCTCTCCGATCTTATCAACC-3', R: 5'- GACTCCTCTTAGAATTCCCGGGGATCC The gene sequence containing the corresponding soybean mutant GSA2N Y158C protein was amplified using the TTATGGTTTCCAAAGAATGGTTGTCTCAGC-3' method (underlined part is the homologous arm). Homologous recombination (ClonExpress II One Step Cloning Kit, Novizan) was then used to construct the corresponding mutant vector (using pCAMBIA1390 as the backbone, Hyg as the selection marker gene, and an expression cassette driven by the ZmUbi promoter-NOS terminator; the complete vector sequence is shown in SEQ ID No. 13, with the insertion site being BamHI). This resulted in the construction of an expression vector for the mutant soybean GSA2N Y158C gene with the corresponding mutation site. The Tianlong No. 1 soybean variety was transformed using the conventional Agrobacterium-mediated transformation method, with the procedure described in the reference (Development of an efficient marker-free soybean transformation method using the novelbacterium). Ochrobactrum haywardense The operation shall be performed in accordance with the method described in H1 [J]. Plant Biotechnol J. 2022; 20(5):977-990.

[0065] The soybean transgenic seedlings obtained in the above steps were transplanted into nutrient soil. When the seedlings reached the 3-leaf stage, leaves of the transgenic seedlings were taken, and DNA samples were extracted using the CTAB method for subsequent testing. The gene-specific primers used in this example were used for amplification, and the PCR products were sequenced to verify the correctness of the amino acids at the mutation sites, thus identifying positive transgenic plants.

[0066] 2. Treatment of transgenic plants expressing the soybean GS A2N Y158C mutant protein with glufosinate.

[0067] The genetically modified soybean seedlings selected through molecular screening were transplanted into large pots in a greenhouse and evenly distributed in the same experimental area. Foliar spraying with glufosinate at the recommended concentration of 4X (200 gae / ha is the generally accepted recommended concentration of glufosinate) was applied. A pneumatic sprayer was used to spray the transgenic positive plants, ensuring even distribution of droplets on the leaf surface. The recipient variety was used as a control. The plants were cultured for approximately 10 days, and their growth was observed.

[0068] from Figure 7 The results showed that wild-type soybean plants died after being sprayed with the recommended 4X concentration of glufosinate; the mutant soybean plants grew well and showed significant resistance to the recommended 4X concentration of glufosinate. In conclusion, the soybean GS A2N Y158C mutant protein exhibits significantly better resistance to glufosinate than the wild type.

[0069] Example 8: Agrobacterium-mediated genetic transformation of rapeseed and identification of transgenic positive plants 1. Construction of rapeseed expression vector for mutant gene and Agrobacterium-mediated transformation

[0070] A gene fragment of the rapeseed GS A2N Y158C mutant (GenScript Biotech; mutant gene sequence shown in SEQ ID No. 17) was synthesized, and using this gene fragment as a template, gene-specific primers F: 5'- ACTAGGTACCTGCAGG TCGACGGATCC ATGAACCTTCTGACCGATCTCGTTAAC-3', R: 5'- GACTCCTCTTAGAATTCCCGGGGATCCThe gene sequence containing the corresponding rapeseed mutant GSA2N Y158C protein was amplified using TCAAGGATTCCAAAGAAGTGTAGTCTCTGC-3' (underlined part is the homologous arm). A corresponding mutant vector was constructed using homologous recombination (ClonExpress II One Step Cloning Kit, Novizan). This vector used the pCAMBIA1390 vector as the backbone, Hyg as the selection marker gene, and the ZmUbi promoter-NOS terminator expression cassette to drive the expression of the target gene. The complete vector sequence is shown in SEQ ID No. 13, with the insertion site being BamHI. An expression vector for the mutant rapeseed GSA2N Y158C gene with the corresponding mutation site was then constructed. The above plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured. Westar rapeseed was transformed using the conventional Agrobacterium-mediated transformation method, following the procedure described in the reference (Study on Agrobacterium-mediated OsWRKY45 gene transformation of rapeseed [J]. Molecular Plant Breeding, 2012, 10(05).). The transgenic rapeseed seedlings obtained in the above steps were transplanted into nutrient soil. When the seedlings reached the 3-leaf stage, leaves of the transgenic seedlings were taken, and DNA samples were extracted using the CTAB method for subsequent testing. The gene-specific primers used in this example were used for amplification, and the PCR products were sequenced to verify the correctness of the amino acids at the mutation sites, thus identifying positive transgenic plants.

[0071] 2. Treatment of transgenic plants expressing the GS A2N Y158C mutant protein in rapeseed with glufosinate.

[0072] Transgenic rapeseed seedlings selected through molecular screening were transplanted into large pots in a greenhouse and evenly distributed in the same experimental area. Foliar spraying with glufosinate at the recommended concentration of 4X (200 gae / ha is the generally accepted recommended concentration of glufosinate) was applied. A pneumatic sprayer was used to spray the transgenic positive plants, ensuring even distribution of droplets on the leaf surface. The recipient variety was used as a control. The plants were cultured for approximately 10 days, and their growth was observed.

[0073] from Figure 8 The results showed that wild-type rapeseed plants died after being sprayed with the recommended 4X concentration of glufosinate; the mutant rapeseed plants grew well and showed significant resistance to the recommended 4X concentration of glufosinate. In conclusion, the GS A2N Y158C mutant protein in rapeseed exhibits significantly better resistance to glufosinate than the wild type.

[0074] sequence list

[0075] SEQ ID No. 1: Wild-type GS protein in rice MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVASEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP* SEQ ID No. 2: Wild-type GS protein from wheat MALLTDLLNLDLTDSTEKIIAEYIWIGGSGMDLRSKARTLPGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGVPIPTNKRYNAVKIFSNPDVAKEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCSIGADKSFGRDIVDSHYKACLFAGVNISGINGEVMPGQWEFQVGPTVGISAGDQVWVARYLLERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKDGGFKVIVDAVEKLKLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTILWKP* SEQ ID No. 3: Wild-type GS protein from maize MACLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNDGRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP* SEQ ID No. 4: Wild-type GS protein from soybean MACLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNDGRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP* SEQ ID No. 5: Wild-type GS protein from Brassica napus MSLLTDLVNLDLSDNTEKIIAEYIWVGGSGMDMRSKARTLPGPVTDPSKLPKWNYDGSSTGQ APGEDSEVILYPQAIFKDPFRRGNNILVMCDTYTPAGEPIPTNKRHAAAQIFSNPDVVAEVPWY GIEQEYTLLQKDVKWPVGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAHYKACLYAGINISGING EVMPGQWEFQVGPSVGISAADEVWIARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTK SMREEGGYEIIKKAIDKLGLRHKEHISAYGEGNERRLTGHHETADINTFKWGVANRGASIRVGRD TEKEGKGYFEDRRPASNMDPYTVTSMIAETTLLWNP* SEQ ID No. 6: Wild-type GS gene of rice SEQ ID No. 7: Wild-type GS gene from wheat SEQ ID No. 8: Zea mays wild-type GS gene SEQ ID No. 9: Soybean wild-type GS gene SEQ ID No. 10: Wild-type GS gene of Brassica napus SEQ ID No. 11: Sequence of the pSC-19 plasmid SEQ ID No. 12 transgenic rice transgenic mutant gene sequence SEQ ID No. 13 transgene vector complete sequence SEQ ID No. 14 Wheat GS mutant gene sequence SEQ ID No. 15 Zea mays GS mutant gene sequence SEQ ID No. 16 Soybean GS mutant gene sequence SEQ ID No. 17 Brassica napus GS mutant gene sequence SEQ ID NO. 18 Rice mutant GS protein MNSLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQ APGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVASEEPWY GIEQEYTLLQKDINWPLGWPVGGFPGPQGPYCCGIGADKSFGRDIVDSHYKACLYAGINISGI NGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTN YSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETADINTFSWGVANRGAS VRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP SEQ ID No. 19: Wheat mutant GS protein MNLLTDLLNLDLTDSTEKIIAEYIWIGGSGMDLRSKARTLPGPVTDPSKLPKWNYDGSSTGQ APGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGVPIPTNKRYNAVKIFSNPDVAKEEPWY GIEQEYTLLQKDINWPLGWPVGGFPGPQGPYCCSIGADKSFGRDIVDSHYKACLFAGVNISGI NGEVMPGQWEFQVGPTVGISAGDQVWVARYLLERITEIAGVVVTFDPKPIPGDWNGAGAHTN YSTESMRKDGGFKVIVDAVEKLKLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGAS VRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTILWKP SEQ ID No. 20: Maize mutant GS protein MNCLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYCCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNDGRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP SEQ ID No. 21 : Mutant GS protein from Glycine max MNLLSDLINLNLSDITDKVIAEYIWVGGSGMDMRSKARTLSGPVKDPSKLPKWNYDGSSTGQAPGQDSEVILYPQAIFKDPFRRGSNILVMCDAYTPAGEPIPTNKRNNAAKIFGHPDVAAEEPWYGLEQEYTLLQKDVQWPLGWPLGGFPGPQGPYCCGTGANKAFGRDIVDSHYKACIYAGINISGINGEVMPGQWEFQVGPSIGISAADELWVARYILERITEIAGVVLSFDPKPIQGDWNGAGAHTNYSTKSMRNDGGYEVIKKAIAKLEKRHKEHIAAYGEGNERRLTGRHETADMNTFVWGVANRGASIRVGRDTEKAGKGYFEDRRPASNMDPYVVTSMIAETTILWKP SEQ ID No. 22: Mutant GS protein from Brassica napus MNLLTDLVNLDLSDNTEKIIAEYIWVGGSGMDMRSKARTLPGPVTDPSKLPKWNYDGSSTGQ APGEDSEVILYPQAIFKDPFRRGNNILVMCDTYTPAGEPIPTNKRHAAAQIFSNPDVVAEVPW YGIEQEYTLLQKDVKWPVGWPIGGFPGPQGPYCCSVGADKSFGRDIVDAHYKACLYAGINIS GINGEVMPGQWEFQVGPSVGISAADEVWIARYILERITEIAGVVVSFDPKPIPGDWNGAGAHT NYSTKSMREEGGYEIIKKAIDKLGLRHKEHISAYGEGNERRLTGHHETADINTFKWGVANRG ASIRVGRDTEKEGKGYFEDRRPASNMDPYTVTSMIAETTLLWNP

Claims

1. A GS mutant protein, characterized in that, The GS mutant protein includes the protein of (a), (b) or (c) as follows: (a) a protein in which the amino acid at position 2 and / or position 158 of a wild-type GS protein of rice or a homologous site of a GS protein of other plants is mutated to have higher glutamine synthetase activity and / or glufosinate resistance; the amino acid sequence of the wild-type GS protein of rice is shown in SEQ ID No. 1; (b) a protein derived from (a) in which the amino acid sequence of (a) is substituted, deleted or added with one or more amino acids and has glutamine synthetase activity and / or glufosinate resistance; (c) a protein having at least 85% or more amino acid sequence identity with any one of the GS mutant proteins of (a) or (b) above, and having higher glutamine synthetase activity and / or glufosinate resistance than the wild-type GS protein of plants.

2. The GS mutant protein according to claim 1, characterized in that, The amino acid sequence of the GS mutant protein is mutated to T, M, Q, L, K, Y, N, G, H, I, P, F or S at the homologous site of position 2 of the wild-type GS protein of rice or other plants, and preferably, the amino acid sequence of the GS mutant protein is mutated to N or H at the homologous site of position 2 of the wild-type GS protein of rice or other plants.

3. The GS mutant protein according to claim 1, characterized in that, The amino acid sequence of the GS mutant protein is mutated to N at the homologous site of position 2 of the wild-type GS protein of rice or other plants, and is mutated to S, G, M, N, A, V, C, W, T, F, Q, L, H, D, A, I or E at the homologous site of position 158 of the wild-type GS protein of rice or other plants; and preferably, the amino acid sequence of the GS mutant protein is mutated to C, A, T, M at the homologous site of position 158 of the wild-type GS protein of rice or other plants.

4. The GS mutant protein according to claim 1 or 2, characterized in that, The other plants include wheat, corn, soybean or rape, and the homologous sites of position 2 and position 158 of the wild-type GS protein of rice are located at position 2 and position 158 of the wild-type GS protein of wheat, corn, soybean or rape, respectively, and the amino acid sequences of the wild-type GS protein of wheat, corn, soybean or rape are shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively.

5. A kind GS Mutant genes are characterized by, The GS mutant protein is encoded by the GS mutant gene of any one of claims 1-4.

6. The method according to claim 5 GS Mutant genes are characterized by, The GS The mutant gene encodes the 2nd amino acid of the wild-type GS protein of rice, wild-type GS protein of wheat, wild-type GS protein of corn, wild-type GS protein of soybean, wild-type GS protein of rape to be mutated into K, the corresponding base is aaa or aag, mutated into S, the corresponding base is tct, tca, tcc, tcg, agt or agc, mutated into G, the corresponding base is ggt, ggc, gga or ggg, mutated into M, the corresponding base is atg, mutated into Y, the corresponding base is tat or tac, mutated into N, the corresponding base is aat or aac, mutated into T, the corresponding base is act, acc, acg or aca, mutated into F, the corresponding base is ttt or ttc, mutated into I, the corresponding base is att, atc or ata, mutated into P, the corresponding base is cct, cca, ccg or ccc, mutated into Q, the corresponding base is caa or cag, mutated into L, the corresponding base is ctt, ctc, cta, ctg, tta or ttg, mutated into H, the corresponding base is cat or cac; the 158th amino acid of the wild-type GS protein of rice, wild-type GS protein of wheat, wild-type GS protein of corn, wild-type GS protein of soybean, wild-type GS protein of rape to be mutated into A, the corresponding base is gct, gcc, gca or gcg; mutated into V, the corresponding base is gtt, gtc, gta or gtg, mutated into C, the corresponding base is tgt or tgc, mutated into W, the corresponding base is tgg, mutated into S, the corresponding base is tct, tca, tcc, tcg, agt or agc, mutated into G, the corresponding base is ggt, ggc, gga or ggg, mutated into M, the corresponding base is atg, mutated into N, the corresponding base is aat or aac, mutated into T, the corresponding base is act, acc, acg or aca, mutated into F, the corresponding base is ttt or ttc, mutated into Q, the corresponding base is caa or cag, mutated into L, the corresponding base is ctt, ctc, cta, ctg, tta or ttg, mutated into H, the corresponding base is cat or cac, mutated into I, the corresponding base is att, atc or ata, mutated into D, the corresponding base is gat or gac, mutated into E, the corresponding base is gaa or gag; as preferred, the base sequence corresponding to the wild-type GS protein mutated into different amino acids is shown in Table 1. GS The gene sequence is shown as SEQ ID No. 6, the wild-type GS gene sequence of wheat is shown as SEQ ID No. 7, the wild-type GS gene sequence of corn is shown as SEQ ID No. 8, the wild-type GS gene sequence of soybean is shown as SEQ ID No. 9, and the wild-type GS gene sequence of rape is shown as SEQ ID No.

10.

7. An expression cassette, a recombinant vector or a recombinant bacterium comprising the GS mutant gene of claim 5 or 6.

8. The GS mutant protein according to any one of claims 1 to 4, the mutant gene according to claim 5 or 6, the expression cassette according to claim 7, the recombinant vector or the recombinant bacterium for use in the breeding of plants resistant to glufosinate-ammonium. GS the use of a mutant gene according to claim 7, a recombinant vector or a recombinant bacterium for breeding plants resistant to glufosinate-ammonium.

9. Use according to claim 8, characterised in that, The application includes a method of gene editing, transgenic, mutagenesis, hybridization, backcrossing or vegetative reproduction to obtain a plant with glufosinate resistance.

10. Use according to claim 9, characterized in that, The plant with glufosinate resistance includes rice, wheat, corn, soybean, rape or other plants.

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