CN117363633A

CN117363633A - Nucleic acid molecule, vector, recombinant bacterium, GS mutant and application thereof

Info

Publication number: CN117363633A
Application number: CN202311296672.7A
Authority: CN
Inventors: 邓龙群; 陈容; 弓亚杰; 袁梦雪; 冯阳; 胥南飞
Original assignee: Gevoto LLC
Current assignee: Gevoto LLC
Priority date: 2023-10-08
Filing date: 2023-10-08
Publication date: 2024-01-09

Abstract

The invention discloses a nucleic acid molecule, a vector, recombinant bacteria, a GS mutant and application thereof, and relates to the technical field of genetic engineering. The original source of the plant glutamine synthetase mutant provided by the invention is a plant, and the plant has glufosinate resistance or the glufosinate resistance is improved through mutation. Plants transformed with the plant glutamine synthetase mutant have glufosinate resistance and can grow and develop normally. Through experiments, the plant glutamine synthetase mutant provided by the invention has good biological enzyme catalytic activity. Therefore, the plant glutamine synthetase mutant provided by the invention enriches a plant glutamine synthetase mutant library, and provides powerful technical support for cultivating glufosinate-resistant plant varieties.

Description

Nucleic acid molecule, vector, recombinant bacterium, GS mutant and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a nucleic acid molecule, a vector, recombinant bacteria, a GS mutant and application thereof.

Background

Glutamine synthetase (Glutamine synthetase, GS) is a key enzyme in nitrogen metabolism in plants, which catalyzes the circulation of glutamate (Glu) with NH ₃ Condensation to form glutamine (Gln), which is involved in the metabolism of nitrogen-containing compounds in plants. Depending on distribution and subcellular localization, higher plant GS (class GSII) isoenzymes can be divided into two classes: one which is located in the cytoplasm is called cytoplasmic GS (GS 1), with a molecular weight of 38-40kDa; another type, designated as apoplast GS (GS 2), is located in chloroplasts (or plastids) and has a molecular weight of 44-45kDa.

Glufosinate (glufosinate ammonium, under the trade name Basta) is a glutamine synthetase (GS 1) inhibitor developed by anget, now bayer, and its active ingredient is phosphinothricin (abbreviated PPT), and its chemical name is (RS) -2-amino-4- (hydroxymethylphosphino) ammonium butyrate. The product is marketed in 1986, and sales increase year by year. The target enzyme for glufosinate is GS, which can normally form lambda-glutamyl phosphate from ATP and glutamate. However, after PPT treatment, PPT binds to ATP first and phosphorylated PPT occupies 8 reaction centers of GS molecules, which changes the spatial configuration of GS, resulting in inhibition of GS activity.

As a result of inhibition of GS by glufosinate, it can lead to disturbance of nitrogen metabolism in plants, excessive accumulation of ammonium, disintegration of chloroplasts, and thus inhibition of photosynthesis in plants, and in severe cases, death of plants.

At present, the main method for cultivating the glufosinate-resistant variety is to introduce the glufosinate-resistant gene from bacteria into crops by using genetic engineering means, so as to cultivate a new transgenic glufosinate-resistant crop variety. The most widely used anti-glufosinate genes in agriculture today are the bar gene from strain Streptomyces hygroscopicus and the pat gene from strain s. The bar gene and the pat gene have 80% homology, and can code glufosinate acetylase, and the enzyme can be used for inactivating glufosinate acetylase. The glufosinate-resistant variety has great use value, wherein resistant rape, corn and the like are commercially planted in a large area.

The glufosinate acetylases encoded by the bar and pat genes can acetylate glufosinate to deactivate it, but it is difficult for the enzyme to completely deactivate glufosinate before it contacts the GS, because many GS are distributed on cell membranes, so that the use of glufosinate on bar and pat gene crops interferes with nitrogen metabolism in plants to varying degrees, as well as normal growth and development of plants. Overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate but is not sufficiently tolerant for commercial use.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nucleic acid molecule, a vector, recombinant bacteria, GS mutant and application thereof so as to solve the technical problems.

The invention is realized in the following way:

in a first aspect, the present invention provides a method for obtaining a protein having glufosinate resistance comprising the steps of:

1) A protein having the reference sequence shown in SEQ ID NO.1 or having an amino acid sequence having at least 85% identity to the reference sequence as a target protein;

2) Comparing the amino acid sequence of the target protein with a reference sequence, and mutating the amino acid sequence of the target protein corresponding to the 60 th amino acid residue of the reference sequence to proline, tryptophan or deleting the proline, the tryptophan;

3) Proteins with increased glufosinate resistance are selected.

In a second aspect, the present invention provides a plant glutamine synthetase mutant having glufosinate resistance, the plant glutamine synthetase mutant being as follows (1) or (2):

(1): it is obtained by mutating the n-th position of wild glutamine synthetase from plant; the position of the nth bit is determined as follows: the wild type glutamine synthetase is aligned with a reference sequence, the nth position of the wild type glutamine synthetase corresponds to the 60 th position of the reference sequence, wherein the amino acid sequence of the reference sequence is shown as SEQ ID NO. 1;

The n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted compared with the wild glutamine synthetase;

(2): which has at least 85% identity with the plant glutamine synthetase mutant shown in (1) and is identical to the amino acid at the n-th position of the plant glutamine synthetase mutant shown in (1).

In a preferred embodiment of the present invention, when the source of the wild-type glutamine synthetase is rice, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted;

when the source of the wild type glutamine synthetase is corn, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted;

when the source of the wild type glutamine synthetase is soybean, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted;

when the source of the wild type glutamine synthetase is rape, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted.

In a third aspect, the invention also provides a nucleic acid molecule encoding a plant glutamine synthetase mutant as described above.

In a fourth aspect, the invention also provides an expression cassette or vector comprising a nucleic acid molecule as described above.

In a fifth aspect, the invention also provides a recombinant bacterium or recombinant cell comprising a nucleic acid molecule as defined above or an expression cassette or vector as defined above; and the recombinant cell is a non-plant cell;

in an alternative embodiment, the recombinant bacterium is an agrobacterium, an escherichia coli or a yeast.

In a sixth aspect, the invention also provides the use of a plant glutamine synthetase mutant, a nucleic acid molecule, an expression cassette or a vector or a recombinant bacterium or recombinant cell for cultivating a plant variety having glufosinate resistance.

In a preferred embodiment of the application of the present invention, the application includes any of the following uses:

(1) Transforming a target plant with a vector containing a coding gene encoding a plant glutamine synthetase mutant;

(2) Modifying the endogenous glutamine synthetase gene of the target plant by a gene editing method to code a plant glutamine synthetase mutant;

(3) Mutagenizing and screening plant cells, tissues, individuals or populations to encode a plant glutamine synthetase mutant;

in an alternative embodiment, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, day lily, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea, or cassava;

In an alternative embodiment, the pasture is selected from gramineous pasture or leguminous pasture;

in an alternative embodiment, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, canola, mustard, blue, canola, green vegetables, or beet;

in an alternative embodiment, the cucurbitaceae plant is selected from cucumber, pumpkin, wax gourd, bitter gourd, luffa, melon, watermelon or melon;

in an alternative embodiment, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;

in an alternative embodiment, the plant is rice, maize, soybean or canola.

In a seventh aspect, the present invention also provides a method of producing a glufosinate herbicide tolerant plant comprising introducing into the genome of a plant of interest a gene encoding a plant glutamine synthetase mutant as described above.

In a preferred embodiment of the present invention, the method of introduction is selected from the group consisting of genetic transformation methods, genome editing methods, and gene mutation methods.

The invention has the following beneficial effects:

(1) The plant glutamine synthetase mutant provided by the invention enriches a plant glutamine synthetase mutant library, and provides powerful technical support for cultivating glufosinate-resistant plant varieties.

(2) The original source of the plant glutamine synthetase mutant is a plant, and the plant has glufosinate resistance or the glufosinate resistance is improved through mutation. Plants transformed with the plant glutamine synthetase mutant have glufosinate resistance and can grow and develop normally.

(3) The plant glutamine synthetase mutant has good biological enzyme catalytic activity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of partial alignment of amino acid sequences of the rice GS1 mutant OT60P, OT60W, OT X and wild-type rice GS1 OsGS1_WT provided in example 1 of the present invention;

FIG. 2 is a partial alignment of amino acid sequences of maize GS1 mutant ZT60P, ZT60W, ZT X and wild-type maize GS1 ZmGS1_WT provided in example 2 of the present invention;

FIG. 3 is a partial alignment of amino acid sequences of soybean GS1 mutant GT60P, GT60W, GT X and wild soybean GS1GmGS1_WT provided in example 3 of the present invention;

FIG. 4 shows the result of partial alignment of amino acid sequences of the canola GS1 mutant BT60P, BT60W, BT X and wild-type canola GS1BnGS1_WT provided in example 4 of the present invention;

FIG. 5 is a schematic diagram of the structure of pADV7 vector provided in Experimental example 1 of the present invention;

FIG. 6 shows the results of E.coli growth on medium containing glufosinate at different concentrations of both the rice GS1 mutant OT60P, OT60W, OT X and the wild-type rice GS1OsGS1_WT provided in Experimental example 1;

FIG. 7 shows the results of E.coli growth on medium containing glufosinate at different concentrations of corn GS1 mutant ZT60P, ZT60W, ZT X and wild-type corn GS1ZmGS1_WT provided in Experimental example 2;

FIG. 8 shows the results of E.coli growth of soybean GS1 mutant GT60P, GT60W, GT X and wild soybean GS1GmGS1_WT on medium containing glufosinate at different concentrations as provided in Experimental example 3 of the present invention;

FIG. 9 shows the results of E.coli growth on medium containing glufosinate at different concentrations of the respective wild-type canola GS1 mutant BT60P, BT60W, BT X and the wild-type canola GS1BnGS1_WT provided in Experimental example 4;

FIG. 10 shows the enzyme kinetic parameters and glufosinate resistance parameters IC of the rice GS1 mutant OT60W, the maize GS1 mutant ZT60W, the soybean GS1 mutant GT60W, the canola GS1 mutant BT60W, the wild-type rice GS1 OsGS1_WT, the wild-type maize GS1 ZmGS1_WT, the wild-type soybean GS1 GmGS1_WT and the wild-type canola GS1 BnGS1_WT ₅₀ ；

FIG. 11 shows amino acid sequence alignment of wild type glutamine synthetase from different plants; in the figure: osGS1_WT: wild type rice glutamine synthetase; zmgs1_wt: corn wild type glutamine synthetase; gmgs1_wt: soybean wild type glutamine synthetase; bnGS1_WT: wild type rape glutamine synthetase.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of plant physiology, plant molecular genetics, cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of one skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); plant physiology (pallidum et al, 2017); the methods are described in the following examples (methods of enzymology) (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M. Weir and C.C. Blackwell, inc.), contemporary molecular biology methods (Current Protocols in Molecular Biology) (F.M. Ausubel et al, 1987), plant molecular genetics (Monica A. Hughes et al), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), each of which is expressly incorporated herein by reference.

Term interpretation:

"deletion mutation" or "deleted" is a deletion mutation (del). If the amino acid sequence of the target protein is deleted, the amino acid residue corresponding to the 60 th amino acid residue of the reference sequence is deleted.

The term "glufosinate" in the present invention, also known as glufosinate, refers to 2-amino-4- [ hydroxy (methyl) phosphono ] ammonium butyrate.

3) Proteins with increased glufosinate resistance are selected.

The inventor researches and discovers that the wild type glutamine synthetase of plant origin is compared with a reference sequence, the amino acid position corresponding to the 60 th position of the reference sequence is mutated, mutated into proline (P), tryptophan (W) or deleted, and the protein with glufosinate resistance can be obtained by screening. The protein has good biological enzyme catalytic activity.

The sequence of SEQ ID NO.1 is as follows:

MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPF

RKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVASEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKS

FGRDIVDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHT

NYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRP

ASNMDPYIVTSMIAETTIIWKP。

the sequence of SEQ ID NO.1 is the amino acid sequence of wild type rice glutamine synthetase.

Comparing the wild type glutamine synthetase from plant with a reference sequence, mutating the amino acid site corresponding to the 60 th site of the reference sequence, namely the n-th site, to proline, tryptophan or deleting, wherein the obtained plant glutamine synthetase mutant has glufosinate resistance, and simultaneously maintains the biological enzyme catalytic activity of the mutant. The plant expressing the plant glutamine synthetase mutant provided by the invention or the recombinant bacteria containing the nucleic acid molecule and the vector for encoding the plant glutamine synthetase mutant can normally grow and develop in the presence of glufosinate, and the plant glutamine synthetase mutant not only can be used for cultivating transgenic crops, but also can be used for cultivating glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybean, corn, wheat, rape, cotton, sorghum and the like, and has wide application prospects.

The reference sequence shown in SEQ ID NO.1 is wild type glutamine synthetase of rice origin.

The sequence alignment method can use Blast website (https:// Blast. Ncbi. Nlm. Nih. Gov/Blast. Cgi) to carry out Protein Blast alignment; the same results can be obtained using other sequence alignment methods or tools well known in the art.

It should be noted that the n-th position of the wild-type glutamine synthetase may be the 60-th position (e.g., corn, soybean, rape, etc.) on its own sequence, but may not be the 60-th position, and the specific position of the n-th position is determined by alignment of the sequences, so long as the position corresponding to the 60-th position of the reference sequence is the n-th position, i.e., the mutation position, of the present invention after alignment with the reference sequence.

The glutamine synthetase mutants of the present invention are synthetic or recombinant proteins, i.e., can be chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria (including but not limited to actinomycetes, etc.), yeasts, plants, animals) using recombinant techniques. Depending on the host used in the recombinant production protocol, the glutamine synthetase mutants of the present invention can be glycosylated or can be non-glycosylated. The glutamine synthetase mutants of the present invention may or may not also include an initial methionine residue.

In a preferred embodiment of the invention, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, day lily, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea or cassava.

All plants' wild-type glutamine synthetases are octamers, generally consisting of 8 subunits of molecular weight 39-45 kD. The plant glutamine synthetase is divided into cytosolic GS (GS 1) and chloroplast GS (GS 2), the GS used in the invention is GS1 type, the enzyme is quite conserved in the evolution process, has higher homology and basically has the same function and structural domain in plant body.

Wild-type glutamine synthetases based on all plants have homology, essentially identical functions and domains in the plant body. Thus, any plant-derived wild-type glutamine synthetase mutant obtained by making the above-described mutation at position 60 has glufosinate resistance. Therefore, the plant glutamine synthetase mutants obtained by mutating wild type glutamine synthetase of any plant origin are all within the scope of the present invention.

Furthermore, it is known and easily achieved by those skilled in the art that a simple amino acid substitution, deletion, or addition is performed in a non-conserved region of the plant glutamine synthetase mutant shown in (1) and the n-th position is maintained as the above-mentioned mutated amino acid, and that the further mutated plant glutamine synthetase mutant has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% etc.) or more identity with the plant glutamine synthetase mutant shown in (1), and that its functions include the enzyme catalytic activity and glufosinate resistance are equivalent to or slightly decreased or slightly increased or greatly increased as those of the plant glutamine synthetase mutant shown in (1). Therefore, such glutamine synthetases should also fall within the scope of the present invention.

In a preferred embodiment of the invention, the pasture is selected from gramineous pasture or leguminous pasture; leguminous forage includes, but is not limited to: red clover, white clover, alfalfa, arrowhead peas, green peas, lupin yellow, white clover, sweet clover, astragalus root, white milk vetch, white butterfly beans, and the like.

In a preferred embodiment of the invention, the brassica vegetables include, but are not limited to, turnip, cabbage, mustard, cabbage mustard, canola, mustard, cabbage, canola, green vegetables, or beet.

In a preferred embodiment of the present invention, the cucurbitaceae plant includes, but is not limited to, cucumber, pumpkin, wax gourd, luffa, melon, watermelon or melon;

in a preferred embodiment of the invention, leguminous plants include, but are not limited to, mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;

in a preferred embodiment of the invention, the plant is rice, maize, soybean or canola.

In a preferred embodiment of the present invention, when the source of the wild-type glutamine synthetase is rice, the amino acid sequence of the wild-type glutamine synthetase of rice is shown as SEQ ID NO. 1. The n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted.

When the source of the wild-type glutamine synthetase is corn, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted. The amino acid sequence of the corn wild type glutamine synthetase is SEQ ID NO.2:

MACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP。

When the source of the wild type glutamine synthetase is soybean, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted; the soybean wild type glutamine synthetase is SEQ ID NO.3:

MSLLSDLINLNLSDTTEKVIAEYIWIGGSGMDLRSKARTLPGPVSDPSKLPKWNYDGSSTGQAPGEDSEVIIYPQAIFRDPFRRGNNILVICDTYTPAGEPIPTNKRHDAAKVFSHPDVVAEETWYGIEQEYTLLQKDIQWPLGWPVGGFPGPQGPYYCGVGADKAFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDEVWAARYILERITEIAGVVVSFDPKPIQGDWNGAGAHTNYSTKSMRNDGGYEVIKTAIEKLGKRHKEHIAAYGEGNERRLTGRHETADINTFLWGVANRGASVRVGRDTEKAGKGYFEDRRPASNMDPYVVTSMIADTTILWKP。

when the source of the wild type glutamine synthetase is rape, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted. The wild type rape glutamine synthetase is shown as SEQ ID NO. 4:

MSLLTDLVNLNLSETTDKIIAEYIWVGGSGMDMRSKARTLPGPVSDPSELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKVFSHPDVVAEVPWYGIEQEYTLLQKDVNWPLGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPAVGISAGDEIWVARFILERITEIAGVVVSFDPKPIPGDWNGAGAHCNYSTKSMREDGGYEIIKKAIDKLGLRHKEHIAAYGEGNERRLTGHHETADINTFLWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYIVTSMIAETTILWKP。

the Similarity and Identity (Identity) of the wild-type glutamine synthetases of partial plant origin are shown in the following table, and the partial results of the sequence alignment are shown in FIG. 11, with the arrow indicating amino acid 60.

The above-mentioned Similarity (Similarity) and Identity (Identity) comparison method is as follows: the amino acid sequence of one species is input to the Blast website (https:// Blast. Ncbi. Nlm. Nih. Gov/Blast. Cgi) for Protein Blast alignment, and the Similarity (Similarity) and Identity (Identity) of this species and other species to be aligned are looked up from the alignment.

The term "nucleic acid molecule encoding a mutant of a plant glutamine synthetase as described above" may be a polynucleotide comprising a mutant protein of the present invention, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or muteins having the same amino acid sequence as the invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the mutein encoded thereby.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

In the case where the present invention provides the above amino acid sequence, a nucleic acid sequence encoding the above plant glutamine synthetase mutant is easily obtained by a person skilled in the art based on the degeneracy of codons. For example, a nucleic acid sequence encoding a mutant of a plant glutamine synthetase as described above may be obtained by mutating a corresponding nucleotide in a nucleic acid sequence encoding a wild type glutamine synthetase. This is readily accomplished by one skilled in the art.

For example, the rice wild-type glutamine synthetase has a nucleic acid sequence of SEQ ID NO.5:

ATGGCTTCTCTCACCGATCTCGTCAACCTCAACCTCTCCGACACCACGGAGAAGATCATCGCCGAGTACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAGGCTAGGACTCTCTCCGGCCCTGTGACTGATCCCAGCAAGCTGCCCAAGTGGAACTACGATGGCTCCAGCACCGGCCAGGCCCCCGGCGAGGACAGTGAGGTCATCCTGTACCCACAGGCTATCTTCAAGGACCCATTCAGGAAGGGAAACAACATCCTTGTCATGTGCGATTGCTACACGCCAGCCGGAGAACCGATCCCCACCAACAAGAGGCACAATGCTGCCAAGATCTTCAGCTCCCCTGAGGTTGCTTCTGAGGAGCCCTGGTACGGTATTGAGCAAGAGTACACCCTCCTCCAGAAGGACATCAACTGGCCCCTTGGCTGGCCTGTTGGTGGCTTCCCTGGTCCTCAGGGTCCTTACTACTGTGGTATCGGTGCTGACAAGTCTTTTGGGCGTGATATTGTTGACTCCCACTACAAGGCTTGCCTCTATGCCGGCATCAACATCAGTGGAATCAACGGCGAGGTCATGCCAGGACAGTGGGAGTTCCAAGTTGGCCCGTCTGTCGGCATTTCTGCCGGTGATCAGGTGTGGGTTGCTCGCTACATTCTTGAGAGGATCACCGAGATCGCCGGAGTCGTCGTCTCATTTGACCCCAAGCCCATCCCGGGAGACTGGAACGGTGCTGGTGCTCACACCAACTACAGCACCAAGTCGATGAGGAACGATGGTGGCTACGAGATCATCAAGTCCGCCATTGAGAAGCTCAAGCTCAGGCACAAGGAGCACATCTCCGCCTACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGACATCAACACCTTCAGCTGGGGAGTTGCCAACCGCGGCGCCTCGGTCCGCGTCGGCCGGGAGACGGAGCAGAACGGCAAGGGCTACTTCGAGGATCGCCGGCCGGCGTCCAACATGGACCCTTACATCGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA。

accordingly, on a sequence basis, a mutant encoding rice plant glutamine synthetase as described above can be obtained by performing a corresponding nucleotide mutation at a codon corresponding to the 60 th position of the encoded amino acid sequence.

The coding nucleic acid sequence of the corn wild type glutamine synthetase is SEQ ID NO.6:

ATGGCCTGCCTCACCGACCTCGTCAACCTCAACCTCTCGGACAACACCGAGAAGATCATCGCGGAATACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAAGCAAGGACCCTCTCCGGCCCGGTGACCGATCCCAGCAAGCTGCCCAAGTGGAACTACGACGGCTCCAGCACGGGCCAGGCCCCCGGCGAGGACAGCGAGGTCATCCTGTACCCGCAGGCCATCTTCAAGGACCCATTCAGGAGGGGCAACAACATCCTTGTGATGTGCGATTGCTACACCCCAGCCGGCGAGCCAATCCCCACCAACAAGAGGTACAACGCCGCCAAGATCTTCAGCAGCCCTGAGGTCGCCGCCGAGGAGCCGTGGTATGGTATTGAGCAGGAGTACACCCTCCTCCAGAAGGACACCAACTGGCCCCTTGGGTGGCCCATCGGTGGCTTCCCCGGCCCTCAGGGTCCTTACTACTGTGGAATCGGCGCCGAAAAGTCGTTCGGCCGCGACATCGTGGACGCCCACTACAAGGCCTGCTTGTATGCGGGCATCAACATCAGTGGCATCAACGGGGAGGTGATGCCAGGGCAGTGGGAGTTCCAAGTCGGGCCTTCCGTGGGTATATCTTCAGGCGACCAGGTCTGGGTCGCTCGCTACATTCTTGAGAGGATCACGGAGATCGCCGGTGTGGTGGTGACGTTCGACCCGAAGCCGATCCCGGGCGACTGGAACGGCGCCGGCGCGCACACCAACTACAGCACGGAGTCGATGAGGAAGGAGGGCGGGTACGAGGTGATCAAGGCGGCCATCGAGAAGCTGAAGCTGCGGCACAGGGAGCACATCGCGGCATACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGACATCAACACGTTCAGCTGGGGCGTGGCCAACCGCGGCGCGTCGGTGCGCGTGGGCCGGGAGACGGAGCAGAACGGCAAGGGCTACTTCGAGGACCGCCGCCCGGCGTCCAACATGGACCCCTACGTGGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA。

the encoding nucleic acid sequence of the soybean wild type glutamine synthetase is SEQ ID NO.7:

ATGTCGCTGCTCTCAGATCTCATCAACCTTAACCTCTCAGACACTACTGAGAAGGTGATCGCAGAGTACATATGGATCGGTGGATCAGGAATGGACCTGAGGAGCAAAGCAAGGACTCTCCCAGGACCAGTTAGCGACCCTTCAAAGCTTCCCAAGTGGAACTATGATGGTTCCAGCACAGGCCAAGCTCCTGGAGAAGACAGTGAAGTGATTATATACCCACAAGCCATTTTCAGGGATCCATTCAGAAGGGGCAACAATATCTTGGTTATCTGTGATACTTACACTCCAGCTGGAGAACCCATTCCCACTAACAAGAGGCACGATGCTGCCAAGGTTTTCAGCCATCCTGATGTTGTTGCTGAAGAGACATGGTATGGTATTGAGCAGGAATACACCTTGTTGCAGAAAGATATCCAATGGCCTCTTGGGTGGCCTGTTGGTGGTTTCCCTGGACCACAGGGTCCATACTACTGTGGTGTTGGCGCTGACAAGGCTTTTGGCCGTGACATTGTTGACGCACATTACAAAGCCTGTCTTTATGCTGGCATCAACATCAGTGGAATTAATGGAGAAGTGATGCCCGGTCAGTGGGAATTCCAAGTTGGACCTTCAGTTGGAATCTCAGCTGGTGACGAGGTGTGGGCAGCTCGTTACATCTTGGAGAGGATCACTGAGATTGCTGGTGTGGTGGTTTCCTTTGATCCCAAGCCAATTCAGGGTGATTGGAATGGTGCTGGTGCTCACACAAACTACAGCACTAAGTCCATGAGAAATGATGGTGGCTATGAAGTGATCAAAACCGCCATTGAGAAGTTGGGGAAGAGACACAAGGAGCACATTGCTGCTTATGGAGAAGGCAACGAGCGTCGTTTGACAGGGCGCCACGAAACCGCTGACATCAACACCTTCTTATGGGGAGTTGCAAACCGTGGAGCTTCAGTTAGGGTTGGGAGGGACACAGAGAAAGCAGGGAAGGGATATTTTGAGGACAGAAGGCCAGCTTCTAACATGGACCCATATGTGGTTACTTCCATGATTGCAGACACAACCATTCTGTGGAAGCCATGA。

the encoding nucleic acid sequence of the wild type rape glutamine synthetase is SEQ ID NO.8:

ATGAGTCTTCTTACAGATCTCGTTAACCTTAACCTCTCAGAGACCACTGACAAAATCATTGCGGAATACATATGGGTTGGAGGTTCAGGAATGGATATGAGAAGCAAAGCCAGGACTCTTCCTGGACCAGTGAGTGACCCTTCGGAGCTACCAAAGTGGAACTATGATGGCTCAAGCACAGGCCAAGCTCCTGGTGAAGACAGTGAAGTCATCTTATACCCTCAAGCCATATTCAAAGATCCTTTCCGTAGAGGCAACAACATTCTTGTCATGTGCGATGCTTACACTCCAGCGGGCGAACCGATCCCAACAAACAAAAGACACGCTGCGGCTAAGGTCTTTAGCCACCCCGATGTTGTAGCTGAAGTGCCATGGTATGGTATTGAGCAAGAGTATACTTTACTTCAGAAAGATGTGAACTGGCCTCTTGGTTGGCCTATTGGCGGCTTCCCCGGTCCTCAGGGACCATACTATTGTAGTGTTGGAGCAGATAAATCTTTTGGTAGAGACATCGTTGATGCTCACTACAAGGCCTGCTTATACGCTGGCATCAATATTAGTGGCATCAACGGAGAAGTCATGCCTGGTCAGTGGGAGTTCCAAGTTGGTCCAGCTGTTGGTATCTCGGCCGGTGATGAAATTTGGGTCGCACGTTTCATTTTGGAGAGGATCACAGAGATTGCTGGTGTGGTGGTATCTTTTGACCCAAAACCGATTCCCGGTGACTGGAATGGTGCTGGTGCTCACTGCAACTATAGTACCAAGTCAATGAGGGAAGATGGTGGTTACGAGATTATTAAGAAGGCAATCGATAAACTGGGACTGAGACACAAAGAACACATTGCAGCTTACGGTGAAGGCAATGAGCGCCGTCTCACGGGTCACCACGAGACTGCTGACATCAACACTTTCCTCTGGGGTGTTGCGAACCGTGGAGCATCAATCCGTGTAGGACGTGACACAGAGAAAGAAGGGAAAGGATACTTTGAGGATAGGAGGCCAGCTTCGAACATGGATCCTTACATTGTGACTTCCATGATTGCAGAGACCACAATCCTCTGGAAACCTTGA。

Such vectors include, but are not limited to, expression vectors, shuttle vectors, and integration vectors.

In the present invention, the term "expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

In an alternative embodiment, the expression vector further comprises a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

In an alternative embodiment, the expression cassette comprises a promoter, a gene encoding a plant glutamine synthetase mutant, and a terminator. In an alternative embodiment, the expression cassette comprises a first promoter, a gene encoding a mutant plant glutamine synthetase, a first terminator, a second promoter, a selectable marker gene, and a second terminator. Wherein the first promoter and the second promoter may be the same or different, and the first terminator and the second terminator may be the same or different.

In a fifth aspect, the invention also provides a recombinant bacterium or recombinant cell comprising a nucleic acid molecule as defined above or an expression cassette or vector as defined above; and the recombinant cells are non-plant cells.

The recombinant cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher non-plant eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast.

(3) The plant cells, tissues, individuals or populations are subjected to mutagenesis and selection to encode a plant glutamine synthetase mutant.

Regarding use (1), in a preferred embodiment of the application of the present invention, the gene editing is selected from at least one of CRISPR/Cas9, TALEN technology, ZFN technology, CRISPR/Cas12 technology, and ARCUS gene editing.

On the basis of the present invention, it is easy for a person skilled in the art to modify a target plant by means of a gene editing technique (e.g., by zinc finger endonuclease (ZFN, zinc-finger nucleic) technique, transcription activator-like effector nuclease (TALEN, transcription activator-like effector nucleases) technique or CRISPR/Cas9, CRISPR/Cas12, ARCUS technique (insertion, removal and/or repair using ARCUS nuclease), mutation breeding technique (e.g., chemical, radiation mutagenesis, etc.) etc. which is conventional in the art, so as to have a gene encoding a plant glutamine synthetase mutant as described above, thereby obtaining glufosinate resistance and enabling normal growth and development, and thus a new variety of plants having glufosinate resistance.

In an alternative embodiment, the plant is subjected to mutagenesis in a physicochemical mutagenesis mode that is mutagenized to a non-lethal dose to obtain plant material.

The above-mentioned non-lethal dose means that the dose is controlled to be within a range of 20% floating above and below the semi-lethal dose.

Physicochemical mutagenesis modes include combinations of one or more of the following physical mutagenesis and chemical mutagenesis modes: physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, gamma-ray mutagenesis, beta-ray mutagenesis, alpha-ray mutagenesis, high-energy particle mutagenesis, cosmic ray mutagenesis, microgravity mutagenesis; chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analogue mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis and intercalating dye mutagenesis; alkylating agent mutagenesis includes ethylcyclomate mutagenesis, diethylsulfate mutagenesis, and ethylenimine mutagenesis.

in an alternative embodiment, the plant is rice, maize, soybean or canola.

In an alternative embodiment, the method of introduction is selected from the group consisting of genetic transformation methods, genome editing methods, or genetic mutation methods.

The above genetic transformation methods include, but are not limited to: individuals with glufosinate resistance are produced by selfing or crossing parent plants with genes of glufosinate resistant plant glutamine synthetase mutants with other plant individuals.

In other embodiments, the methods of transformation described above include, but are not limited to, agrobacterium-mediated gene transformation, gene gun transformation, and pollen tube channel.

In a preferred embodiment of the invention, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, day lily, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea or cassava;

in a preferred embodiment of the invention, the pasture is selected from gramineous pasture or leguminous pasture;

in a preferred embodiment of the invention, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, canola, mustard, engine blue, canola, green vegetables or beet;

In a preferred embodiment of the invention, the cucurbitaceae plant is selected from cucumber, pumpkin, wax gourd, bitter gourd, luffa, melon, watermelon or melon;

in a preferred embodiment of the invention, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

This example provides three rice (Oryza sativa) glutamine synthetase (GS 1) mutants. The rice GS1 mutants are respectively named as OT60P, OT W and OT60X, which are obtained by mutating the 60 th amino acid residue T of wild rice glutamine synthetase (named as OsGS1_WT, the amino acid sequence is shown as SEQ ID NO.1 and the encoding nucleotide sequence is shown as SEQ ID NO. 5) into P (proline), W (tryptophan) and deleting. The amino acid sequence alignment of the rice GS1 mutant OT60P, OT60W, OT X and the wild-type rice GS1 is shown in fig. 1, in which: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each rice GS1 mutant was at the position encoding amino acid 60, the codons for the corresponding amino acids were as shown in the following table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.

Amino acids	P	W	Deletion of
				Codons	CCA	TGG	Without any means for

The rice GS1 mutants OT60P, OT W and OT60X and the nucleic acid molecules encoding the same provided in this example can be obtained by chemical synthesis.

Example 2

This example provides three maize (Zea mays) GS1 mutants. It is obtained by mutating the 60 th site (corresponding to the 60 th site of the reference sequence (SEQ ID NO. 1)) of wild-type corn GS1 itself (named ZmGS1_WT, the amino acid sequence is shown as SEQ ID NO.2, the coding nucleotide sequence is SEQ ID NO. 6) from the amino acid residue T (threonine) to P (proline), W (tryptophan) and deleting. The obtained corn GS1 mutants were named ZT60P, ZT W and ZT60X, respectively.

The amino acid sequence alignment of maize GS1 mutant ZT60P, ZT W and ZT60X and wild-type maize GS1 is shown in figure 2, in which: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each maize GS1 mutant was at the position encoding amino acid 60, the codons for the corresponding amino acids are shown in the following table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.

Amino acids	P	W	Deletion of
				Codons	CCA	TGG	Without any means for

The maize GS1 mutants ZT60P, ZT W and ZT60X and the nucleic acid molecules encoding them provided in this example can both be obtained by chemical synthesis.

Example 3

This example provides three soybean (Glycine max) GS1 mutants. The mutant was obtained by mutating the 60 th position (corresponding to the 60 th position of the reference sequence (SEQ ID NO. 1)) of wild-type soybean GS1 itself ((named GmGS1_WT, the amino acid sequence shown as SEQ ID NO.3, the coding nucleotide sequence shown as SEQ ID NO. 7) from the amino acid residue T (threonine) to P (proline), W (tryptophan) and deleting the same.

The amino acid sequence alignment of soybean GS1 mutants GT60P, GT W and GT60X and wild type soybean GS1 is shown in fig. 3, in which: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each soybean GS1 mutant was at the position encoding amino acid 60, the codons for the corresponding amino acids were as shown in the following table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.

Amino acids	P	W	Deletion of
				Codons	CCA	TGG	Without any means for

The soybean GS1 mutants GT60P, GT W and GT60X and the nucleic acid molecules encoding them provided in this example can be obtained by chemical synthesis.

Example 4

This example provides three canola (Brassica napus) GS1 mutants, which are obtained from the wild-type canola GS1 itself (designated as bngs1_wt, amino acid sequence shown in SEQ ID No.4, encoding nucleotide sequence of SEQ ID No. 8) at position 60 (corresponding to position 60 of the reference sequence (SEQ ID No. 1)) by mutation of amino acid residue T (threonine) to P (proline), W (tryptophan) and deletion, respectively. The obtained rape GS1 mutants were named BT60P, BT W and BT60X, respectively.

The amino acid sequence alignment of the rape GS1 mutant BT60P, BT W and BT60X and the wild type rape GS1 is shown in FIG. 4, in which: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each canola GS1 mutant is at the position encoding amino acid 60, the codons for the corresponding amino acids are shown in the following table, and the nucleotides at the remaining positions are identical to the corresponding wild-type coding sequence.

Amino acids	P	W	Deletion of
				Codons	CCA	TGG	Without any means for

The rape GS1 mutant BT60P, BT W and BT60X and the nucleic acid molecules encoding the same can be obtained by a chemical synthesis method.

Experimental example 1

The rice GS1 mutants OT60P, OT W and OT60X provided in example 1 were tested for glufosinate resistance by the following method:

According to the sequence of the nucleic acid molecule provided in example 1, coding genes encoding rice GS1 mutant OT60P, OT W and OT60X are synthesized by adopting a chemical synthesis method, enzyme cleavage sites (Pac 1 and Sbf 1) are introduced into two ends of the coding genes, the coding genes are connected to an expression vector (such as pADV7 vector with a structure shown in FIG. 5) subjected to the same enzyme cleavage treatment under the action of a ligase after enzyme cleavage, and then glutamine synthetase-deficient E.coli obtained by knocking out glutamine synthetase from Tianyuxing grass biotechnology Co., ltd, on the basis of E.coli DH5 alpha, is transformed respectively. After verification, positive clones are selected, inoculated to M9 culture medium containing glufosinate with different concentrations for growth, and defective E.coli growth is observed. Glufosinate resistance was examined with wild-type rice GS1 (wild-type rice GS1 transformed with glutamine synthetase-deficient escherichia coli) as a negative control, and with GS1 mutants OT60P (T60P, amino acid T at position 60 of rice GS1 was mutated to P), OT60W (T60W), OT60X (T60X, amino acid T at position 60 of rice GS1 was deleted). The results are shown in FIG. 6.

On a culture medium containing 0mM glufosinate (KP 0), the defective strains of the coding genes of the wild-type rice GS1 (OsGS 1-WT) and rice GS1 mutants OT60P, OT W and OT60X can grow normally, which shows that GS1 coded by OT60P, OT W and OT60X has normal GS1 enzyme activity;

Coli transformed with wild-type rice GS1 failed to grow on 10mM glufosinate (KP 10) but the growth of the transformed rice mutants OT60P, OT W and OT60X was significantly better than the negative control, indicating that the single mutants containing OT60P, OT W and OT60X were significantly better than the wild-type.

These results demonstrate that single mutants of OT60P, OT W and OT60X are able to increase the tolerance to glufosinate (to higher concentrations of glufosinate) of wild-type glutamine synthetase and that both single mutants have resistance to glufosinate.

Experimental example 2

Referring to the detection method of experimental example 1, the glufosinate resistance of maize GS1 mutants ZT60P, ZT60W and ZT60X provided in example 2 was verified, and the results are shown in fig. 7:

transformation of a defective strain encoding wild-type maize GS1 (zmgs1_wt) and maize GS1 mutants ZT60P, ZT W and ZT60X encoding genes both grew on a medium containing 0mM glufosinate (KP 0), indicating that GS1 encoded by ZT60P, ZT W and ZT60X has normal GS1 enzyme activity;

coli transformed with wild-type rice GS1 could not grow on a medium containing 10mM glufosinate (KP 10), but the escherichia coli transformed with maize mutants ZT60P, ZT W and ZT60X could still grow normally, demonstrating that the ability of the single mutant containing ZT60P, ZT W and ZT60X to resist glufosinate was significantly better than that of the wild-type.

These results demonstrate that single mutants of ZT60P, ZT W and ZT60X are able to increase the tolerance to glufosinate (to higher concentrations of glufosinate) of wild-type glutamine synthetases and that both single mutants have resistance to glufosinate.

Experimental example 3

Referring to the test method of experimental example 1, glufosinate resistance of soybean GS1 mutants GT60P, GT W and GT60X provided in example 3 was verified, and the results are shown in fig. 8: transformation of defective strains encoding the wild type soybean GS1 (gmgs1_wt) and soybean GS1 mutants GT60P, GT W and GT60X, both grown normally, on a medium containing 0mM glufosinate (KP 0), indicating that GS1 encoded by both GT60P, GT W and GT60X has normal GS1 enzyme activity;

coli transformed with wild-type soybean GS1 grew little on the medium containing 5mM glufosinate (KP 5), but the growth of the transformed soybean mutants GT60P, GT W and GT60X was significantly better than the negative control, indicating that the single mutants containing GT60P, GT W and GT60X were significantly better than the wild-type.

These results demonstrate that single mutants of GT60P, GT W and GT60X are able to increase the tolerance to glufosinate (to higher concentrations of glufosinate) of wild-type glutamine synthetases and that both single mutants have resistance to glufosinate.

Experimental example 4

Referring to the test method of experimental example 1, the glufosinate resistance of the rape GS1 mutants BT60P, BT W and BT60X provided in example 4 was verified, and the results are shown in fig. 9:

on a culture medium containing 0mM glufosinate (KP 0), the defective strains of the coding genes of the wild rape GS1 (BnGS 1-WT) and the rape GS1 mutants BT60P, BT W and BT60X can grow normally, which shows that GS1 coded by BT60P, BT W and BT60X has normal GS1 enzyme activity;

coli transformed with wild-type canola GS1 was unable to grow on media containing 5mM glufosinate (KP 5), but the growth of the coli transformed with canola mutants BT60P, BT W and BT60X was significantly better than that of the negative control, indicating that the single mutants containing BT60P, BT W and BT60X were significantly better than the wild-type.

These results demonstrate that both single mutants of BT60P, BT60W and BT60X have resistance to glufosinate.

Experimental example 5

The enzyme kinetic parameters of OT60W provided in example 1, ZT60W provided in example 2, GT60W mutant provided in example 3 and BT60W mutant provided in example 4 and enzyme kinetic parameters in the presence of glufosinate were measured separately against wild type rice gs1 osgs1_wt, wild type maize gs1 zmgs1_wt, wild type soybean gs1 gmgs1_wt and wild type canola gs1 bngs1_wt as follows:

1. And (3) constructing a carrier:

the nucleic acid sequence encoding the mutant is cloned into a prokaryotic expression vector pET32a, and the cloning is verified by sequencing.

2.6His protein purification:

the mutant enzyme protein was purified by 6His and the concentration was determined using the Bradford protein concentration determination kit using standard methods and the protein was stored in a protein stock solution.

3. Enzyme activity determination:

(1) Instrument and reagents: enzyme-labeled instrument (De-Fe: HBS-1096A), glufosinate, substrate L-sodium glutamate (CAS: 6106-04-3).

(2) Principle of measurement

Glutamine Synthetase (GS) is the first enzyme to convert inorganic nitrogen to organic nitrogen, catalyzes the binding of ammonia from different sources to glutamate to form glutamine, and liberates Pi, as shown in the following specific reaction scheme:

the invention adopts a phosphomolybdic blue method to measure GS enzyme activity, and the principle of the method is as follows: under suitable acidic conditions, phosphoric acid (Pi) reacts with ammonium molybdate to form ammonium phosphomolybdate, which in turn is reduced by reducing agents (e.g., vitamin C, stannous chloride, etc.) to blue phosphomolybdenum blue, the blue shade of which is proportional to the phosphorus content.

(2) The operation steps are as follows:

the reaction was carried out in a Tris-HCl buffer system at pH7.5 in a total reaction volume of 30uL. The glutamine synthetase enzyme activity determination reaction liquid comprises the following components: 200mM Tris-HCl (pH 7.5), 1.67mM ATP,30mM ammonium chloride, 20mM MgCl ₂ 10mM sodium L-glutamate. After 30ul of reaction solution is uniformly mixed, preheating is carried out for 5min at 35 ℃, mutant protein solution is added for reaction, after reaction is carried out for 30min at 35 ℃, 100 μl of color reaction D solution [ D=2A+B ] is added; and (3) solution A: 12% (W/V) ascorbic acid in 1mol/L hydrochloric acid solution, solution B: 2% (W/V) aqueous solution of ammonium molybdate tetrahydrate]Color was developed, left to stand for 5 minutes, and 100. Mu.l of a reaction termination F solution (2% sodium citrate, 2% acetic acid aqueous solution) was added thereto, and left to stand for 15 minutes, 200. Mu.l of the light absorption value was measured at 660 nm.

The results are shown in FIG. 10. As can be seen from the results of fig. 10:

the Km values of the GS1 mutants were slightly higher than those of the wild-type controls osgs1_wt, zmgs1_wt, gmgs1_wt and bngs1_wt, indicating that the GS mutants reduced sensitivity to glufosinate inhibitors while slightly reduced sensitivity to normal substrate (L-sodium glutamate). V of GS1 mutant _max All were higher than the wild type control, indicating that these mutants had improved enzymatic ability. Wild type controls are sensitive to glufosinate, IC for wild type rice GS1, wild type maize GS1, wild type soybean GS1 and wild type canola GS1 ₅₀ 0.006mM, 0.005mM and 0.007mM, respectively.

Mutant IC ₅₀ IC's of OT60W, ZT60W, GT60W, BT60W, all significantly higher than wild-type controls ₅₀ Far higher than the wild-type control, indicating that the mutant is less sensitive to glufosinate. From mutant IC ₅₀ And wild type IC ₅₀ It can also be seen in the multiple relationship of OT60W, ZT60W, GT60W, BT60W IC ₅₀ Corresponding to wild type GS1 IC ₅₀ 796.33 times, 10373.5 times, 105.4 times, 13308.5 times, these values also indicate the enzymatic activity of the mutantsSex was far higher than wild type control. These data illustrate the mechanism of mutant resistance to glufosinate by enzyme kinetics.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for obtaining a protein having glufosinate resistance comprising the steps of:

3) Proteins with increased glufosinate resistance are selected.

2. A plant glutamine synthetase mutant having glufosinate resistance, wherein said plant glutamine synthetase mutant is represented by (1) or (2):

(1): it is obtained by mutating the n-th position of wild glutamine synthetase from plant; the position of the nth bit is determined as follows: the wild type glutamine synthetase is aligned with a reference sequence, the n-th position of the wild type glutamine synthetase corresponds to the 60-th position of the reference sequence, wherein the amino acid sequence of the reference sequence is shown as SEQ ID NO. 1;

3. The plant glutamine synthetase mutant according to claim 2, wherein when the source of said wild type glutamine synthetase is rice, the amino acid at the n-th position of the plant glutamine synthetase mutant is proline, tryptophan or deleted;

when the source of the wild-type glutamine synthetase is soybean, the n-th amino acid of the plant glutamine synthetase mutant is proline, tryptophan or deleted;

4. A nucleic acid molecule encoding a plant glutamine synthetase mutant according to any one of claims 2-3.

5. An expression cassette or vector comprising the nucleic acid molecule of claim 4.

6. A recombinant bacterium or recombinant cell comprising the nucleic acid molecule of claim 4 or the expression cassette or vector of claim 5; and the recombinant cell is a non-plant cell;

preferably, the recombinant bacteria are agrobacterium, escherichia coli or yeast.

7. Use of a plant glutamine synthetase mutant according to any one of claims 2-3, a nucleic acid molecule according to claim 4, an expression cassette or vector according to claim 5 or a recombinant bacterium or recombinant cell according to claim 6 for the cultivation of a plant variety having glufosinate resistance.

8. The use according to claim 7, wherein the use comprises any one of the following uses:

(1) Transforming a plant of interest with said vector comprising a gene encoding a plant glutamine synthetase mutant;

preferably, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy chrysanthemum, grape, strawberry, sugarcane, tobacco, brassica vegetable, cucurbitaceae, leguminous plant, pasture, tea or cassava;

preferably, the pasture is selected from gramineous pasture or leguminous pasture;

preferably, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, blue, canola, green vegetables or beet;

Preferably, the cucurbitaceae plant is selected from cucumber, pumpkin, white gourd, balsam pear, luffa, melon, watermelon or melon;

preferably, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;

preferably, the plant is rice, maize, soybean or canola.

9. A method of producing a glufosinate herbicide tolerant plant comprising introducing into the genome of a plant of interest a gene encoding a plant glutamine synthetase mutant of any one of claims 2-3.

10. The method of producing a glufosinate herbicide tolerant plant of claim 9 wherein the method of introduction is selected from genetic transformation methods, genome editing methods or genetic mutation methods.