WO2022142936A1 - Plant-derived glufosinate-ammonium-resistant glutamine synthase mutant, nucleic acid molecule, and applications - Google Patents

Abstract

Provided are a plant-derived glufosinate-ammonium-resistant glutamine synthase mutant, a nucleic acid molecule, and applications. Compared with wild-type glutamine synthases, the mutant has a mutation at position 68, which turns to D, E, G, H, N, P, Q, V or deletion after the mutation, and such mutation gives a glutamine synthase glufosinate-ammonium resistance. The glutamine synthase mutant can be used for breeding new plant varieties having glufosinate-ammonium resistance.

Description

Plant-derived glufosinate-resistant glutamine synthase mutants, nucleic acid molecules, and applications

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure requires a Chinese patent with the application number CN 202011626161.3 and the title of "Plant-derived Glufosinate-resistant Glutamine Synthetase Mutants, Nucleic Acid Molecules and Applications" filed with the China Patent Office on December 31, 2020 Priority of the application, the entire contents of which are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the technical field of genetic engineering, in particular, to a plant-derived glutamine synthase mutant with glufosinate-ammonium resistance, a nucleic acid molecule and applications.

Background technique

Glutamine synthetase (GS) is a key enzyme in plant nitrogen metabolism. It catalyzes the condensation of glutamate (Gln) and NH3 to form glutamine (Glu) in the glutamate synthetase cycle, and is involved in plant nitrogen-containing Metabolism of compounds. According to the distribution and subcellular localization, the isoenzymes of higher plant GS (which belong to GSII) can be divided into two types: one is located in the cytoplasm called cytoplasmic GS (GS1) with a molecular weight of 38-40kDa; the other is located in the cytoplasm. The chloroplast (or plastid) is called plastid-type GS (GS2), with a molecular weight of 44-45kDa.

Glufosinate (glufosinate, glufosinate ammoniμM, trade name Basta) is a glutamine synthase (GS1) inhibitor developed by Aventis (now Bayer), its active ingredient is phosphinothricin (referred to as PPT), chemical name It is (RS)-2-amino-4-(hydroxymethyl phosphinyl) ammonium butyrate. The product was launched in 1986, and sales have increased year by year. The target enzyme of glufosinate-ammonium is GS. Under normal circumstances, GS can form λ-glutamyl phosphate from ATP and glutamate. However, after PPT treatment, PPT is first combined with ATP, and the phosphorylated PPT occupies the eight reaction centers of GS molecule, which changes the spatial configuration of GS and inhibits the activity of GS. PPT inhibits all known forms of GS.

The result of glufosinate-ammonium inhibition of GS can lead to disorder of nitrogen metabolism in plants, excessive accumulation of ammonium, disintegration of chloroplasts, inhibition of photosynthesis, and ultimately plant death.

At present, the main method of cultivating glufosinate-resistant varieties is to use genetic engineering methods to introduce the glufosinate-resistant gene from bacteria into crops, so as to cultivate new varieties of transgenic glufosinate-resistant crops. At present, the most widely used glufosinate-resistant genes in agriculture are the bar gene from the strain Streptomyces hygroscopicus and the pat gene from the strain S. viridochromogenes. Bar gene and pat gene share 80% homology, and both can encode glufosinate acetylase, which can inactivate glufosinate acetylation. Glufosinate-resistant varieties have great use value, among which resistant rape and corn have been commercialized in large areas.

However, due to the anti-GM wave, the acceptance of GM crops in the world is still low. Even in the Americas, where the GM crops are planted with the largest area, GM is mainly limited to a few crops such as corn, soybean, and cotton. In particular, the bar gene and pat gene are derived from microorganisms, rather than from the crops themselves, which are more likely to cause consumer resistance.

The glufosinate acetylase encoded by Bar and pat genes can acetylate and inactivate glufosinate, but it is difficult for the enzyme to completely inactivate glufosinate before the glufosinate is exposed to GS, because many GS are distributed in the Therefore, when glufosinate-ammonium is applied to bar gene and pat gene crops, it will interfere with the nitrogen metabolism of plants to varying degrees, and at the same time affect the normal growth and development of plants. Overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate, but the degree of tolerance is insufficient for commercial application.

In view of this, the present disclosure is hereby made.

SUMMARY OF THE INVENTION

The present disclosure provides a glutamine synthase mutant with glufosinate resistance, which is shown in the following (1) or (2):

(1): it is obtained by mutating the n-th position of a plant-derived wild-type glutamine synthetase; the position of the n-th position is determined by comparing the wild-type glutamine synthase with the reference sequence. Right, the nth position of the wild-type glutamine synthetase corresponds to the 68th position of the reference sequence, wherein the amino acid sequence of the reference sequence is as shown in SEQ ID NO.1;

The amino acid at position n of the glutamine synthase mutant is X, where X=D, E, G, H, N, P, Q, V or deletion.

(2): It is at least 85% identical to the glutamine synthetase mutant shown in (1), and is at the n-th position with the glutamine synthase mutant shown in (1). Same amino acid and glufosinate resistance.

The research of the present disclosure found that the wild-type glutamine synthetase derived from plants was compared with the reference sequence, and the amino acid position corresponding to the 68th position of the reference sequence, that is, the n-th position, was mutated to mutate to D, E, G, H, N, P, Q, V or deletion, the obtained glutamine synthase mutant has glufosinate resistance, while maintaining its own biological enzyme catalytic activity. Plants or recombinant bacteria transformed with the plant glutamine synthase mutant provided by the present disclosure can grow and develop normally in the presence of glufosinate-ammonium, and the plant glutamine synthase mutant is not only used for transgenic crop cultivation, but also It can be used to cultivate glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybean, corn, wheat, rapeseed, cotton and sorghum, etc., and has broad application prospects.

The above reference sequence is a wild-type glutamine synthase derived from rice.

The sequence alignment method can use the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to perform Protein Blast alignment; other sequence alignment methods or tools well known in the art can also be used to obtain the same result.

It is believed, without being bound by theory, that position n of wild-type glutamine synthase may also be position 68 on its own sequence (eg, corn, wheat, soybean, canola, etc.), but may not be position 68, The position of the nth position is determined according to the aforementioned sequence alignment. As long as it is aligned with the reference sequence, the position corresponding to the 68th position of the reference sequence is the nth position described in the present disclosure, that is, the mutation site.

Optionally, in some embodiments of the present disclosure, the plants include, but are not limited to, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato , cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon , tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, grass, lawn Any of grass, tea and tapioca.

It is believed, without being bound by theory, that all plant wild-type glutamine synthetases are homologous and have substantially the same functions and domains in plants. Therefore, any plant-derived wild-type glutamine synthase mutant obtained by mutating the above-mentioned 68th position has glufosinate resistance. Therefore, the glutamine synthetase mutant obtained by the above-mentioned mutation of any plant-derived wild-type glutamine synthase belongs to the protection scope of the present disclosure.

In addition, those skilled in the art know and can easily realize that, in the non-conserved region of the glutamine synthetase mutant shown in (1), simple amino acid substitution or deletion or addition is performed, and the nth position is maintained after the above-mentioned mutation. amino acid, and the glutamine synthetase mutant obtained by further mutation has at least 85% (for example, 85%, 86%, 87%, 88%, 89% of the glutamine synthetase mutant shown in (1)) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) or more identity, and its functions include enzymatic activity and glufosinate resistance Compared with the glutamine synthase mutant shown in (1), it is equivalent to or slightly decreased, or slightly increased or greatly increased, etc. Therefore, such glutamine synthase should also fall within the scope of the present disclosure.

It should be noted that X=deletion means that the amino acid at position n of the wild-type glutamine synthetase is deleted, that is, deletion mutation.

In some embodiments, for different plant-derived glutamine synthetases, the nth position is mutated to D, E, G, H, N, P, Q, V or deleted, and it is mutated to other The amino acid also makes glutamine synthase resistant to glufosinate.

For example, optionally, in some embodiments of the present disclosure, when the plant is rice or corn, X=A, C, D, E, F, G, H, I, K, L, M, N , P, Q, R, T, V, W, Y or delete;

When the plant is soybean, X=D, E, G, H, I, K, M, N, P, Q, V, Y or deletion;

When the plant is wheat, X=D, E, G, H, N, P, Q, V or deletion;

When the plant is rape, X=A, C, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y or deletion.

In some embodiments, for different plant-derived glutamine synthetases, mutating the nth position to D, E, G, H, N, P, Q, V and other amino acids other than deletions will also make Glutamine synthase is glufosinate-resistant.

Optionally, in some embodiments of the present disclosure, when the plant is rice, the rice wild-type glutamine synthase is SEQ ID NO.1:

Optionally, in some embodiments of the present disclosure, when the plant is maize, the maize wild-type glutamine synthetase is SEQ ID NO.2:

Optionally, in some embodiments of the present disclosure, when the plant is soybean, the soybean wild-type glutamine synthetase is SEQ ID NO.3:

Optionally, in some embodiments of the present disclosure, when the plant is wheat, the wheat wild-type glutamine synthetase is SEQ ID NO.4:

Optionally, in some embodiments of the present disclosure, when the plant is rape, the rape wild-type glutamine synthase is SEQ ID NO.5:

The similarity and identity of some plant-derived wild-type glutamine synthetases are shown in the following table, and the partial results of the sequence alignment are shown in Figure 13, and the arrow indicates the 68th amino acid.

The alignment method of the above similarity (Similarity) and identity (Identity) is: input the amino acid sequence of a species into the Blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for Protein Blast Align, find the similarity (Similarity) and identity (Identity) of this species and other species that need to be aligned from the alignment results.

The present disclosure provides an isolated nucleic acid molecule encoding the glufosinate-resistant glutamine synthase mutant of any of the above.

In the case where the present disclosure provides the above-mentioned amino acid sequences, those skilled in the art can easily obtain nucleic acid sequences encoding the above-mentioned glutamine synthase mutants according to the degeneracy of codons. For example, the nucleic acid sequence encoding the above-mentioned glutamine synthase mutant can be obtained by making corresponding nucleotide mutations in the nucleic acid sequence encoding wild-type glutamine synthase. This is easily accomplished by those skilled in the art.

For example, the encoding nucleic acid sequence of rice wild-type glutamine synthase is SEQ ID NO.6:

Accordingly, on the basis of the sequence, the corresponding nucleotide mutation is performed at the codon corresponding to the 68th position of the encoded amino acid sequence to obtain the rice glutamine synthase mutant encoding the above.

The coding nucleic acid sequence of corn wild-type glutamine synthetase is SEQ ID NO.7:

The coding nucleic acid sequence of soybean wild-type glutamine synthetase is SEQ ID NO.8:

The coding nucleic acid sequence of wheat wild-type glutamine synthetase is SEQ ID NO.9:

The coding nucleic acid sequence of rape wild-type glutamine synthetase is SEQ ID NO.10:

The present disclosure provides a vector containing the nucleic acid molecule as described above.

The present disclosure provides a recombinant bacteria or a recombinant cell containing the above-mentioned nucleic acid molecule or the above-mentioned vector.

The present disclosure provides the glufosinate-resistant glutamine synthetase mutant as described in any one of the above, the nucleic acid molecule as described above, the vector as described above, or the recombinant bacteria or recombinant cells as described above when cultivating Application of glufosinate-ammonium-resistant plant varieties.

Optionally, in some embodiments of the present disclosure, it comprises: transforming a plant of interest with a vector containing a gene encoding the glutamine synthase mutant.

Optionally, in some embodiments of the present disclosure, it includes: modifying the endogenous glutamine synthase gene of the target plant to encode the glutamine synthase mutant.

Optionally, in some embodiments of the present disclosure, it includes mutagenizing and screening plant cells, tissues, individuals or populations to encode the glutamine synthase mutant.

On the basis that the present disclosure provides glutamine synthetase mutants, those skilled in the art can easily imagine that through conventional transgenic technology, gene editing technology (such as zinc finger endonucleases (ZFN, zinc-finger nucleases) in the art technology, transcription activator-like effector nucleases (TALEN, transcription activator-like effector nucleases) technology or CRISPR/Cas9), mutagenesis breeding technology (such as chemical, radiation mutagenesis, etc.), etc. The gene encoding the glutamine synthase mutant as described above is then used to obtain glufosinate-ammonium resistance and to be able to grow and develop normally, so as to obtain a new plant variety with glufosinate-ammonium resistance. Therefore, no matter what technology is adopted, as long as it utilizes the glutamine synthase mutant provided by the present disclosure to impart glufosinate resistance to plants, it falls within the protection scope of the present disclosure.

Optionally, in some embodiments of the present disclosure, the plants of interest include but are not limited to wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, Potato, cotton, soybean, canola, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter squash, bitter gourd, loofah, vegetable melon, watermelon, Melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, pasture, Any of lawn grass, tea and cassava.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present disclosure more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 provides rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, Results of partial alignment of amino acid sequences of OsY and OsX and wild-type rice GS1 OsGS1_WT.

Fig. 2 The partial ratio of amino acid sequences of soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX and wild-type soybean GS1 GmGS1_WT provided in Example 2 of the present disclosure to the results.

Figure 3 provides the maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, Results of partial alignment of amino acid sequences of ZmY and ZmX with wild-type maize GS1 ZmGS1_WT.

Figure 4 shows the partial alignment results of the amino acid sequences of wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX and wild-type wheat GS1 TaGS1_WT provided in Example 2 of the present disclosure.

Fig. 5 Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and Partial alignment of amino acid sequences of BnX and wild-type rape GS1 BnGS1_WT.

FIG. 6 is a schematic structural diagram of the pADV7 vector provided in Experimental Example 1 of the present disclosure.

FIG. 7 is provided in Experimental Example 1 of the present disclosure. The rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, Growth results of E. coli OsT, OsV, OsW, OsY and OsX and wild-type rice GS1 OsGS1_WT on media containing different concentrations of glufosinate.

Figure 8 provides the transformation example 2 of the present disclosure provided the soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX and wild-type soybean GS1 Growth results of GmGS1_WT Escherichia coli on media containing different concentrations of glufosinate.

Fig. 9 is the maize GS1 mutant ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, Growth results of E. coli ZmT, ZmV, ZmW, ZmY and ZmX and wild-type maize GS1 ZmGS1_WT on media containing different concentrations of glufosinate.

Figure 10 is provided in Experimental Example 4 of the present disclosure for the transformation of wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX and wild-type wheat GS1 TaGS1_WT provided in Example 4 in E. coli containing different concentrations Growth results on glufosinate-ammonium medium.

Fig. 11 The Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, Growth results of BnV, BnW, BnY and BnX and wild-type canola GS1 BnGS1_WT E. coli on media containing different concentrations of glufosinate.

Figure 12 Rice GS1 mutant OsP, soybean GS1 mutant GmQ, corn GS1 mutant ZmV, wheat GS1 mutant TaG, rape GS1 mutant BnE, wild-type rice GS1 OsGS1_WT, wild-type soybean GS1 provided in Experimental Example 6 of the present disclosure Enzyme kinetic parameters and glufosinate resistance parameters IC50 of GmGS1_WT, wild-type maize GS1 ZmGS1_WT, wild-type wheat GS1 TaGS1_WT and wild-type rape GS1 BnGS1_WT.

Figure 13 is the amino acid sequence alignment results of different plant wild-type glutamine synthases; in the figure: TaGS1_WT: wheat wild-type glutamine synthase; OsGS1_WT: rice wild-type glutamine synthase; ZmGS1_WT: maize wild-type type glutamine synthase; GmGS1_WT: soybean wild-type glutamine synthase; BnGS1_WT: rape wild-type glutamine synthase.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If the conditions are not specified in the examples, it is carried out in accordance with the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Some embodiments of the present disclosure provide a plant-derived glufosinate-resistant glutamine synthase mutant, a nucleic acid molecule, and uses. The glutamine synthase mutant provided by the present disclosure is originally derived from plants, and has glufosinate resistance after mutation. Plants transformed with the glutamine synthase mutant not only have glufosinate resistance, but also can grow normally. and development.

The features and properties of the present disclosure will be further described in detail below with reference to the embodiments.

Example 1

The rice (Oryza sativa) glutamine synthase (GS1) mutant provided in this example is composed of the wild-type rice glutamine synthase itself (named OsGS1_WT, the amino acid sequence is shown in SEQ ID NO. The amino acid residue S at the 68th position of the nucleotide sequence of SEQ ID NO.6) is mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y or deletion, the obtained rice GS1 mutants were named OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX.

Amino acids of rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY, OsX and wild-type rice GS1 The sequence alignment is shown in Figure 1, in the figure: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each rice GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

amino acid	A	C	D	E	F
a	GCC	TGC	GAT	GAG	TTC
amino acid	G	H	I	K	L
a	GGT	CAC	ATC	AAG	CTC
amino acid	M	N	P	Q	R
a	ATG	AAC	CCC	CAG	CGC
amino acid	T	V	W	Y	delete
a	ACC	GTC	TGG	TAC	none

The rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX provided in this example and coding Their nucleic acid molecules can be obtained by chemical synthesis.

Example 2

The soybean (Glycine max) GS1 mutant provided in this example is derived from the wild-type soybean GS1 itself ((named GmGS1_WT, the amino acid sequence is shown in SEQ ID NO.3, and the encoding nucleotide sequence is SEQ ID NO.8) The 68th position of (corresponding to the 68th position of the reference sequence (SEQ ID NO. 1)) is mutated from the amino acid residue S to D, E, G, H, I, K, M, N, P, Q, V, Y or deletion.The obtained rice soybean GS1 mutants were named GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX, respectively.

The amino acid sequence alignment of soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY, GmX and wild-type soybean GS1 is shown in Figure 2, in the figure: the arrow The indicated positions are mutation sites.

The coding sequences of soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX provided in this example correspond to SEQ ID NO.3.

In this example, the coding sequence of each soybean GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

The soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX provided in this example and the nucleic acid molecules encoding them can be obtained by chemical synthesis.

Example 3

The maize (Zea mays) GS1 mutant provided in this example is composed of wild-type maize GS1 itself (named ZmGS1_WT, the amino acid sequence is shown in SEQ ID NO.2, and the coding nucleotide sequence is SEQ ID NO.7). Position 68 (corresponding to position 68 of the reference sequence (SEQ ID NO. 1)) was mutated from amino acid residue S to A, C, D, E, F, G, H, I, K, L, M, N , P, Q, R, T, V, W, Y or delete. The resulting maize GS1 mutants were named ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX, respectively.

Amino acids of maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY, ZmX and wild-type maize GS1 The sequence alignment is shown in Figure 3, in the figure: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each maize GS1 mutant is at the position encoding the 68th amino acid, and the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

amino acid	A	C	D	E	F
a	GCC	TGC	GAT	GAG	TTC
amino acid	G	H	I	K	L
a	GGT	CAC	ATC	AAA	CTC
amino acid	M	N	P	Q	R
a	ATG	AAC	CCA	CAA	AGG
amino acid	T	V	W	Y	delete
a	ACC	GTC	TGG	TAC	none

The maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX and coding Their nucleic acid molecules can be obtained by chemical synthesis.

Example 4

The wheat (Triticum aestivum) GS1 mutant provided in this example is derived from wild-type wheat GS1 itself (named TaGS1_WT, the amino acid sequence is shown in SEQ ID NO.4, and the coding nucleotide sequence is SEQ ID NO.9). Position 68 (corresponding to position 68 of the reference sequence (SEQ ID NO. 1)) results from mutation of amino acid residue S to D, E, G, H, N, P, Q, V or deletion. The obtained wheat GS1 mutants were named TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX, respectively.

The amino acid sequence alignment of wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV, TaX and wild-type wheat GS1 is shown in Figure 4. In the figure, the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each wheat GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

The wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX provided in this example and the nucleic acid molecules encoding them can be obtained by chemical synthesis.

Example 5

The Brassica napus GS1 mutant provided in this example is derived from the wild-type Brassica napus GS1 itself (named BnGS1_WT, the amino acid sequence is shown in SEQ ID NO.5, and the coding nucleotide sequence is SEQ ID NO.10). Position 68 (corresponding to position 68 of the reference sequence (SEQ ID NO. 1)) was mutated from amino acid residue S to A, C, D, E, F, G, H, I, K, L, M, N , P, Q, T, V, W, Y or delete. The resulting rapeseed GS1 mutants were named BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX, respectively.

Amino acid sequence ratio of rapeseed GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY, BnX and wild-type rapeseed GS1 As shown in Figure 5, in the figure: the position indicated by the arrow is the mutation site.

In this example, the coding sequence of each rapeseed GS1 mutant is at the position encoding the 68th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

Brassica napus GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX and their encoding Nucleic acid molecules can be obtained by chemical synthesis.

Experimental example 1

Detection of rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX provided in Example 1 Glufosinate-ammonium resistance, as follows:

According to the sequence of the nucleic acid molecule provided in Example 1, the method of chemical synthesis was used to synthesize the rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, The coding genes of OsR, OsT, OsV, OsW, OsY and OsX were introduced with restriction enzyme sites (Pac1 and Sbf1) at both ends. After restriction enzyme digestion, they were connected to the expression vector treated with the same restriction enzyme under the action of ligase ( For example, the pADV7 vector, whose structure is shown in Figure 6), was then transformed into glutamine synthase-deficient E. coli. After verification, positive clones were picked and inoculated on M9 medium containing different concentrations of glufosinate for growth. , to observe the growth of defective E. coli. Using the wild-type rice GS1 mutant as a negative control, the GS1 mutants containing OsA (S68A, the amino acid S at position 68 of rice GS1 was mutated to A), OsC (S68C), OsD (S68D), OsE (S68E), and OsF were detected. (S68F), OsG(S68G), OsH(S68H), OsI(S68I), OsK(S68K), OsL(S68L), OsM(S68M), OsN(S68N), OsP(S68P), OsQ(S68Q), OsR (S68R), OsT (S68T), OsV (S68V), OsW (S68W), OsY (S68Y) and OsX (S68Δ) glufosinate resistance. The results are shown in Figure 7.

On the medium containing 0 mM glufosinate-ammonium (KP0), transform coding wild-type rice GS1 (OsGS1_WT) and rice GS1 mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX coding genes of the deficient strains can grow normally, indicating that OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL , OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX encoded GS1 all have normal GS1 enzymatic activity;

E. coli transformed with wild-type rice GS1 could not grow on medium containing 10 mM glufosinate-ammonium (KP10), but transformed rice mutants OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL , OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX E. coli growth was significantly better than the negative control, indicating that OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, The ability of single mutants of OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX to resist glufosinate was significantly better than wild type; at better glufosinate concentration (20mM, KP20) On the medium, E. coli transformed with rice GS1 mutants OsA, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsV, OsW, OsY and OsX also had Significant growth.

These results indicate that the single mutants of OsA, OsC, OsD, OsE, OsF, OsG, OsH, OsI, OsK, OsL, OsM, OsN, OsP, OsQ, OsR, OsT, OsV, OsW, OsY and OsX are resistant to weeds The ability of ammonium phosphine.

Experimental example 2

Referring to the detection method of Experimental Example 1, it was verified that the soybean GS1 mutants GmD (S68D, the amino acid S at position 68 of soybean GS1 was mutated to D), GmE (S68E), GmG (S68G), GmH (S68H) provided in Example 2 , GmI(S68I), GmK(S68K), GmK(S68M), GmN(S68N), GmP(S68P), GmQ(S68Q), GmV(S68V), GmK(S68Y) and GmX(S68Δ) glufosinate resistance sex. The results are shown in Figure 8.

According to the results in Figure 8, it can be seen that:

Transformation of wild-type soybean GS1 (GmGS1_WT) and soybean GS1 mutants GmD, GmE, GmG, GmH, GmI, GmK, GmN, GmP, GmQ, GmV and GmX on medium containing 0 mM glufosinate-ammonium (KP0) The deficient strains encoding the genes could grow normally, indicating that GS1 encoded by GmD, GmE, GmG, GmH, GmI, GmK, GmN, GmP, GmQ, GmV and GmX all had normal GS1 enzyme activity;

On medium containing 2 mM glufosinate-ammonium (KP2), E. coli transformed with wild-type soybean GS1 could not grow substantially, but soybean mutants GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP were transformed , GmQ, GmV, GmY and GmX E. coli growth was significantly better than the negative control, indicating that the single mutant antibody containing GmD, GmE, GmG, GmH, GmI, GmK, GmM, GmN, GmP, GmQ, GmV, GmY and GmX The ability of glufosinate-ammonium was significantly better than that of the wild type; E. coli transformed with soybean GS1 mutants GmG and GmQ still grew significantly on the medium with higher glufosinate concentration (20mM, KP20).

These results indicate that the single mutants of GmD, GmE, GmG, GmH, GmI, GmK, GmN, GmP, GmQ, GmV and GmX all have the ability to resist glufosinate, and the soybean GS1 mutants GmG and GmQ are resistant to glufosinate more capable.

Experimental example 3

With reference to the detection method of Experimental Example 1, it was verified that the maize GS1 mutants ZmA (S68A, the amino acid S at position 68 of maize GS1 was mutated to A), ZmC (S68C), ZmD (S68D), ZmE (S68E) provided in Example 3 , ZmE(S68F), ZmG(S68G), ZmH(S68H), ZmI(S68I), ZmK(S68K), ZmL(S68L), ZmM(S68M), ZmN(S68N), ZmP(S68P), ZmQ(S68Q) , ZmR (S68R), ZmT (S68T), ZmV (S68V), ZmW (S68W), ZmY (S68Y) and ZmX (S68Δ) glufosinate resistance. The results are shown in Figure 9.

According to the results in Figure 9, it can be seen that:

On medium containing 0 mM glufosinate-ammonium (KP0), transform coding wild-type maize GS1 (ZmGS1_WT) and maize GS1 mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, The defective strains of ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX can grow normally, indicating that ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL , ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX encoded GS1 all have normal GS1 enzymatic activity;

On medium containing 2 mM glufosinate-ammonium (KP2), E. coli transformed with wild-type maize GS1 were essentially incapable of growing, but maize mutants ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK were transformed , ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX E. coli growth was significantly better than the negative control, indicating that ZmA, ZmC, ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, The single mutants of ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmR, ZmT, ZmV, ZmW, ZmY and ZmX were significantly more resistant to glufosinate-ammonium than wild-type; at higher glufosinate concentrations (20mM, KP20 ), the E. coli transformed with the maize GS1 mutants ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmV, ZmW, ZmY and ZmX also grew significantly .

These results indicate that the single mutants of ZmD, ZmE, ZmF, ZmG, ZmH, ZmI, ZmK, ZmL, ZmM, ZmN, ZmP, ZmQ, ZmV, ZmW, ZmY and ZmX all have glufosinate resistance.

Experimental example 4

With reference to the detection method of Experimental Example 1, it was verified that the wheat GS1 mutants TaD (S68D, the amino acid S at position 68 of maize GS1 was mutated to D), TaE (S68E), TaG (S68G), TaH (S68H) provided in Example 4 , TaN (S68N), TaP (S68P), TaQ (S68Q), TaV (S68V) and TaX (S68Δ) glufosinate resistance. The results are shown in Figure 10.

According to the results in Figure 10, it can be seen that:

Transformation of genes encoding wild-type wheat GS1 (TaGS1_WT) and wheat GS1 mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX on medium containing 0 mM glufosinate-ammonium (KP0) All strains can grow normally, indicating that GS1 encoded by TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX all have normal GS1 enzyme activity;

E. coli transformed with wild-type wheat GS1 were essentially unable to grow on medium containing 1 mM glufosinate-ammonium (KP1), but transformed wheat mutants TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX The growth of Escherichia coli was significantly better than that of the negative control, indicating that the single mutants containing TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX had significantly better glufosinate resistance than the wild type; E. coli transformed with wheat GS1 mutant TaN still grew significantly on the medium with phosphine concentration (10 mM, KP10).

These results indicated that the single mutants of TaD, TaE, TaG, TaH, TaN, TaP, TaQ, TaV and TaX all had glufosinate resistance, and the wheat GS1 mutant TaN had stronger glufosinate resistance.

Experimental example 5

With reference to the detection method of Experimental Example 1, it is verified that the rapeseed GS1 mutants BnA (S68A, the amino acid S at position 68 of corn GS1 is mutated to A), BnC (S68C), BnD (S68D), BnE (S68E) provided in Example 5 , BnF(S68F), BnG(S68G), BnH(S68H), BnI(S68I), BnK(S68K), BnL(S68L), BnM(S68M), BnN(S68N), BnP(S68P), BnQ(S68Q) , BnT (S68T), BnV (S68V), BnW (S68W), BnY (S68E) and BnX (S68Δ) glufosinate resistance. The results are shown in Figure 11.

According to the results in Figure 11, it can be seen that:

On medium containing 0 mM glufosinate-ammonium (KP0), transformation encoding wild-type rapeseed GS1 (BnGS1_WT) and rapeseed GS1 mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX coding genes of defective strains can grow normally, indicating that BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM , BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX encoded GS1 all have normal GS1 enzymatic activity;

On medium containing 2 mM glufosinate-ammonium (KP2), E. coli transformed with wild-type rape GS1 could not grow substantially, but the rape mutants BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK were transformed , BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX E. coli growth was significantly better than the negative control, indicating that BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, The single mutants of BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY, and BnX were significantly more resistant to glufosinate-ammonium than wild-type; in medium with higher glufosinate concentration (20mM, KP20) E. coli transformed with the rapeseed GS1 mutants BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnV, BnW and BnY also showed significant growth.

These results indicate that single mutants of BnA, BnC, BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnT, BnV, BnW, BnY and BnX are all glufosinate-resistant GS1 mutants BnD, BnE, BnF, BnG, BnH, BnI, BnK, BnL, BnM, BnN, BnP, BnQ, BnV, BnW and BnY had stronger glufosinate resistance.

Experimental example 6

Detect the enzyme kinetic parameters of OsP provided in Example 1, GmQ provided in Example 2, ZmV provided in Example 3, TaG provided in Example 4 and BnE mutant provided in Example 5 and the enzyme kinetic parameters in the presence of glufosinate-ammonium. Enzyme kinetic parameters were compared with wild-type rice GS1 OsGS1_WT, wild-type soybean GS1 GmGS1_WT, wild-type maize GS1 ZmGS1_WT, wild-type wheat GS1 TaGS1_WT and wild-type rape GS1 BnGS1_WT as controls, the methods are as follows:

Vector construction:

The nucleic acid sequence encoding the above mutant was cloned into the prokaryotic expression vector pET32a, and the clone was verified by sequencing.

6His protein purification:

The mutant enzyme protein was purified by 6His and standard methods, the concentration was determined with the Bradford method protein concentration assay kit, and the protein was stored in protein stock solution.

Enzyme activity assay:

1. Instruments and reagents: microplate reader (Deutsche Bronze: HBS-1096A), glufosinate-ammonium, substrate L-sodium glutamate (CAS: 6106-04-3).

2. Operation steps:

The components of the glutamine synthase enzyme activity assay reaction solution were: 100 mM Tris-HCl (pH7.5), 5 mM ATP, 10 mM sodium L-glutamate, 30 mM hydroxylamine, 20 mM MgCl ₂ . After mixing 100 μl of the reaction solution, it was preheated at 35°C for 5 minutes, and then 1 μl of mutant protein solution (protein concentration of 200ug/ml) was added to start the reaction. After 60 minutes of reaction at 35°C, 110 μl of reaction stop solution (55g/L FeCl ₃ · 6H ₂ was added) O, 20g/L trichloroacetic acid, 2.1% concentrated hydrochloric acid) to terminate the reaction and let stand for 10min. Centrifuge at 5000Xg for 10min, take 200μl and measure the light absorption value at 500nm.

The results are shown in Figure 12.

According to the results in Figure 12, it can be seen that:

Compared with the wild-type controls OsGS_WT, GmGS1_WT, ZmGS1_WT, TaGS1_WT and BnGS1_WT, the Km values of the GS1 mutants were slightly higher, indicating that the GS mutants reduced the sensitivity to glufosinate-ammonium inhibitors while slightly reducing the sensitivity to glufosinate-ammonium inhibitors. normal substrate sensitivity. The V _max of the GS1 mutants was higher than that of the wild-type control, indicating that the enzymatic catalytic ability of these mutants was improved. The wild-type control was sensitive to glufosinate-ammonium with IC50s of 7.93 μM, 13.55 μM, 8.92 μM, 7.22 μM and _1.5 μM, respectively. The IC50s of the mutants were significantly higher _than those of the wild-type control. The _IC50 was much higher than the wild-type control, indicating that the mutant was less sensitive to glufosinate. It can also be seen from the fold relationship between the mutant IC ₅₀ and the wild-type IC ₅₀ that the IC ₅₀ of OsP, GmQ, ZmV, TaG and BnE are 63.05 times, 32.34 times, 36.69 times and 23.83 times that of the corresponding wild type GS1 IC ₅₀ , respectively. and 15.83 times, these values also indicate that the enzyme activity of the mutant is much higher than that of the wild-type control. These data illustrate the mechanism of glufosinate resistance of the mutants enzymatically.

The above descriptions are only optional embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims (10)

1. A glutamine synthase mutant with glufosinate resistance, characterized in that it is shown in the following (1) or (2):

   (1): it is obtained by mutating the n-th position of a plant-derived wild-type glutamine synthetase; the position of the n-th position is determined by comparing the wild-type glutamine synthase with the reference sequence. Right, the nth position of the wild-type glutamine synthetase corresponds to the 68th position of the reference sequence, wherein the amino acid sequence of the reference sequence is as shown in SEQ ID NO.1;

   The amino acid at the nth position of the glutamine synthase mutant is X, X=D, E, G, H, N, P, Q, V or deletion;

   (2): It is at least 85% identical to the glutamine synthetase mutant shown in (1) and has the same amino acid at the n-th position as the glutamine synthase mutant shown in (1). , and glufosinate-ammonium resistance.
2. The glufosinate-resistant glutamine synthase mutant of claim 1, wherein the plant is selected from the group consisting of wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, Green beans, broad beans, peas, lentils, sweet potatoes, potatoes, cotton, soybeans, canola, sesame, peanuts, sunflowers, radishes, carrots, turnips, beets, cabbage, mustard greens, kale, cauliflower, kale, cucumber, zucchini, pumpkin, Winter gourd, bitter gourd, loofah, vegetable gourd, watermelon, melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, day lily, grape, strawberry, Any of sugar beet, sugar cane, tobacco, alfalfa, pasture, lawn grass, tea and cassava.
3. The glutamine synthase mutant with glufosinate-ammonium resistance according to claim 1 or 2, wherein when the plant is rice or corn, X=A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y or delete;

   When the plant is soybean, X=D, E, G, H, I, K, M, N, P, Q, V, Y or deletion;

   When the plant is wheat, X=D, E, G, H, N, P, Q, V or deletion;

   When the plant is rape, X=A, C, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y or deletion.
4. An isolated nucleic acid molecule, characterized in that it encodes the glufosinate-resistant glutamine synthase mutant of any one of claims 1-3.
5. A vector, characterized in that it contains the nucleic acid molecule of claim 4 .
6. A recombinant bacteria or recombinant cell, characterized in that it contains the nucleic acid molecule of claim 4 or the vector of claim 5.
7. The glufosinate-resistant glutamine synthetase mutant of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5, or the recombination of claim 6 The application of bacteria or recombinant cells in the cultivation of plant varieties with glufosinate-ammonium resistance.
8. The use according to claim 7, characterized in that it comprises: transforming a target plant with a vector, wherein the vector contains an encoding gene encoding the glutamine synthase mutant.
9. The application according to claim 7, characterized in that it comprises: modifying the endogenous glutamine synthase gene of the target plant to encode the glutamine synthase mutant.
10. The application according to claim 8 or 9, characterized in that it comprises: mutagenizing and screening plant cells, tissues, individuals or populations to encode the glutamine synthase mutant;

    Preferably, the target plant is selected from wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato, cotton, soybean, rape, sesame, peanut, Sunflower, radish, carrot, turnip, beet, cabbage, mustard greens, kale, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon, tomato, eggplant, pepper, kidney bean, cowpea, Any of edamame, leeks, green onions, onions, leeks, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grapes, strawberries, beets, sugarcane, tobacco, alfalfa, pasture, lawn grass, tea, and cassava.

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