GAT_656, a protein associated with glyphosate resistance, and its related biomaterials and applications

Abstract

This invention discloses a glyphosate resistance-related protein, GAT_656, and its related biomaterials and applications. The protein GAT_656 is one of the following: a) a) a protein with the amino acid sequence shown in Sequence 1; b) a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 1; c) a protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1; d) a protein with 75% or more identity to the amino acid sequence shown in Sequence 1 and with the same function. The protein GAT_656 and its related biomaterials of this invention can be used to breed novel glyphosate-resistant crop varieties, providing an efficient and stable solution for glyphosate resistance in crops, contributing to the improvement of agricultural production efficiency and sustainable development, and possessing significant application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a protein GAT_656 associated with glyphosate resistance and its related biomaterials and applications. Background Technology

[0002] Weeds are wild plants that compete with crops for water, light, soil nutrients, and space, and can also serve as hosts for various plant diseases. Controlling weed growth is crucial for achieving optimal crop yields. Using herbicides not only reduces the labor involved in weeding but also minimizes economic losses caused by weeds. Glyphosate is a low-cost, highly efficient, easily degradable, and broad-spectrum herbicide with minimal environmental impact. Its mechanism of action involves inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) during shikimic acid metabolism in plants, thus blocking the biosynthesis of aromatic amino acids and ultimately leading to plant death. Introducing a mutated EPSPS gene into plants is the mechanism of action for most transgenic glyphosate-resistant crops. However, glyphosate is not selective for either weeds or crops, and long-term accumulation of glyphosate in crops can negatively impact growth and yield. Therefore, exploring new glyphosate resistance mechanisms is essential.

[0003] Another method to induce glyphosate resistance in crops is through the expression of functional enzyme genes that can modify or degrade glyphosate. Studies have found that the N-acetyltransferase (GAT) gene, which exhibits highly efficient glyphosate resistance, is another commonly used glyphosate-resistant gene. This gene demonstrates strong glyphosate resistance in prokaryotic expression systems, tolerating up to 300 mM glyphosate in *E. coli* BL21. The encoded N-acetyltransferase loses its herbicide activity by acetylifying glyphosate. Acetylated glyphosate is no longer an effective inhibitor of EPSPS, thus preventing glyphosate accumulation in crops and having no impact on the crop's growth cycle. This gene was first obtained through genomic library screening in soils heavily contaminated with glyphosate and has been validated in crops such as maize and cotton. For example, co-expression of the gat and gr79-epsps genes in maize and cotton showed higher glyphosate resistance compared to single-gene expression. The use of genome mining technology to find more proteins and their encoding genes similar to N-acetyltransferases provides an important strategy for developing new glyphosate-resistant crops. Summary of the Invention

[0004] One object of the present invention is to provide a protein.

[0005] The protein provided by this invention is the protein shown in a), b), c), or d) below:

[0006] a) The amino acid sequence is that of the protein shown in Sequence 1;

[0007] b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 1;

[0008] c) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1.

[0009] d) Proteins that have 75% or more of the same amino acid sequence as shown in Sequence 1 and have the same function.

[0010] In this invention, the protein with the amino acid sequence shown in Sequence 1 is named GAT_656.

[0011] In the protein described in b) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] In the protein described in c) above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0013] In the protein described in d) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0014] The 75% or higher identity can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.

[0015] The proteins described in a), b), c), or d) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0016] Another objective of this invention is to provide biomaterials related to the GAT_656 protein.

[0017] The biomaterial related to the GAT_656 protein provided by this invention is any one of the following A1) to A8):

[0018] A1) The nucleic acid molecule encoding the GAT_656 protein described above;

[0019] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0020] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0021] A4) A recombinant vector containing the expression cassette described in A2);

[0022] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0023] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0024] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0025] A8) Recombinant microorganisms containing the recombinant vector described in A4).

[0026] In the above-mentioned biological materials, the nucleic acid molecule is a gene as shown in 1) or 2) below:

[0027] 1) Its coding sequence is the DNA molecule shown in sequence 2;

[0028] 2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the GAT_656 protein described above.

[0029] Those skilled in the art can readily mutate the nucleotide sequence encoding the GAT_656 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the GAT_656 nucleotide sequence of this invention, as long as they encode the GAT_656 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0030] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in Sequence 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] The 75% or higher identity can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher.

[0032] In the aforementioned biological materials, the expression cassette (GAT_656 gene expression cassette) refers to DNA capable of expressing the GAT_656 protein in host cells. This DNA may include not only a promoter for initiating GAT_656 transcription but also a terminator for terminating GAT_656 transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0033] In some embodiments, the expression cassette sequentially includes a promoter for initiating GAT_656 transcription, a nucleic acid molecule encoding the GAT_656 protein, and a terminator for terminating GAT_656 transcription.

[0034] In some specific embodiments, the promoter used to initiate GAT_656 transcription is the T5 promoter. The nucleotide sequence of the T5 promoter is shown in Sequence 3.

[0035] In some specific embodiments, the terminator used to terminate GAT_656 transcription is the lambda t0 terminator. The nucleotide sequence of the lambda t0 terminator is shown in Sequence 4.

[0036] In the above-mentioned biological materials, the vector refers to a vector that can carry the above-mentioned nucleic acid molecules into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0037] The recombinant vector refers to a recombinant DNA molecule constructed by ligating the aforementioned nucleic acid molecule to the vector in vitro. Existing expression vectors can be used to construct recombinant vectors containing the GAT_656 gene expression cassette.

[0038] In some embodiments, the recombinant vector is the 656-pQE80L recombinant vector. The 656-pQE80L recombinant vector is obtained by linking a nucleic acid molecule encoding the GAT_656 protein between the T5 promoter and the lambda t0 terminator of the pQE-80L vector (with the 6xHis tag removed).

[0039] In some specific embodiments, the nucleotide sequence of the 656-pQE80L recombinant vector is shown in Sequence 5.

[0040] The microorganisms mentioned above can be yeast, bacteria, algae, or fungi. Specifically, the bacteria can be Escherichia coli, such as Escherichia coli M15.

[0041] The recombinant microorganisms refer to those obtained by manipulating and modifying the genes of a target microorganism, resulting in a functional change. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0042] In some embodiments, the recombinant microorganism is a recombinant bacterium obtained by transferring the above-mentioned recombinant vector into a host bacterium.

[0043] In some specific implementations, the host bacterium is Escherichia coli M15.

[0044] Another object of the present invention is to provide new uses for the above-mentioned GAT_656 protein or the above-mentioned biomaterial.

[0045] This invention provides the use of the above-described GAT_656 protein or the above-described biological material in any of the following B1)-B4):

[0046] B1) Regulates glyphosate tolerance in microorganisms or plants;

[0047] B2) Prepare products that regulate glyphosate tolerance in microorganisms or plants;

[0048] B3) Cultivate glyphosate-resistant microorganisms or plants;

[0049] B4) Prepare products for cultivating glyphosate-resistant microorganisms or plants.

[0050] In the above applications, the microorganism can be bacteria.

[0051] Furthermore, the bacteria may specifically be Escherichia coli.

[0052] Furthermore, the Escherichia coli may specifically be Escherichia coli M15.

[0053] Another objective of this invention is to provide a recombinant Escherichia coli.

[0054] The recombinant Escherichia coli provided by this invention is obtained by modifying Escherichia coli as follows: introducing and expressing a nucleic acid molecule encoding the above-mentioned GAT_656 protein.

[0055] The final objective of this invention is to provide a method for constructing glyphosate-resistant Escherichia coli.

[0056] The method for constructing glyphosate-resistant Escherichia coli provided by the present invention includes the following steps: introducing and expressing a nucleic acid molecule encoding the above-mentioned GAT_656 protein into Escherichia coli to obtain glyphosate-resistant Escherichia coli.

[0057] The method described above for introducing and expressing a nucleic acid molecule encoding the GAT_656 protein involves introducing an expression vector containing a nucleic acid molecule encoding the GAT_656 protein into Escherichia coli.

[0058] In some embodiments, the expression vector containing the nucleic acid molecule encoding the GAT_656 protein is a 656-pQE80L recombinant vector. The 656-pQE80L recombinant vector is obtained by linking the nucleic acid molecule encoding the GAT_656 protein between the T5 promoter and the lambda t0 terminator of a pQE-80L vector (with the 6xHis tag removed).

[0059] In some specific embodiments, the nucleotide sequence of the 656-pQE80L recombinant vector is shown in Sequence 5.

[0060] This invention identifies the candidate protein GAT_656, associated with glyphosate resistance, by mining potential glyphosate resistance proteins in public genomic databases. Validation of GAT_656 protein function using E. coli resistance experiments revealed that GAT_656 can enhance E. coli tolerance to glyphosate. The protein GAT_656 and related biomaterials of this invention have the value of further exploring their high-level glyphosate tolerance potential in plant transgenic validation, providing important application prospects for the development of glyphosate-resistant plants and possessing extremely high practical value. This invention provides an efficient and stable solution for glyphosate resistance in crops, contributing to improved agricultural production efficiency and sustainable development. Attached Figure Description

[0061] Figure 1 The vector map for the 656-pQE80L recombinant plasmid.

[0062] Figure 2 The vector map of the positive control GAT_HA plasmid.

[0063] Figure 3 The vector map of the positive control GAT_LA plasmid.

[0064] Figure 4 The vector map of the negative control pQE-80L plasmid. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] The GAT_HA and GAT_LA vectors used in the following examples are described in the literature “Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho HJ, Duck N, Wong J, Liu D, Lassner MW. Discovery and directed evolution of a glyphosate tolerance gene. Science. 2004 May 21; 304(5674):1151-4. doi:10.1126 / science.1096770.PMID:15155947.”.

[0068] Example 1: Obtaining the novel glyphosate resistance protein GAT_656 and its encoding gene

[0069] To delve deeper into potential glyphosate resistance proteins, this invention uses protein sequences annotated with the PF00583 (Acetyltransferase family) domain from the InterPro database (https: / / www.ebi.ac.uk / interpro) as the base dataset for analysis. Multiple sequence alignment (MSA) was performed using MAFFT software to identify highly conserved regions within the proteins. After MSA, a Hidden Markov Model (HMM) was constructed based on the alignment results using HMMER software. The constructed HMM model will be used for iterative searches in publicly available genomic databases (such as NCBI, UniProt, Ensembl, etc.) to identify potential glyphosate resistance proteins. The search process employs the following iterative strategy:

[0070] 1. Initial search: Use the HMM model to scan the target genome database and identify protein sequences that match the PF00583 domain.

[0071] 2. Results Analysis and Screening: The search results are classified and their quality is evaluated, and sequences with low confidence and those unrelated to the PF00583 domain are removed.

[0072] 3. Model update: Add high-quality screening results to the training dataset and update the HMM model to improve the accuracy and coverage of the next round of search.

[0073] 4. Multiple iterations: Repeat the search and screening process until the number and quality of candidate proteins reach a stable level.

[0074] The final discovery revealed a novel glyphosate resistance protein with the amino acid sequence of sequence 1, which was named GAT_656 protein. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene sequence encoding the GAT_656 protein shown in sequence 2.

[0075] Example 2: Functional Verification of the Novel Glyphosate Resistance Protein GAT_656

[0076] I. Construction of prokaryotic expression vectors

[0077] The 6xHis tag was removed from the pQE-80L vector (Beijing Qingke Biotechnology Co., Ltd., vector number 26) to obtain the pQE-80L vector with the 6xHis tag removed.

[0078] The GAT_656 gene fragment shown in sequence 2 was ligated between the T5 promoter and lambda t0 terminator of the pQE-80L vector with the 6xHis tag removed to obtain the 656-pQE80L recombinant vector.

[0079] The vector pattern of the 656-pQE80L recombinant vector is as follows: Figure 1 As shown.

[0080] The nucleotide sequence of the 656-pQE80L recombinant vector is shown in Sequence 5.

[0081] II. Obtaining Escherichia coli transformed with 656-pQE80L vector

[0082] The 656-pQE80L recombinant vector was transformed into M15 competent E. coli cells, and E. coli transfected with the 656-pQE80L vector were identified. The specific transformation steps are as follows:

[0083] 1. In a clean bench, aspirate 15 μL of M15 E. coli competent cells (Bomaide, BC203-02) into a 0.2 mL PCR tube that has been sterilized by high temperature, add 1 μL of 656-pQE-80L vector plasmid, cover the tube, and place it on ice for 30 min.

[0084] 2. Place the PCR tube in the PCR instrument and heat shock it at 42°C for 1 minute.

[0085] 3. Quickly place on ice for 5 minutes.

[0086] 4. After the ice bath, add 50 μL of blank LB liquid medium to the PCR tube in a clean bench, place it on a shaker at 37°C, and incubate at 220 rpm for 30 min to activate the strain.

[0087] 5. Inoculate the activated bacterial culture onto ampicillin-resistant solid LB medium, label it, and incubate it in a 37°C incubator for 24 hours.

[0088] 6. Select 5 single colonies from the growth region of the strain, streak them on fresh ampicillin-resistant LB agar, and perform first-generation sequencing. The sequencing primer sequences are as follows:

[0089] F: 5'-ATTTATTTGCTTTGTGAGCG-3';

[0090] R: 5'-ACCGAGCGTTCTGAACAAAT-3'.

[0091] 7. Sequencingly sequenced single-clone colonies were inoculated into 500 μL of ampicillin-resistant liquid LB medium using a sterilized 10 μL pipette tip. The medium was incubated overnight at 37°C with shaking at 220 rpm. Then, 500 μL of 50% glycerol was added, mixed thoroughly, and stored at -20°C. This yielded *E. coli* transformed with the 656-pQE80L recombinant vector.

[0092] Following the methods described in steps 1-7 above, GAT_HA vector, GAT_LA vector, and empty vector pQE-80L were respectively transformed into M15 Escherichia coli competent cells, and Escherichia coli transformed with GAT_HA vector, GAT_LA vector, and pQE-80L empty vector were obtained after identification.

[0093] E. coli transgenic with GAT_HA vector and E. coli transgenic with GAT_LA vector were used as positive controls.

[0094] pQE-80L empty vector E. coli was used as a negative control.

[0095] GAT_HA, GAT_LA, and pQE-80L sequencing can share primers. The sequencing primer sequences are as follows:

[0096] F: 5'-TTTATTTGCTTTGTGAGCGG-3';

[0097] R: 5'-TGAGGTCATTACTGGATCTA-3'.

[0098] III. Identification of glyphosate resistance in *E. coli* strained with the 656-pQE80L recombinant vector

[0099] 1. Preparation of reagents

[0100] Reagents required for the experiment: M9 low-salt medium (M9 liquid screening medium, M9 solid screening medium), 20% glyphosate amine salt solution, 20% glucose solution, IPTG (0.5M), MgSO4 (1M), CaCl2 (1M), proline (1mg / mL), thiamine (1mg / mL).

[0101] The reagent preparation methods are as follows:

[0102] 20% glyphosate amine salt solution: Weigh 20g of glyphosate amine salt and dissolve it in 100mL of pure water. Sterilize the solution by filtration using a 0.22µm cell filter and a 50mL disposable syringe in a laminar flow hood.

[0103] 20% glucose solution: Weigh 20g of glucose solution and dissolve it in 100mL of pure water, then autoclave at 120℃ for 15min.

[0104] IPTG (0.5M): Weigh 1.79g of powder and dissolve it in 15ml of pure water. Sterilize the solution by filtration using a 0.22um cell filter and a 50mL disposable syringe in a laminar flow hood.

[0105] MgSO4 (1M): Weigh 6.02g of magnesium sulfate powder and dissolve it in 50ml of pure water. Sterilize the solution by filtration using a 0.22um cell filter and a 50mL disposable syringe in a laminar flow hood.

[0106] CaCl2 (1M): Weigh 5.55g of calcium chloride powder and dissolve it in 50ml of pure water. Sterilize the solution by filtration using a 0.22um cell filter and a 50mL disposable syringe in a laminar flow hood.

[0107] Proline (1 mg / mL): Weigh 0.05 g of proline powder and dissolve it in 50 ml of pure water. Sterilize the solution by filtration using a 0.22 μm cell filter and a 50 mL disposable syringe in a laminar flow hood.

[0108] Thiamine (1 mg / mL): Weigh 0.05 g of thiamine powder and dissolve it in 50 ml of pure water. Sterilize the solution by filtration using a 0.22 μm cell filter and a 50 mL disposable syringe in a laminar flow hood.

[0109] The preparation method for 1L of M9 liquid screening medium is as follows: Weigh 11.3g of M9 low-salt medium powder (Sangon Biotech (Shanghai) Co., Ltd., main components: Na2HPO4, KH2PO4, NH4Cl and NaCl) and dissolve it in 979mL of pure water. Adjust the pH to 7.0 and autoclave at 120℃. Add 20mL of separately sterilized 20% glucose solution, 2mL of filtration-sterilized MgSO4 (1M), 100uL of filtration-sterilized CaCl2 (1M), 10mL of filtration-sterilized proline and 10mL of filtration-sterilized thiamine. Then add 1mL of ampicillin (50mg / mL) and 1mL of kanamycin (50mg / mL) to obtain 1×M9 liquid screening medium.

[0110] Preparation method of 1L M9 solid screening medium: Weigh 11.3g of M9 low-salt medium powder (Sangon Biotech (Shanghai) Co., Ltd., main components: Na2HPO4, KH2PO4, NH4Cl and NaCl) and dissolve it in 979mL of pure water. After the powder is completely dissolved, adjust the pH to 7.0, add 10g of agar, and autoclave at 120℃. After sterilization, add the same amount and concentration of glucose, MgSO4, CaCl2, proline, thiamine, ampicillin and kanamycin as when preparing the liquid medium.

[0111] 2. Validation of GAT_656 protein expression in prokaryotes

[0112] 1) Inoculate the positive monoclonal bacterial culture (transformed with 656-pQE80L recombinant vector Escherichia coli) from step 2 above into a new ampicillin and kanamycin resistant LB liquid medium, and incubate overnight at 37°C with shaking at 220 rpm to revive the strain and obtain the activated positive monoclonal bacterial culture.

[0113] 2) In a clean bench, pipette 500 μL of pre-prepared M9 liquid screening medium into autoclaved 2 mL centrifuge tubes, then pipette 20 μL of activated positive monoclonal bacterial solution into the corresponding centrifuge tubes, label them, and adjust the OD. 600 The value is 0.1.

[0114] 3) Incubate at 37℃ with shaking at 220 rpm until OD reaches 100°C. 600 The value is 0.8-1.0.

[0115] 4) In the clean bench, add 500 μL of OD 600 Add 1 μL of IPTG (0.5 M) to the bacterial culture with a pH of 0.8-1.0 to a final concentration of 1 mM, and induce at 37°C and 220 rpm for 4-16 h.

[0116] 5) The expression of GAT_656 protein was detected by SDS-PAGE electrophoresis after different induction times.

[0117] The results showed that *E. coli* transformed with the 656-pQE80L recombinant vector could normally express the 16 kDa GAT_656 protein after ITPG induction. Specifically, GAT_656 protein expression began 4 hours after IPTG induction, and the protein expression level increased with increasing induction time, reaching a stable state after 16 hours of induction.

[0118] 3. Identification of glyphosate resistance in *E. coli* strained with the 656-pQE80L recombinant vector

[0119] In a clean bench, 3 μL of *E. coli* strains expressing a 16 kDa recombinant vector (656-pQE80L) were simultaneously inoculated onto M9 solid selection medium at 0 mM and 1 mM glyphosate concentrations using a 10 μL pipette for glyphosate resistance identification. The medium was sealed twice with sealing film, and the corresponding strains were labeled. The medium was then incubated at 37°C for 72 h. *E. coli* strains transgenic with GAT_HA and GAT_LA vectors served as positive controls. *E. coli* strains transgenic with the empty pQE-80L vector served as negative controls.

[0120] The results showed that both *E. coli* strains transfected with the 656-pQE80L recombinant vector and those transfected with the pQE-80L empty vector exhibited turbidity in M9 solid selection medium containing 0 mM glyphosate, yet both strains grew normally. However, *E. coli* transfected with the pQE-80L empty vector showed clear bacterial culture in M9 solid selection medium containing 1 mM glyphosate, but the strains failed to grow. In contrast, *E. coli* transfected with the 656-pQE80L recombinant vector showed turbidity in M9 solid selection medium containing 1 mM glyphosate, but the degree of turbidity was less than that in M9 solid selection medium containing 0 mM glyphosate. This indicates that expression of the GAT_656 protein in *E. coli* enhances the resistance of *E. coli* to glyphosate.

[0121] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein is a protein as shown in a) or b) or c) or d) below: a) The amino acid sequence is that of the protein shown in Sequence 1; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 1; c) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1. d) Proteins that have 75% or more of the same amino acid sequence as shown in Sequence 1 and have the same function.

2. The biomaterial related to the protein of claim 1 is any one of A1) to A8) below: A1) A nucleic acid molecule encoding the protein of claim 1; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganisms containing the nucleic acid molecules described in A1); A6) Recombinant microorganisms containing the expression cassette described in A2); A7) Recombinant microorganisms containing the recombinant vector described in A3); A8) Recombinant microorganisms containing the recombinant vector described in A4).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is a gene as shown in 1) or 2) below: 1) Its coding sequence is the DNA molecule shown in sequence 2; 2) Having 75% or more identity with the nucleotide sequence defined in 1), and encoding a DNA molecule of the protein of claim 1.

4. The biomaterial according to claim 2, characterized in that: The expression cassette sequentially includes a promoter, the nucleic acid molecule as described in claim 3, and a terminator.

5. The biomaterial according to claim 4, characterized in that: The promoter is the T5 promoter; the terminator is the lambda t0 terminator.

6. The biomaterial according to claim 2, characterized in that: The recombinant vector is an expression vector containing the expression cassette of claim 4 or 5.

7. The biomaterial according to claim 2, characterized in that: The recombinant microorganism is a recombinant bacterium obtained by transferring the recombinant vector of claim 6 into a host bacterium.

8. The use of the protein of claim 1 or the biomaterial of any one of claims 2-7 in any one of the following B1)-B4): B1) Regulates glyphosate tolerance in microorganisms or plants; B2) Prepare products that regulate glyphosate tolerance in microorganisms or plants; B3) Cultivate glyphosate-resistant microorganisms or plants; B4) Prepare products for cultivating glyphosate-resistant microorganisms or plants.

9. A recombinant Escherichia coli, obtained by modifying Escherichia coli by introducing and expressing a nucleic acid molecule encoding the protein of claim 1.

10. A method for constructing glyphosate-resistant Escherichia coli, comprising the following steps: introducing and expressing a nucleic acid molecule encoding the protein of claim 1 into Escherichia coli to obtain glyphosate-resistant Escherichia coli.

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