Glyphosate-resistant epsps protein and uses thereof

Abstract

The application discloses an anti-glyphosate EPSPS protein and application thereof. The amino acid sequence of the EPSPS protein coded by the gene is shown as SEQ ID NO. 2, the nucleotide sequence optimized for tobacco codon is shown as SEQ ID NO. 3, and the EPSPS protein is fused with a chloroplast signal peptide. The gene is introduced into Nicotiana benthamiana by an agrobacterium-mediated method, and the obtained transgenic plant shows extremely high glyphosate resistance and can tolerate a concentration of up to 36000 mg / L, which is significantly better than that of a traditional CP4-EPSPS transgenic plant. The gene is screened from microorganisms by using artificial intelligence assisted evolution analysis, structure prediction and semantic clustering, and has the characteristics of high-efficiency expression and strong resistance, and can be widely applied to herbicide-resistant breeding of various crops, thereby reducing production cost and environmental risk.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-glyphosate EPSPS protein and its applications. Background Technology

[0002] Glyphosate, a widely used non-selective herbicide, is extensively applied in agricultural production due to its high efficiency, low toxicity, and broad applicability. Its mechanism of action primarily involves competitively inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants, interfering with the synthesis of aromatic amino acids and thus affecting plant growth and development. The core conserved domain of this enzyme (EPSP_synthase, PF00275) typically consists of approximately 400-500 amino acid residues, containing key catalytic sites and substrate-binding regions; its typical sequence is shown in SEQ ID NO.1. However, with the increasing use of glyphosate, weeds have gradually developed resistance, leading to a continuous decline in its herbicidal efficacy. This necessitates increasing herbicide application rates during crop cultivation to maintain weed control. This phenomenon not only increases production costs but also negatively impacts the environment and ecosystems. Therefore, improving crop tolerance to glyphosate is of paramount importance.

[0003] Currently, the mainstream strategy for conferring glyphosate resistance to crops is to introduce exogenous EPSPS genes derived from microorganisms (such as Agrobacterium tumefaciens CP4 strain) using transgenic technology. These genes express EPSPS enzymes that are insensitive to glyphosate, thus ensuring normal crop growth after glyphosate application. However, with the increasing prevalence of glyphosate-resistant weeds and the strengthening of weed resistance, the effectiveness of glyphosate-based weed control is gradually declining, requiring increased dosages to maintain efficacy. This, in turn, significantly increases the risk of crop damage. Therefore, developing novel and highly efficient glyphosate-resistant genes and crop varieties has become an urgent priority. Summary of the Invention

[0004] In view of the lack of glyphosate-resistant EPSPS genes in existing technologies, this invention provides a new PbmEPSPS gene predicted with the assistance of artificial intelligence technology. This gene has significantly higher glyphosate tolerance than natural EPSPS and is conducive to efficient expression in plants.

[0005] To achieve the above and related objectives, the present invention provides a protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] The nucleic acid molecule encoding the protein described above has the nucleotide sequence shown in SEQ ID NO.3.

[0007] A recombinant expression vector containing the nucleic acid molecules described above is also within the scope of protection of this invention.

[0008] As a preferred technical solution for recombinant expression vectors, it also includes a chloroplast signal peptide sequence as shown in SEQ ID NO.4.

[0009] A host cell comprising the recombinant expression vector described above.

[0010] A transgenic plant comprising the protein described above, or the recombinant expression vector described above.

[0011] As a preferred technical solution for genetically modified plants, the plant is tobacco, corn, soybean, rice, cotton or wheat.

[0012] In addition, the present invention also provides a method for cultivating glyphosate-resistant plants, comprising the following steps: Step S1. Obtain the protein or its encoding nucleic acid sequence described above through artificial intelligence-assisted prediction and screening; Step S2. Construct a recombinant expression vector containing the nucleic acid sequence; Step S3. Introduce the recombinant expression vector into plant cells or tissues; Step S4. Screening and regeneration to obtain transgenic plants expressing the protein.

[0013] Application of the proteins, nucleic acid molecules, or recombinant expression vectors described above in the preparation of glyphosate-resistant plants.

[0014] This invention utilizes artificial intelligence-assisted prediction and screening to obtain a novel glyphosate-resistant EPSPS protein (amino acid sequence shown in SEQ ID NO.2) and its optimized coding sequence (nucleotide sequence shown in SEQ ID NO.3), significantly improving the glyphosate tolerance level of transgenic plants. Experimental results show that transgenic plants obtained by introducing this gene into Nicotiana benthamiana can grow normally under glyphosate treatment at concentrations up to 36,000 mg / L, with a tolerance concentration equivalent to more than 8 times the commonly recommended field dose, far exceeding the tolerance threshold of traditional CP4-EPSPS transgenic plants, and also significantly superior to control non-transgenic plants and empty-gene plants. This high level of resistance effectively reduces the risk of increased herbicide use due to the evolution of weed resistance, reducing agricultural production costs and environmental toxicological pressure. Furthermore, this invention employs codon optimization and chloroplast signal peptide targeting design (SEQ ID NO.4) specifically for tobacco, improving the expression efficiency and stability of the exogenous protein in plant cells. Gene expression analysis shows that the PbmEPSPS transcription level is significantly upregulated in the high-resistant lines, highly consistent with phenotypic tolerance. Compared to traditional methods of creating resistance genes that rely on empirical screening, this invention utilizes AI-driven evolutionary analysis, structure prediction, and semantic space clustering techniques to significantly improve the discovery efficiency of novel high-affinity resistance variants, expand the diversity of herbicide resistance gene sources, and provide a more efficient and sustainable molecular tool for glyphosate-resistant breeding of various important crops such as maize, soybean, rice, and cotton. Attached Figure Description

[0015] Figure 1 A schematic diagram of the three-dimensional structure of the PbmEPSPS protein predicted using AlphaFold3.

[0016] Figure 2 Electrophoresis diagram of PCR molecular identification of transgenic tobacco plants. Note: M is the DNA molecular weight standard; CK is the genomic DNA of tobacco lines transgenic with the empty vector ptf101; lanes 1-7 are the genomic DNA of different transgenic tobacco lines transgenic with ptf101-PbmEPSPS; lane 8 is the ptf101-PbmEPSPS recombinant plasmid DNA (positive control).

[0017] Figure 3 Phenotypic comparison between the control group (CK) and the transgenic tobacco (OE) after 5 days of treatment with 12000 mg / L glyphosate.

[0018] Figure 4Phenotypic comparison of different strains 5 days after treatment with 36000 mg / L glyphosate. Note: CK- is the tobacco strain transgenic with empty ptf101 vector; CK+ is the tobacco strain transgenic with ptf101-CP4EPSPS (traditional control); OE is the transgenic tobacco strain transgenic with ptf101-PbmEPSPS.

[0019] Figure 5 Results of relative expression levels of exogenous genes in transgenic tobacco (qRT-PCR). Note: CK is the tobacco line transgenic with the empty vector ptf101; OE is the transgenic tobacco line transgenic with ptf101-PbmEPSPS. Detailed Implementation

[0020] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0021] Example 1: AI-assisted screening of EPSPS genes First, the EPSPS protein sequence (accession number: ARW80140.1) of Agrobacterium tumefaciens strain CP4 was retrieved from the gene database and used as a reference sequence. Then, BLAST software was used to perform a homology search using this reference sequence; simultaneously, HMMER software was used to perform a domain homology search using the EPSP_synthase conserved domain model (PF00275). The selection criteria were set as follows: sequences with an E value < 1e-40 in the BLAST search, or sequences containing the PF00275 domain and an E value < 0.01 in the HMMER search. Multiple sequence alignment was performed on these sequences, and a phylogenetic tree centered on the CP4 EPSPS protein was constructed. Candidate sequences belonging to the same clade as the reference protein and potentially orthologous were selected based on evolutionary distance; these sequences are more evolutionarily related and are expected to have higher functional conservation. The second step, based on structural features, utilizes Alphafold3 for efficient structure prediction of candidate sequences and calculates structural similarity (TM-score) using tools such as TM-align to verify functional similarity at the three-dimensional conformation level. When the TM-score is between 0.4 and 0.8, we consider that this protein is structurally similar to CP4 EPSPS, but may have some functional differences. The third step involves innovative integration, using a protein language model to transform the sequences into high-dimensional feature vectors, which are then visualized using t-SNE / UMAP dimensionality reduction. In the semantic space, complementary "dark horse" sequences and functional clustering patterns are identified. Cluster 2 ("dark horse" sequence region): This region contains sequences related to glyphosate resistance that have not been discovered by traditional screening methods. Although they may be missed in traditional screening, they have high structural and functional similarity to glyphosate-resistant proteins in the semantic space. Finally, the PbmEPSPS protein from Paracoccaceae bacterium was screened out, and its amino acid sequence is shown in SEQ ID NO.2. This protein has a high similarity in structure and function to CP4 EPSPS in semantic space, and has the potential to become a new glyphosate resistance gene. Figure 1 As shown.

[0022] Example 2: Synthesis of the full sequence of the glyphosate-resistant EPSPS gene The candidate protein sequences selected above (amino acid sequences as shown in SEQ ID NO.2) were then subjected to global codon optimization based on tobacco codon bias, while the GC content was finely adjusted to achieve the highest expression efficiency. The optimized coding region nucleotide sequence is shown in SEQ ID NO.3. Then, the chloroplast signal peptide and gene nucleotide sequence (as shown in SEQ ID NO.4) were synthesized artificially.

[0023] Example 3: Construction of plant expression vectors (1) Using a cloning vector containing the synthetic herbicide-resistant gene EPSPS as a template, homologous arm sequences were introduced at both ends of the gene by PCR amplification. The primer sequences used are as follows: forward primer (F): as shown in SEQ ID NO.8; reverse primer (R): as shown in SEQ ID NO.9. The target band was separated and recovered by gel electrophoresis, and the high-purity PbmEPSPS gene fragment was obtained using a gel purification kit. At the same time, the plant binary expression vector ptf101 was digested with MluI restriction endonuclease, and the linearized vector backbone fragment was recovered. The nucleotide sequence of the ptf101 plant expression vector is shown in SEQ ID NO.7.

[0024] (2) The purified PbmEPSPS gene fragment was ligated in vitro with the linearized ptf101 vector backbone to construct a recombinant plant expression vector, named ptf101-PbmEPSPS. The recombinant plant expression vector includes a constitutive CaMV 35S promoter, a PbmEPSPS gene coding sequence connected downstream of it, and a terminator sequence in its T-DNA region, wherein the PbmEPSPS gene is expressed in plant cells under the drive of the CaMV 35S promoter.

[0025] (3) The constructed recombinant plasmid was transformed into competent E. coli cells, and single clones were selected for expansion culture and plasmid was extracted. Further verification was performed by sequencing of the target gene to ensure the integrity and accuracy of the inserted sequence.

[0026] Example 4: Obtaining and Identifying Genetically Modified Tobacco (1) Select a single clone of Agrobacterium GV1301 carrying the ptf101-PbmEPSPS recombinant vector, inoculate it into 5 mL of LB liquid medium containing 20 mg / L Rif and 50 mg / L Kan, and culture overnight at 28°C with shaking. The next day, inoculate the activated bacterial solution into LB liquid medium containing the same antibiotic at a ratio of 1:50, and continue to culture until the OD600 reaches 0.6-0.8. Then, collect the bacteria by centrifugation at 6000 rpm for 5 minutes, take the supernatant and resuspend it in 1 / 2 MS0 liquid medium, adjust the OD600 value of the bacterial solution to 0.3-0.35, and use it for later use.

[0027] (2) Using Nicotiana benthamiana as the recipient plant, sterile seedlings approximately 30 days old were selected, and leaf discs approximately 0.8 cm × 0.8 cm in size were cut as explants. Under aseptic conditions, the leaf discs were immersed in the Agrobacterium tumefaciens solution for 3-5 minutes. After removal, excess bacterial solution was absorbed with sterile filter paper, and then the explants were placed in a co-culture medium (1×MS basal medium, containing 3% sucrose, 1% agar powder, pH 5.8) and incubated in the dark at 25°C for 2 days.

[0028] (3) After co-culture, the explants were transferred to shoot induction medium (1×MS basal medium containing 3% sucrose, 2.0 mg / L 6-BA, 0.5 mg / L IAA, 500 mg / L carbenicillin, 20 mg / L hygromycin, 1% agar powder, pH 5.8). Subculture was performed every 2-3 weeks. When the adventitious shoots grew to 1-1.5 cm, they were cut and transferred to rooting induction medium (1×MS, 3% sucrose, 0.5 mg / L IAA, 500 mg / L carbenicillin, 20 mg / L hygromycin, 1% agar powder, pH 5.8) for rooting induction.

[0029] (4) Transplant the well-rooted tissue culture seedlings into sterilized horticultural substrate, maintain appropriate humidity, apply compound fertilizer, and continue cultivation until the plants flower and bear fruit. Finally, 10 T0 generation transgenic plants were successfully obtained, and the seeds were harvested smoothly.

[0030] (5) T0 generation seeds were sown to obtain T1 generation plants. Genomic DNA was extracted from leaves using the CTAB method. Using non-transgenic tobacco as a negative control, PCR amplification was performed using specific primers (the left primer is located inside the PbmEPSPS gene, primer sequence SEQ ID NO. 5; the right primer is located inside the vector 3×flag, primer sequence SEQ ID NO. 6). The reaction system was 20 μL, including: 10 μL of 2×Taq Master Mix, 1 μL of DNA template, 1 μL each of forward and reverse primers, and 7 μL of ddH2O. The amplification conditions were: 94℃ pre-denaturation for 4 minutes; 94℃ denaturation for 40 seconds, 58℃ annealing for 40 seconds, and 72℃ extension for 40 seconds, for a total of 35 cycles; and a final extension at 72℃ for 5 minutes. Agarose gel electrophoresis showed that most transgenic lines could amplify specific bands of the expected size, such as... Figure 2 As shown.

[0031] Example 5: Glyphosate resistance identification Glyphosate spraying experiment on T1 generation transgenic tobacco: Group 1: After 5 days of treatment with 12000 mg / L glyphosate, the ptf101 (CK) transgenic line died, while the ptf101-PbmEPSPS (OE) transgenic plants grew normally. Figure 3 As shown; Group 2: After 5 days of treatment with 36000 mg / L glyphosate, the herbicide tolerance of the ptf101-PbmEPSPS (OE) tobacco line was significantly better than that of the ptf101-CP4EPSPS (CK+) line and the empty (CK-) line. Figure 4 As shown.

[0032] from Figure 3 and Figure 4 It can be seen that the PbmEPSPS gene provided by this invention significantly enhances the tolerance of transgenic tobacco to glyphosate. After 5 days of treatment with 12000 mg / L glyphosate (e.g., ...), Figure 3 As shown in the figure), the control group (CK, including non-transgenic or empty-gene plants) suffered severe wilting, yellowing, and even complete death of leaves, while the transgenic line (OE) overexpressing PbmEPSPS maintained bright green leaves, normal plant morphology, and robust growth, with no obvious signs of damage; under higher concentrations of glyphosate (36000 mg / L) treatment (such as... Figure 4 As shown in the figure, the control group further differentiated: the empty negative control (CK-) exhibited varying degrees of poisoning symptoms such as leaf curling, chlorosis, necrosis, and stunted growth; while the traditional CP4-EPSPS positive control (CK+) also showed mild leaf curling; and the OE group plants maintained good overall growth, with intact leaves and upright stems, showing no significant difference from untreated plants. This intuitive phenotypic comparison clearly verifies the excellent resistance performance of PbmEPSPS protein under high concentrations of glyphosate, with a tolerance threshold far higher than that of the traditional CP4 variant. This fully demonstrates the efficient expression of the novel EPSPS sequence obtained through AI-assisted screening in plant cells, providing reliable experimental evidence for developing more resistant herbicide-tolerant crops.

[0033] Example 6: Gene Expression Analysis Tobacco leaves transfected with empty vector (CK) and ptf101-PbmEPSPS (OE) were harvested. Total RNA was extracted from the leaves, reverse transcribed, and analyzed by quantitative real-time PCR to detect the relative expression level of the transgene. We used 1 mg of total RNA and performed reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara (Beijing, China) Co., Ltd.; catalog number RR047A). Subsequently, quantitative RT-PCR (RT-qPCR) was performed on a Roche Light Cycler 2.0 instrument using Light Cycler software (version 4.1.1.21). The simplified procedure is as follows: 1 μL of cDNA was added to 5 μL of SYBRPremix Ex Taq™, followed by 0.8 μL of 10 mM specific primers (forward and reverse), and finally, the total volume was brought to 10 μL using DNase-free water. (The process involved 2...) −ΔΔCt Relative expression levels were calculated using a method that utilizes the tobacco internal control gene Actin as a reference gene. Each tissue underwent at least three biological replicates. The primer sequences used are as follows: PbmF (SEQ ID NO.10): 5'-TCTGTACACAACGCTGCAAG-3' PbmR (SEQ ID NO.11): 5'-CTTTCACCCTCAGCTCTTTC-3' Tobacco ActinF (SEQ ID NO.12): 5'-GTATGGGTCAGAAAGATGC-3' Tobacco ActinR (SEQ ID NO.13): 5'-AGGACAGCCTGAATAGCA-3' qRT-PCR results showed that the transgenic tobacco lines with high glyphosate tolerance exhibited pbmEPSPS gene expression, with significantly higher relative expression levels than non-transgenic tobacco (CK). This result is consistent with the herbicide tolerance characteristics of transgenic tobacco. Figure 5 As shown.

[0034] The relative expression level of exogenous PbmEPSPS gene in the tobacco lines transfected with the ptf101-PbmEPSPS gene (OE group) of this invention was significantly higher than that in the control group (CK, i.e., tobacco lines transfected with empty ptf101). qRT-PCR was used to detect this, with the tobacco Actin gene as an internal control, and 2... −ΔΔCtThe relative expression level was calculated, and the results showed that the abundance of PbmEPSPS transcripts in the OE group was significantly upregulated. This high expression characteristic is highly consistent with the excellent tolerance phenotype of transgenic plants under high concentrations of glyphosate treatment, indicating that the targeted design of the SEQ ID NO.3 sequence optimized for tobacco codons combined with the chloroplast signal peptide (SEQ ID NO.4) effectively enhanced the transcription and accumulation of exogenous proteins in plant cells, ensuring sufficient EPSPS enzyme levels to competitively resist glyphosate inhibition, thereby maintaining the normal operation of the aromatic amino acid synthesis pathway. These results further confirm that the PbmEPSPS protein (SEQ ID NO.2) obtained through AI screening not only possesses high resistance potential in terms of structure and function, but also achieves efficient and stable transcriptional expression in the plant expression system, laying the molecular foundation for its subsequent application in other crops.

[0035] SEQ ID NO.1: Conserved domain sequence of EPSP_synthase ARRSEALTGEIRIPGDKSISHRSFMFGGLASGETRITGLLEGEDVINTGRAMQAMGAKIRKEGDVWIINGVGNGCLLQPEAALDFGNAGTGARLTMGLVGTYDMKT SFIGDASLSKRPMGRVLNPLREMGVQVEAADGDRMPLTLIGPKTANPITYRVPMASAQVKSAVLLAGLNTPGVTTVIEPVMTRDHTEKMLQGFGADLTVETDKDGVR HIRITGQGKLVGQTIDVPGDPSSTAFPLVAALLVEGSDVTIRNVLMNPTRTGLILTLQEMGADIEVLNARLAGGEDVADLRVRASKLKGVVVPPERAPSMIDEYPV LAIAASFAEGETVMDGLDELRVKESDRLAAVARGLEANGVDCTEGEMSLTVRRGRPDGKGLGGGTVATHLDHRIAMSFLVMGLAAEKPVTVDDSNMIATSFPEFMDMM SEQ ID NO.2: Amino acid sequence of the PbmEPSPS protein of this invention MSGHGPAKPMTSRKGGALKGVANVPGDKSVSHRSLIFGAMTVGETKIEGLLEGQDVLDTAKAMRAFGAEVERTGEGQWRVHGVGVGGFAEPEDVIDCGNSGTGVRLIMGAMASSPIAATFTGDASLRSRPMGRITDPISLFGARSFGRVEGRLPLTIIGAQNPGPVEYRSPVPSAQVKSAVLLAGLNAPGITTLIEAEATRDHTERMLKGFGAKVETEVTPDGRYIHLTGQPELTPIDITVPRDPSSAAFPVCAALIAEGSDVLVPNISLNPTRAGLYTTLQEMGADLTFENEREEGGEPVADLRAKFSPDMKGIEVPPERAASMIDEFPVLSVVAANATGATKMLGVKELRVKESDRIDAMARGLEDMGVTVEETEDTFTVHGMGAGGVPGGGIAEARLDHRIAMSFLVLGFSTQKPVSVDDGSPIDTSFPIFEPLMQELGASVARSNS SEQ ID NO.3: The coding region sequence of PbmEPSPS codon-optimized for tobacco SEQ ID NO.4: Chloroplast signal peptide sequence ATGGCTTACTCTATGCTCTCCTCTGCCACCGTGGTTTAGCTCACCGGCTCA AGCGGCCATGGTTGCTCCATTCACAGGCTTGAAGTCATCCGCTGCATTCC CCAGTCACTCGCAAGACCGACACTGACATTACTTCCATGGCAAGCAATGGA GGAAGAGTTA ACTCG SEQ ID NO.5: PCR identification primer F 5'-TTTGTTACTAACCGATCGACATCAC-3' SEQ ID NO.6: PCR identification primer R 5'-CCGTCATGGTCTTTGTAGTC-3' SEQ ID NO.7: Nucleotide sequence of the ptf101 plant expression vector AAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACAC CAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGAGCGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTT TAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAATCGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA SEQ ID NO.8: Forward primer (F): GGGCCCAGGCCTACGCGT SEQ ID NO.9: Reverse primer (R): TCATCCTTGTAATCACGCGT SEQ ID NO.10: PbmF: 5'-TCTGTACACAACGCTGCAAG-3' SEQ ID NO.11: PbmR: 5'-CTTTCACCCTCAGCTCTTTC-3' SEQ ID NO.12: Tobacco ActinF: 5'-GTATGGGTCAGAAAGATGC-3' SEQ ID NO.13: Tobacco ActinR: 5'-AGGACAGCCTGAATAGCA-3' The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a glyphosate-resistant EPSPS protein, a nucleic acid molecule encoding said protein, or a recombinant expression vector containing said nucleic acid molecule in the preparation of glyphosate-resistant tobacco, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2.

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