High tolerance of glyphosate of esps synthase and its cloning, expression and application

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a novel EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) mutant with high glyphosate tolerance, and further discloses application of a coding gene thereof in the field of novel glyphosate-tolerant crop cultivation. A 5-enolpyruvylshikimate-3-phosphate synthase is cloned from a marine strain Halomonas sp., and then through three rounds of directed evolution technology, multiple mutants with significantly improved glyphosate tolerance are obtained. The mutant coding gene is transferred into tobacco, and the tobacco is endowed with high resistance to herbicides, which proves that the mutant has high application value in the cultivation of herbicide-tolerant plants.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a novel highly glyphosate-tolerant EPSP synthase (5-enol shikimate-3-phosphate synthase), and further disclosing the application of its encoding gene in the field of cultivating novel glyphosate-tolerant crops. Background Technology

[0002] During crop cultivation, weeds compete with crops for limited sunlight, water, and nutrients, severely impacting crop yields. The ever-increasing cost of manual weeding further reduces crop profits. Glyphosate (trade name Roundup), chemically known as N-(phosphonomethyl)glycine (GLP), is structurally similar to glycine and is a derivative of glycine. Glyphosate is a non-selective, highly effective herbicide characterized by its high efficiency, broad spectrum, and easy degradation. It is currently the most widely produced and used broad-spectrum herbicide globally, and also the most used pesticide worldwide. Research shows that the combined use of glyphosate and glyphosate-resistant crops can effectively solve the problem of weed control; therefore, effectively improving the glyphosate resistance of crops is also crucial.

[0003] Glyphosate, the world's most consumed herbicide, boasts advantages such as broad spectrum, high efficiency, low residue, and low price. It works by competitively inhibiting 5-enolshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimic acid metabolic pathway, leading to the disruption of aromatic amino acid synthesis and ultimately plant death. Since the first commercial planting of glyphosate-tolerant genetically modified crops, the global planting area of ​​such crops has been continuously expanding, resulting in a steady increase in glyphosate usage (Benbrook 2016). Furthermore, the combined planting strategy of glyphosate and glyphosate-tolerant genetically modified crops has further boosted glyphosate sales, making it the most widely used pesticide globally (Duke 2018).

[0004] Currently, glyphosate-resistant crops have been the dominant herbicide-resistant crops globally for over 20 years, with glyphosate-resistant genetically modified soybeans showing the fastest growth and yielding significant economic, social, and environmental benefits. At present, with my country vigorously promoting the industrialization of bio-breeding research, how to scientifically advance the industrialization of herbicide-resistant and insect-resistant genetically modified crops is of great significance. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide an EPSP synthase with high glyphosate resistance, which has high glyphosate resistance. The second technical problem to be solved by the present invention is to provide the application of the above-mentioned highly glyphosate-tolerant EPSP synthase and its encoding gene and mutant.

[0006] To solve the above-mentioned technical problems, the present invention provides an EPSP synthase with high glyphosate tolerance, wherein the EPSP synthase has at least 95% identity with the amino acid sequence shown in SEQ ID No: 1; Preferably, the highly glyphosate-tolerant EPSP synthase comprises the amino acid sequence shown in SEQ ID No: 1; Preferably, the amino acid sequence of the EPSP synthase is shown in SEQ ID No: 1.

[0007] The present invention also discloses a gene encoding the EPSP synthase with high glyphosate tolerance, comprising the nucleotide sequence shown in SEQ ID No: 2; Preferably, its nucleotide sequence is shown in SEQ ID No: 2.

[0008] The present invention also discloses a highly glyphosate-resistant EPSP synthase mutant, which is obtained by mutating amino acids at specific sites based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1.

[0009] Specifically, the highly glyphosate-tolerant EPSP synthase mutant includes: mHoEPSPS: The mutant mHoEPSPS is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating G at position 112 to A; and / or, mHoEPSPS-2: The mutant mHoEPSPS-2 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating M at position 155 to V, I at position 285 to F, and D at position 326 to E; and / or, mHoEPSPS-9: The mutant mHoEPSPS-9 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating Y at position 11 to F, A at position 199 to V, L at position 297 to P, and I at position 428 to N; and / or, mHoEPSPS-15: The mutant mHoEPSPS-15 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating A at position 9 to E, S at position 136 to T, V at position 271 to A, V at position 381 to E, and C at position 432 to G; and / or, mHoEPSPS-16: The mutant mHoEPSPS-16 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating I at position 168 to N, H at position 169 to L, and P at position 210 to T; and / or, mHoEPSPS-2S: The mutant mHoEPSPS-2 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating M at position 155 to V, L at position 297 to P, I at position 429 to L, and F at position 439 to S.

[0010] The present invention also discloses an expression vector for high glyphosate tolerance, wherein the expression vector contains the gene encoding the EPSP synthase for high glyphosate tolerance, or the gene encoding the mutant.

[0011] The present invention also discloses a transgenic cell line with high glyphosate tolerance, wherein the transgenic cell line contains the gene encoding the EPSP synthase with high glyphosate tolerance, or the gene encoding the mutant.

[0012] The present invention also discloses a genetically engineered bacterium with high glyphosate tolerance, wherein the genetically engineered bacterium contains the gene encoding the EPSP synthase with high glyphosate tolerance, or the gene encoding the mutant.

[0013] The present invention also discloses a transgenic plant with high glyphosate tolerance, wherein the transgenic plant contains the gene encoding the EPSP synthase with high glyphosate tolerance, or the gene encoding the mutant. Preferably, the genetically modified plant is genetically modified tobacco.

[0014] This invention also discloses the application of the highly glyphosate-tolerant EPSP synthase, or the gene encoding the highly glyphosate-tolerant EPSP synthase, or the gene encoding the mutant, in the field of cultivating highly glyphosate-tolerant transgenic plants.

[0015] Specifically, the plants include soybeans, corn, rapeseed, cotton, wheat, rice, or sugarcane.

[0016] This invention screened a novel EPSPS enzyme with high glyphosate resistance from marine bacteria, possessing the amino acid sequence shown in SEQ ID NO: 1, and identified its encoding gene as shown in SEQ ID NO: 2. Sequence structure analysis and sequence comparison analysis showed that this EPSPS synthase belongs to type II EPSP synthase. This enzyme was then transferred into plants to cultivate novel transgenic plants with high glyphosate tolerance.

[0017] This invention involves extensive screening of marine bacteria. Activated strains were streaked onto 2216E resistant plates containing 200 mmol / L glyphosate for initial screening. Strains exhibiting good growth were selected for secondary screening. Single colonies of the initially screened strains were further streaked onto M9 basal medium (purchased from Qingdao Rishui Biotechnology Co., Ltd.) containing 0, 100, 200, and 300 mmol / L glyphosate. One strain capable of growing on M9 basal salt medium with high glyphosate concentrations was identified. Preliminary identification using 16S rRNA gene sequencing and comparison analysis revealed that this resistant strain belongs to the genus *Halomonas*. Halomonas Based on whole-genome sequencing results and annotations, the gene structure of glyphosate target enzyme EPSPS was analyzed.

[0018] The present invention describes a glyphosate-resistant EPSP synthase. To develop genes with higher glyphosate resistance, a random mutant library was constructed using error-prone PCR. The HoEPSPS encoding gene was used as a template for random mutation. From over 10,000 mutants, a single-point mutant exhibiting higher glyphosate resistance activity was screened. Glyphosate resistance experiments showed that this mutant had significantly stronger glyphosate resistance activity than the wild type, and this mutant was named mHoEPSPS. To further improve the glyphosate resistance of mHoEPSPS, a second round of directed evolution was conducted using mHoEPSPS as a template and an error-prone PCR library. From over 12,000 mutants, five mutants exhibiting improved glyphosate resistance were screened. These random mutants were used as parents in the next round of DNA shuffling to increase the screening concentration. From over 6,000 mutants, a mutant with further improved resistance, mHoEPSPS-2S, was obtained.

[0019] This invention, through enzyme activity and kinetic parameter determination of wild-type and mutant EPSPS, shows that mHoEPSPS-2S exhibits the best performance. k cat 293.33 min -1 , K i / K m The value was 0.23. Based on the kinetic parameters, mHoEPSPS-2S not only exhibits high glyphosate resistance but also maintains a strong affinity for PEP. These characteristics will provide possibilities for its use in the cultivation of transgenic crops.

[0020] This invention constructs a highly glyphosate-tolerant... EPSPSThe gene expression vector was used to construct glyphosate-resistant transgenic tobacco through leaf disc transformation. Glyphosate resistance experiments proved that the transgenic plants could tolerate four times the commercially recommended dose (0.5% of the commercially recommended dose for general weeds) of Roundup, showing high application potential. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 The growth curves of the mutant mHoEPSPS and wild-type HoEPSPS obtained in the first round of screening in 200 mM glyphosate medium are shown. Figure 2 SDS-PAGE analysis results of CP4 and different mutants are shown; lane 1 is purified CP4; lanes 2-5 are the results of purified random mutants 2, 9, 15, and 16, respectively. Figure 3 The results of SDS-PAGE analysis of the mHoEPSPS-2S protein are shown. Figure 4 The results are the ITC reaction curves; Figure 5 The results are for verifying tobacco resistance in transgenic mHo22-2 genes. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0023] In the following embodiments of the present invention, a new EPSPS enzyme encoding gene with high glyphosate resistance was screened from marine bacteria for the first time, and the enzyme was transferred into plants to cultivate new transgenic plants with high glyphosate tolerance.

[0024] Example 1: Screening, Identification and Analysis of Glyphosate-Tolerant Strains In this embodiment, the test strain of marine strain preserved in the laboratory was activated and then streaked onto glyphosate resistance plates for initial screening.

[0025] During the initial screening process, single colonies of activated strains were inoculated onto 2216E medium (high-salt medium, purchased from Qingdao Rishui Biotechnology Co., Ltd.) containing 200 mmol / L glyphosate, and strains with good growth were selected for the next stage of rescreening.

[0026] During the secondary screening process, single colonies of the initially screened strains were further streaked onto M9 basal medium (purchased from Qingdao Rishui Biotechnology Co., Ltd.) containing 0, 100, 200, and 300 mmol / L glyphosate. A strain that could grow on M9 basal salt medium with high glyphosate concentration was screened out. Genomic DNA was extracted using a bacterial genomic DNA extraction kit and sent to Shanghai Panoson Biotechnology Co., Ltd. for sequencing analysis.

[0027] Based on whole-genome sequencing results and annotations, the gene structure of glyphosate target enzyme EPSPS was further analyzed. The results showed that the gene encoding EPSPS is 2202 bp in length, with a GC content of 60%. It is located upstream and downstream of functional genes unrelated to the shikimic acid pathway, encoding an EPSPS consisting of 733 amino acids with a molecular weight of 78.25 kDa, which is significantly different from the previously reported molecular weight of EPSPS.

[0028] In this embodiment, further BLAST analysis of the enzyme revealed that it encodes a bifunctional enzyme. The N-terminus is predicted to be prebenzoic acid dehydratase (PDT; EC 4.2.1.51), with a length of 272 amino acid residues and a molecular weight of 29.45 kDa; the C-terminus is predicted to be EPSPS (as shown in SEQ ID No. 2), with a length of 461 amino acid residues. In this invention, the C-terminal predicted EPSPS portion (named HoEPSPS) was cloned and expressed, and it was directionally modified.

[0029] Because this enzyme has a multifunctional domain, this embodiment selects the latter half of EPSPS and performs multiple sequence alignment with some classic and identified EPSPS, constructs a phylogenetic tree using MEGA 6 software, and analyzes the sequence homology of different enzymes.

[0030] In this embodiment, HoEPSPS, as shown in SEQ ID No. 2, belongs to type II EPSPS in terms of classification. Its identity with CP4-EPSPS, Iv-EPSPS, G2-EPSPS, and G10-EPSPS is shown in Table 1 below.

[0031] Table 1. Identity comparison results

[0032] The results in the table above are 41.47%, 22.09%, 22.49%, and 22.49%, respectively, indicating that HoEPSPS is a novel glyphosate-resistant gene.

[0033] Example 2: Construction of HoEPSPS mutants and libraries In this embodiment, the following primers were designed based on the gene sequence to amplify the EPSPS encoding gene: Forward primer F (5'-aro) A H.sp. - EcoR I): 5'-GGAATTCAtGttgaacaaaaccagttatcaggcgg-3'; Reverse primer R (3'-aro) A H.sp. - Xho I): 5'-CCGCTCGAGttagtggtcattggcacc-3'.

[0034] After PCR amplification of the target fragment, use Eco RI and Xho After digestion with enzyme I, the plasmid was ligated into the vector pGEX-6p-1, which had been digested with the same enzyme, to obtain the recombinant plasmid pGEX-6p- HoEPSPS It was then transformed into Escherichia coli BL21(DE3) to express EPSP synthase with high glyphosate tolerance.

[0035] In this embodiment, a random mutant library was constructed using the error-prone PCR method, with pGEX-6p- HoEPSPS Use it as a template for PCR amplification.

[0036] In this embodiment, the specific PCR reaction procedure is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 57℃ annealing for 30 sec, 72℃ extension for 90 sec, for a total of 30 cycles; 25℃ incubation for 5 min.

[0037] In this embodiment, the specific PCR reaction system (50 μL) consists of: 1 μL dNTPs (2.5 mM), 1 μL dTTPs (2.5 mM), 1 μL dCTPs (2.5 mM), 1 μL each of forward and reverse primers, 1 μL MnCl2 (25 mM), 1 μL template, and 1 μL... Taq DNA polymerase (5 U / µL), add H2O to 50 μL.

[0038] In this embodiment, it is introduced via PCR. Bam HI / Xho I restriction enzyme site, after PCR amplification of the target fragment, use... Bam HI / Xho After digestion with enzyme I, it is ligated into the vector pGEX-6p-1, which has been digested with the same enzyme.

[0039] In this embodiment, mutant resistance was randomly verified, with wild-type HoEPSPS and commercially available CP4-EPSPS selected as controls. The constructed pGEX-6p-1 and pGEX-6p- CP4 pGEX-6p- HoEPSPSpGEX-6p- mHoEPSPS plasmid transformation to E. coli DH5α competent cells were selected, and single colonies of transformants were inoculated into ampicillin-resistant LB cells. The cells were incubated overnight at 37°C, washed twice to remove nutrients from the LB cells, and the bacterial concentration was adjusted. OD 600 Up to 1.0, transfer 2% of the culture medium to deep-well plates containing 500 μL of M63 basal salt medium (with glyphosate added to a concentration of 280 mM), with three replicates per group. After incubation at 37°C and 200 rpm for 3 days, take 200 μL for analysis. OD 600 Numerical values ​​were used to select mutant strains with growth advantages for growth curve monitoring to verify glyphosate resistance. Bioscreen's fully automated growth curve analysis was employed to determine... OD 600 The numerical values ​​were set to be measured over a 3-day period, with measurements taken every 2 hours. OD 600 The values ​​are: temperature 37℃, speed 200rpm.

[0040] It can be seen that, as Figure 1 As shown in Figure a, mHoEPSPS is more tolerant to glyphosate than HoEPSPS.

[0041] Since mHoEPSPS exhibits higher glyphosate tolerance, there is still room for improvement in its resistance. To develop genes with higher glyphosate tolerance, this embodiment conducts a second round of directed evolution on mHoEPSPS to further improve its glyphosate resistance level, using the same method as the random mutant construction described above.

[0042] In this embodiment, mutant resistance was randomly verified, with CP4-EPSPS and mHoEPSPS selected as controls. The method was the same as the resistance verification method described above.

[0043] Specifically, the highly glyphosate-tolerant EPSP synthase mutants include: mHoEPSPS: The mutant mHoEPSPS is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, with the G at position 112 mutated to A; the amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS is shown in SEQ ID No: 3. mHoEPSPS-2: The mutant mHoEPSPS-2 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating G at position 112 to A, M at position 155 to V, I at position 285 to F, and D at position 326 to E; the amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-2 is shown in SEQ ID No: 4. mHoEPSPS-9: The mutant mHoEPSPS-9 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating Y at position 11 to F, G at position 112 to A, A at position 199 to V, L at position 297 to P, and I at position 428 to N; the amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-9 is shown in SEQ ID No: 5. mHoEPSPS-15: The mutant mHoEPSPS-15 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating A at position 9 to E, G at position 112 to A, S at position 136 to T, V at position 271 to A, V at position 381 to E, and C at position 432 to G; the amino acid sequence of the highly glyphosate-tolerant EPSP synthase mutant mHoEPSPS-15 is shown in SEQ ID No: 6. mHoEPSPS-16: The mutant mHoEPSPS-16 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating G at position 112 to A, I at position 168 to N, H at position 169 to L, P at position 210 to T, and I at position 429 to L; the amino acid sequence of the highly glyphosate-tolerant EPSP synthase mutant mHoEPSPS-16 is shown in SEQ ID No: 7.

[0044] It can be seen that, as Figure 1 As shown in Figure b, the mutants mHoEPSPS-2 and mHoEPSPS-16 showed significantly higher tolerance to 300mM glyphosate than mHoEPSPS and exhibited stronger growth performance than CP4-EPSPS.

[0045] Example 3: Third Round of Directed Evolution Based on DNA Shuffling In this embodiment, mHoEPSPS-2, 9, 15, and 16, which exhibited the best resistance performance in the second round of directed evolution, were selected as parents for the second round of DNA shuffling-based directed evolution. In this embodiment, the recombinant plasmid pGEX-6p- was used respectively. mHoEPSPSUsing templates -2, 9, 15, and 16, different mutant genes were amplified using Taq DNA polymerase and specific primers carrying the homologous arm of the pGEX-6p-1 vector.

[0046] Because ultrasound generates strong mechanical shearing, cavitation, and high temperatures, it breaks hydrogen bonds and disrupts the double helix structure of DNA in aqueous solutions. Based on this characteristic of ultrasound, a method for DNA fragmentation was used. Specific processing conditions were as follows: the instrument power was set to 600W, the frequency to 25kHz, and 600µL of sample (PCR stock solution) was placed on ice. Ultrasound treatment was performed under ice bath conditions, with 15s of sonication followed by an 8s interval, for a total of one minute per cycle. Samples were collected after each cycle for testing, and this process was repeated ten times.

[0047] In this embodiment, the size was detected by 1.5% agarose gel electrophoresis until the fragment size was 100-200 bp. The processed DNA fragment was centrifuged at 12000 rpm for 10 min, the supernatant was collected, and 1 µL of the fragment was used as a template. PCR amplification was performed using two pairs of primers (pGEX-6p-1 universal primers and specific primers carrying the homologous arms of the vector) to detect whether the template DNA fragment was completely fragmented.

[0048] In this embodiment, the specific PCR reaction system (50 μL) is as follows: 1 μL of sonicated fragments, 1 μL each of forward and reverse primers, 1 μL of dNTPs (10 mM), and 1 μL of... FastPfu DNA polymerase, 5 μL 5× FastPfu Add H2O to the buffer to a final volume of 50 μL.

[0049] In this embodiment, the specific PCR reaction procedure is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 sec, annealing at 52℃ for 30 sec, extension at 72℃ for 90 sec, for a total of 30 cycles; incubation at 25℃ for 5 min. If the test result is negative, then proceed to primer-free PCR reaction.

[0050] The product obtained by gel extraction and recovery of the 100-200bp fragment obtained by ultrasonic fragmentation was used as a template for primerless PCR reaction.

[0051] In this embodiment, the specific PCR system (50 μL) is as follows: 40 μL DNA fragments, 1 μL dNTPs (10 mM), 1 μL Taq DNA polymerase (5 U / µL), 5 μL 10×PCR Buffer, and H2O added to 50 μL.

[0052] In this embodiment, the specific PCR reaction procedure was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 40℃ annealing for 30 sec, 72℃ extension for 20 sec + 1 sec / cycle, for a total of 70 cycles; 72℃ incubation for 10 min; 15℃ for 5 min. After the PCR reaction was completed, the product size was detected by 1% agarose gel electrophoresis.

[0053] In this embodiment, primerless PCR stock solution was used as a template, and specific primers carrying the vector and homologous arms of the target gene were used to amplify the recombinant full-length gene.

[0054] Specific PCR reaction mixture (50 μL): 5 μL primer-free PCR product, 1 μL each of forward and reverse primers, 1 μL dNTPs (10 mM), 1 μL... Taq DNA polymerase (5 U / µL), 5 μL 10×PCR Buffer, add H2O to 50 μL. The specific PCR reaction program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 52℃ annealing for 30 sec, 72℃ extension for 90 sec, for a total of 30 cycles; 25℃ incubation for 5 min.

[0055] In this embodiment, the target fragment was amplified by PCR and then purified and recovered by agarose gel electrophoresis. The recovered recombinant target gene and pGEX-6P-1 vector were subjected to restriction endonuclease assays. Bam HI / Xho I was subjected to double digestion, followed by ligation with T4 DNA ligase to obtain the recombinant plasmid pGEX-6p- mHoEPSPS-shuffling It was then transformed into Escherichia coli DH5α.

[0056] The method for verifying resistance to random mutants is the same as the aforementioned embodiment.

[0057] Specifically, mHoEPSPS-2S: The mutant mHoEPSPS-2 is based on the EPSP synthase with the amino acid sequence shown in SEQ ID No: 1, by mutating G at position 112 to A, M at position 155 to V, L at position 297 to P, I at position 429 to L, and F at position 439 to S; the amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-2S is shown in SEQ ID No: 8.

[0058] It can be seen that, as Figure 1 As shown in Figure c, the mutant mHoEPSPS-2S obtained from the third round of directed evolution showed significantly stronger growth performance than mHo22 and CP4-EPSPS in 320mM glyphosate medium.

[0059] In summary, the specific amino acid substitution structures in the present invention are shown in Table 2 below.

[0060] Table 2 Amino Acid Substitution

[0061] Example 4: EPSPS Expression in Highly Glyphosate-Tolerant Species Continue transforming the selected mutant plasmids into... E. coli For BL21(DE3), pick a single colony containing the recombinant plasmid and inoculate it into 10 mL of LB broth containing 100 μg / mL ampicillin. Incubate overnight at 37°C with shaking at 180 rpm. Transfer 1% of the inoculum to a large Erlenmeyer flask containing 500 mL of LB broth and incubate at 37°C for 2-3 hours. OD 600 When the concentration reached 0.6, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mmol / L, and the cells were induced at 18℃ and 180 rpm for 16 h. After induction, the cells were collected by centrifugation at 7500 rpm for 10 min, washed with pre-cooled Hepes buffer (pH 7.0), centrifuged twice to remove the supernatant, and then fully resuspended in 100 mL of fresh Hepes buffer. The cells were then disrupted using a low-temperature high-pressure homogenizer (Guangzhou Juneng Nanobiotechnology Co., Ltd.), and the cell lysis buffer was collected. After centrifugation at 12000 rpm and 4℃ for 60 min, the supernatant was collected and purified by GST affinity chromatography. The results are shown in the appendix. Figure 2-3 As shown.

[0062] As can be seen from the SDS-PAGE electrophoresis, the protein without GST was successfully purified; its size was approximately 48.7 kDa, consistent with the predicted value.

[0063] Example 5: Determination of enzyme activity and kinetic parameters of EPSP Determination methods Enzyme activity is mainly determined based on the amount of inorganic Pi generated by the EPSPS enzyme-catalyzed reaction. Since malachite green can react with inorganic phosphorus, the product has a maximum absorption peak at 660 nm. The reaction is terminated by sodium citrate. This indirect method is used to determine the amount of inorganic Pi generated.

[0064] Preparation of inorganic phosphorus standard curve: Prepare 10 mM inorganic phosphorus standard solution, and take 0-200 μL into 21 1.5 mL centrifuge tubes respectively. Add Hepes buffer to make up to 1 mL, so that the final concentration of inorganic phosphorus in the 20 centrifuge tubes are 0, 0.01, 0.02... 0.19, 0.2 mmol / L respectively.

[0065] Take 20 μL of each concentration of inorganic phosphorus standard solution into a centrifuge tube, incubate in a 28°C water bath for 4 min, then add 800 μL of MAT solution, incubate at room temperature for 1 min, then quickly add 100 μL of 34% trisodium citrate solution (SC), incubate at room temperature for 30 min, and then take 200 μL into a 96-well plate to determine OD. 660 value.

[0066] The design included one control group (without shikimic acid-3-phosphate S3P) and three parallel experimental groups. The concentration of inorganic phosphorus was plotted on the x-axis, showing the effects of different concentrations of inorganic phosphorus standard solutions. OD 660 The inorganic phosphorus standard curve is obtained by plotting the values ​​on the ordinate.

[0067] k cat(gly) Measurement To simulate the intracellular environment of plants, the glyphosate concentration was set to 1 mM, the concentration obtainable by plant meristems. Ethylene glycol and KCl were added to aid in the simulation. The 20 μL reaction system consisted of 100 mM KCl and 5% ethylene glycol. The concentration of shikimic acid-3-phosphate (S3P) was fixed at 1 mM. Different PEP concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) were set. Protein was added to a final concentration of 0.3 μM, and Hepes buffer was added to bring the reaction volume to 20 μL. The reaction was incubated at 28°C for 4 min. 800 μL of MAT solution was added, and after incubation for 1 min, 100 μL of 34% trisodium citrate (SC) solution was added. After incubation at room temperature for 30 min, 200 μL was transferred to a 96-well plate and measured using a preheated microplate reader. OD 660 Values. Plotting PEP concentration on the x-axis and relative reaction rate on the y-axis yields... k cat(gly) The measurement results.

[0068] K m(PEP) Measurement The concentration of shikimic acid-3-phosphate (S3P) in the system was fixed at 1 mM. Different PEP concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) were set. Purified protein with a final concentration of 0.3 μM was added to a 20 μL reaction system. The reaction was carried out at 28 °C for 4 min. 800 μL of MAT solution was added, and after standing for 1 min, 100 μL of 34% trisodium citrate (SC) solution was added. After standing at room temperature for 30 min, 200 μL was transferred to a 96-well plate, and the OD was measured using a preheated microplate reader. 660 Values. Plotting PEP concentration on the x-axis and relative reaction rate on the y-axis yields... K m(PEP) The measurement results.

[0069] K m(S3P) Measurement The concentration of PEP in the system was fixed at 1 mM. Different S3P concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) were set. Purified protein with a final concentration of 0.3 μM was added to a 20 μL reaction system. The reaction was carried out at 28℃ for 4 min. 800 μL of MAT solution was added, and after standing for 1 min, 100 μL of 34% trisodium citrate (SC) solution was added. After standing at room temperature for 30 min, 200 μL was transferred to a 96-well plate, and the OD was measured using a preheated microplate reader. 660 Values. Plotting S3P concentration on the x-axis and relative reaction rate on the y-axis yields... K m (S3P) The measurement results.

[0070] Half-inhibitory dose ( IC 50 ) Measurement Add different concentrations (10) to the above reaction system -5 10 -4 10 - 3 10 -2 10 -1 The data were obtained by plotting glyphosate concentrations (1, 10, and 100 mM) on a logarithmic scale with relative activity rate on the ordinate to obtain the half-inhibitory dose. IC 50 The results of the value determination.

[0071] Suppression constant K i Measurement The concentration of shikimic acid-3-phosphate (S3P) in the system was fixed at 1 mM. 20 μL reaction systems were prepared with different PEP concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 mM, respectively). Glyphosate was added to a final concentration of 100 μM. The system without glyphosate was used as a control to measure the relative enzyme activity. A graph was plotted with substrate concentration on the x-axis and relative activity on the y-axis to obtain the results. K i The measurement results.

[0072] Isothermal titration calorimetry (ITC) is a technique used to quantitatively study various biomolecular interactions. It directly measures the heat released or absorbed during biomolecular binding, and the experimental data is presented as a thermogram, providing information on the amount of substances in the reaction (titer endpoint) and the properties of the reactants (enthalpy change). Analysis of the graph reveals the type and number of reactions occurring in the reaction vessel, the concentration of each species in the solution, and provides accurate and complete thermodynamic information on biomolecular interactions: the binding constant (…). K a) Reaction chemical quantity (n), enthalpy ( H ) and entropy (Δ S ).

[0073] Use the same buffer solution from the same batch used for protein purification to ensure consistency of composition. Perform filtration and degassing to avoid the influence of impurities and air bubbles on the experiment. After rinsing the sample cell with pure water, rinse the titration needle and sample cell with buffer solution. Prepare 0.1 mM mHo22 protein and 25 mM glyphosate solution; prepare 0.3 mM nHo22-2 protein and 50 mM glyphosate solution. Draw 600 μL of mHo22 protein into the sample cell, and draw approximately 60 μL of glyphosate solution into the titration needle. Set the isothermal titration calorimeter parameters as follows: temperature 40℃, rotation speed 150 rpm, 25 titrations, 2 μL each time, and a reaction interval of 90 s.

[0074] In this embodiment, each mutant k cat(gly) The measurement results are shown in Table 3 below.

[0075] Table 3 HoEPSPS and mutants k cat(gly)

[0076] It can be seen that the random mutant mHoEPSPS-2 k cat(gly) The value was the highest, 1.23 times that of mHoEPSPS. Therefore, we ultimately chose the mutant mHoEPSPS-2 for in-depth enzyme activity assays, including resistance indicators, and the results are shown in Table 4 below.

[0077] Table 4. mHoEPSPS-2 Index Test Results

[0078] The above inhibition kinetic analysis showed that mHoEPSPS-2 significantly enhanced resistance to glyphosate. To further explore the molecular mechanism of this enhanced resistance, the direct binding ability between the enzyme and the inhibitor was quantitatively analyzed by isothermal titration (ITC). The dissociation constant between the inhibitor and the enzyme... K d The value () represents the concentration of free inhibitor at which 50% of the enzyme is bound to the inhibitor. A smaller value indicates a stronger affinity between the inhibitor and the enzyme. See the attached ITC reaction curve for details. Figure 4 As shown.

[0079] In this embodiment, the ITC measurement results showed that mHoEPSPS-2 has an effect on glyphosate. K d 3.53×10 -2 μM, while mHoEPSPS K d It is 5.64 × 10 -3 μM. The association constant between the enzyme and the inhibitor. K a )and K d Conversely, a higher value indicates stronger affinity. mHoEPSPS-2 has a higher affinity for glyphosate. K a 2.84×10 4 1 / M, while mHoEPSPS K a It is 1.77 × 10 5 1 / M. Therefore, whether K d still K a The values ​​all indicate that mHoEPSPS-2 has a 5.26-fold lower affinity for glyphosate compared to mHoEPSPS.

[0080] Furthermore, the enzyme thermokinetic parameters shown in Table 5 below also indicate the enthalpy (Δ) of glyphosate binding to the enzyme. H ) and entropy (Δ S All of these parameters decreased. Therefore, the weakened binding ability of glyphosate to the mutant mHo22-2 is the fundamental reason for its increased resistance.

[0081] Table 5 Enzyme thermokinetic parameters

[0082] Example 6: Construction of an expression vector for the EPSP synthase gene in plants with high glyphosate tolerance In this embodiment, the enzyme with the best activity was selected. mHoEPSPS-2, mHoEPSPS-2S Gene transgenic verification was performed by introducing [a gene] at the 5' end of the gene fragment. Kpn I. Restriction site, introduced at the 3' end Bam HⅠ restriction enzyme site, after PCR amplification of the target fragment, use Kpn I and Bam After HⅠ restriction enzyme digestion, the plasmid was ligated into the plant expression vector pCAMBIA1300S, which was also digested with the same enzyme, to obtain the recombinant plasmid pCAMBIA1300S- mHoEPSPS-2 pCAMBIA1300S- mHoEPSPS-2S They were then transferred into Agrobacterium and used to transform the model plant tobacco.

[0083] Example 7: Constructing glyphosate-resistant transgenic tobacco using the leaf disc method. In this embodiment, sterile tobacco seedlings were cultivated by immersing mature "Yanyan 97" seeds in clean water for 5 minutes, filtering with gauze, and rinsing again with clean water. The seeds were then soaked in 70% alcohol for 8 seconds, 2% sodium hypochlorite for 10 minutes, and rinsed five times with sterile water. The seedlings were inoculated onto MS germination medium and cultured alternately at 25°C, light intensity of 1000-1500 lux, 16 hours of light, and 8 hours of darkness.

[0084] Seeds germinate after one week, and sterile seedlings emerge after two weeks. Seedlings are ready for transformation when they have 5-6 leaves. Using a sterile punch, take approximately 0.5 cm leaf samples (avoiding veins) and transfer them to a pre-medium, epidermal side up. Culture for 1 day under 18 hours of light and 6 hours of darkness. In a liquid solution containing rifampicin, tetracycline, and kanamycin, culture a small amount of Agrobacterium tumefaciens containing the target gene at 28°C for 3 days, then transfer to 50 mL of medium for further culture. Suspend Agrobacterium in the liquid co-culture medium, adjust the OD600 value to 0.5-1.0, and keep on ice. Infect the pre-cultured leaf discs with Agrobacterium solution for approximately 30 minutes. Gently remove each leaf disc one by one with tweezers, remove excess solution, and place them in MS co-medium, epidermal side up. Place a filter paper on the surface of the co-medium. Seal with sealing film and culture at 23°C in the dark for 3 days. First, wash the leaf discs after co-culturing with sterile water, then wash them with an aqueous solution containing cephalosporin, and blot dry the surface moisture with filter paper. Transfer to selection medium, gently press the edges of the leaf discs into the medium, and culture at 24℃, light intensity of 1000-1500 lux, alternating between 12h light and 12h darkness for about 2 weeks. In the selection medium, callus will grow from the edges of the explants, and buds will emerge from the callus. When the buds reach 3mm in length, transfer to rooting medium, placing 2-3 seedlings per bottle. Once the roots reach 3cm-4cm in length, the seedlings can be hardened off and transplanted. Cut leaves from the regenerated seedlings, extract DNA using the CTAB method, and verify glyphosate resistance in transgenic positive seedlings using hygromycin resistance gene-specific primers.

[0085] In this embodiment, when the transgenic tobacco plants reached the 4-6 leaf stage, 1.7%-2.0% Roundup (41% glyphosate isopropylamine salt, Monsanto) was sprayed onto the leaves of mHo22-2 transgenic tobacco plants, and the phenomena were observed after 7 days. Wild-type plants, the "WT" line, exhibited wilting, yellowing, and even death when treated with 0.5% Roundup; while the mHo22-2 transgenic tobacco plants grew normally and showed no inhibited phenotypes when treated with 2% Roundup (see attached figure). Figure 5 (As shown).

[0086] The resistance experiment in this example shows that the mHo22-2 transgenic tobacco can tolerate four times the commercially recommended dose (0.5% of the commercially recommended dose for general weeds) of Roundup, with normal growth and no growth inhibition, demonstrating great commercial value.

[0087] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

[0088] Sequence List: SEQ ID No.1 HoEPSPS amino acid sequence: MLNKTSYQAEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSGTAMRLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAMIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPAPTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVINILQLMGADLTLQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIIIDDCANVATSFPDFVALAQRIGMSVEVEGANDH

[0089] SEQ ID No.2 HoEPSPS nucleotide sequence: SEQ ID No.3 mHoEPSPS amino acid sequence: MLNKTSYQAEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAMRLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAMIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPAPTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVINILQLMGADLTLQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIIIDDCANVATSFPDFVALAQRIGMSVEVEGANDH

[0090] SEQ ID No.4 mHoEPSPS-2 amino acid sequence: MLNKTSYQAEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAMRLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAVIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPAPTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVFNILQLMGADLTLQNEREVGGEPVADIRIRYAPLKGIDIPVEQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIIIDDCANVATSFPDFVALAQRIGMSVEVEGANDH

[0091] SEQ ID No.5 Amino acid sequence of mHoEPSPS-9: MLNKTSYQAEFHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAMRLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAMIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYVEGETRVREPAPTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVINILQLMGADLTPQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEINIDDCANVATSFPDFVALAQRIGMSVEVEGANDH

[0092] SEQ ID No.6 Amino acid sequence of mHoEPSPS-15: MLNKTSYQEEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAMRLFAGLLAGQAFDSELTGDETLTKRPMGRVADPLRLMGAMIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPAPTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIALEHVGINPTRIGVINILQLMGADLTLQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTEVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIIIDDGANVATSFPDFVALAQRIGMSVEVEGANDH

[0093] SEQ ID No.7 Amino acid sequence of mHoEPSPS-16: MLNKTSYQAEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAMRLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAMIDTAEGGRPPLKNLGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPATTRDHTERMLNGFGYEVSREGDTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVINILQLMGADLTLQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNAYGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIILDDCANVATSFPDFVALAQRIGMSVEVEGANDH

[0094] SEQ ID No.8 Amino acid sequence of mHoEPSPS-2S: MLNKTSYQAEYHMQPHGKVTYRVQPGGKAQGRLRVPGDKSMSHRSIMLGALAEGVTEVKGFLEGEDSLATLQAFREMGVAIEGPHQGRVTIHGVGMHGLKAPAGPLYVGNSATAM RLFAGLLAGQAFDSELTGDESLTKRPMGRVADPLRLMGAVIDTAEGGRPPLKIHGGKALKGIHYDMPMASAQVKSCLLLAGLYAEGETRVREPAPTRDHTERMLNGFGYEVSREG DTCWLQGGGRLTAGPIDVPSDISSATFFLVAAAITPGSDIVLEHVGINPTRIGVINILQLMGADLTPQNEREVGGEPVADIRIRYAPLKGIDIPVDQVPLAIDEFPALFIAASNA YGTTRLRGAEELRVKESDRIQAMADGLAVLGVEHTVVEDGIDIVGNGNDSGPSYGGGRIDSLGDHRIAMAFAIASLRAGAEIILDDCANVATSSPDFVALAQRIGMSVEVEGANDH

[0095] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A glyphosate-resistant EPSP synthase mutant, characterized in that, The mutant is obtained by mutating amino acids at specific sites based on the EPSP synthase with the amino acid sequence shown in SEQ ID No:

1. The mutant is: mHoEPSPS: The amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS is shown in SEQ ID No: 3; or, mHoEPSPS-2: The amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-2 is shown in SEQ ID No: 4; or, mHoEPSPS-9: The amino acid sequence of the highly glyphosate-tolerant EPSP synthase mutant mHoEPSPS-9 is shown in SEQ ID No: 5; or, mHoEPSPS-15: The amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-15 is shown in SEQ ID No: 6; or, mHoEPSPS-16: The amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-16 is shown in SEQ ID No: 7; or, mHoEPSPS-2S: The amino acid sequence of the highly glyphosate-resistant EPSP synthase mutant mHoEPSPS-2S is shown in SEQ ID No:

8.

2. An expression vector with high glyphosate tolerance, characterized in that, The expression vector contains a gene encoding the mutant of claim 1.

3. A transgenic cell line highly tolerant to glyphosate, characterized in that, The transgenic cell line contains the gene encoding the mutant of claim 1.

4. A genetically engineered bacterium with high glyphosate tolerance, characterized in that, The genetically engineered bacteria contains a gene encoding the mutant of claim 1.

5. A transgenic plant with high tolerance to glyphosate, characterized in that, The transgenic plant contains a gene encoding the mutant of claim 1.

6. The transgenic plant with high glyphosate tolerance according to claim 5, characterized in that, The genetically modified plant is genetically modified tobacco.

7. The application of the gene encoding the mutant of claim 1 in the field of breeding highly glyphosate-tolerant transgenic plants.

8. The application according to claim 7, characterized in that, The plants mentioned include soybeans, corn, rapeseed, cotton, wheat, rice, or sugarcane.

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