Application of rice EPSPS protein point mutation in improving glyphosate resistance of rice

Abstract

The invention relates to the technical field of rice breeding, in particular to application of rice EPSPS protein point mutation in improving glyphosate resistance of rice. Point mutation is located at the 410th site and the 414th site of the rice EPSPS protein, the amino acid at the 410th site mutates from V to D, and the amino acid at the 414th site mutates from A to D and the like. V410D / A414D and P227Q / L509V mutation modes generated by natural variation of the rice EPSPS gene are reported for the first time, the physical and chemical properties and the protein structure of the rice EPSPS gene are found to be changed, and transgenosis verifies that the two mutations can remarkably improve the glyphosate resistance of the rice, and a new target is provided for molecular design of glyphosate-resistant rice.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, and in particular to application of point mutation of rice EPSPS protein in improving rice resistance to glyphosate. Background Art

[0002] As of March 2025, 534 weed populations worldwide have developed resistance to herbicides, making weed control increasingly difficult. Currently, expanding the use of other highly effective herbicides in rice fields, combined with the planting of resistant rice varieties, has become a new strategy for effectively controlling weeds in rice fields. Glyphosate, known as the "once-in-a-century herbicide," boasts high efficiency, low toxicity, and long-lasting effectiveness. It is commonly used for weed control in non-arable land and, since its commercial application, has gradually become the world's best-selling herbicide. Because the EPSPS gene is highly conserved in plants, glyphosate is lethal to nearly all plants. By breeding glyphosate-resistant varieties of major crops such as rice, soybeans, corn, cotton, and rapeseed, the large-scale application of glyphosate in crops has been significantly expanded, effectively addressing the challenge of controlling resistant weeds. This patent obtains natural glyphosate-resistant rice materials through screening, clarifies the mutation type of the EPSPS gene of resistant rice, analyzes its physical and chemical properties and the secondary and tertiary structures of the protein, verifies the glyphosate-resistant function of EPSPS allelic variation, and provides genetic resources and germplasm materials for the cultivation of glyphosate-resistant rice. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides the use of a point mutation of rice EPSPS protein in improving the glyphosate resistance of rice. The point mutation of rice EPSPS protein improves the glyphosate resistance of rice.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides an application of a point mutation of a rice EPSPS protein in improving rice resistance to glyphosate, wherein the point mutation is located at positions 410 and 414 of the rice EPSPS protein, wherein the amino acid at position 410 mutates from V to D, and the amino acid at position 414 mutates from A to D;

[0006] Or the point mutation is located at positions 227 and 509 of the rice EPSPS protein, the amino acid at position 227 mutates from P to Q, and the amino acid at position 509 mutates from L to V;

[0007] or a point mutation at position 509 of the rice EPSPS protein, where the amino acid at position 509 is mutated from L to V;

[0008] Or the point mutation is located at positions 54 and 509 of the rice EPSPS protein, the amino acid at position 54 mutates from E to K, and the amino acid at position 509 mutates from L to V;

[0009] Or the point mutation is located at positions 251 and 401 of the rice EPSPS protein, the amino acid at position 251 mutates from Q to K, and the amino acid at position 401 mutates from V to I;

[0010] The amino acid sequence of the rice EPSPS protein is shown in SEQ ID No. 1.

[0011] Preferably, the gene sequence of the rice EPSPS protein is shown as SEQ ID No. 2.

[0012] Preferably, the rice variety includes Nipponbare.

[0013] Beneficial effects of the present invention:

[0014] This paper reports for the first time the V410D / A414D and P227Q / L509V mutation patterns produced by natural variation of the rice EPSPS gene, discovers that its physicochemical properties and protein structure are altered, and verifies through genetic modification that these two mutations can significantly improve rice's resistance to glyphosate, providing new targets for the molecular design of glyphosate-resistant rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0016] Figure 1 Screening of natural rice materials for glyphosate resistance, where a shows the phenotype of natural rice materials 7 days after glyphosate treatment, with red arrows marking surviving rice and blue arrows marking dead rice; b shows the phenotype of second-generation rice 21 days after glyphosate treatment, with the first column representing the untreated control group (#N), the second column representing the Nipponbare-treated group, and columns 3-6 representing the second-generation rice material treatment groups (#1-#10, representing different individual plants);

[0017] Figure 2 Comparison of amino acid sequences of EPSPS from natural materials of Nipponbare and rice;

[0018] Figure 3 The phenotype of transgenic rice 21 days after glyphosate treatment;

[0019] Figure 4 Schematic diagram of the T-DNA region of the transgenic plant expression vector. DETAILED DESCRIPTION

[0020] The present invention provides an application of a point mutation of a rice EPSPS protein in improving rice resistance to glyphosate, wherein the point mutation is located at positions 410 and 414 of the rice EPSPS protein, wherein the amino acid at position 410 is mutated from V to D, and the amino acid at position 414 is mutated from A to D; or the point mutation is located at positions 227 and 509 of the rice EPSPS protein, wherein the amino acid at position 227 is mutated from P to Q, and the amino acid at position 509 is mutated from L to V; or the point mutation is located at positions 410 and 414 of the rice EPSPS protein, wherein the amino acid at position 410 is mutated from V to D, and the amino acid at position 414 is mutated from A to D; The point mutations are located at positions 54 and 509 of the rice EPSPS protein, where the amino acid at position 509 is mutated from L to V; or the point mutations are located at positions 54 and 509 of the rice EPSPS protein, where the amino acid at position 54 is mutated from E to K, and the amino acid at position 509 is mutated from L to V; or the point mutations are located at positions 251 and 401 of the rice EPSPS protein, where the amino acid at position 251 is mutated from Q to K, and the amino acid at position 401 is mutated from V to I; the amino acid sequence of the rice EPSPS protein is shown in SEQ ID No. 1.

[0021] SEQ ID No. 1:

[0022] MAATMASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEAL KALGLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLM AAPLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGK KHLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0023] In the present invention, the gene sequence of the rice EPSPS protein (i.e., EPSPS gene) is shown in SEQ ID No. 2, specifically as follows:

[0024]

[0025] In the present invention, the rice varieties preferably include Nipponbare.

[0026] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] Experimental methods and steps

[0029] 1.1 Test materials

[0030] The experimental materials were collected from the Chunhua Experimental Base of Hunan Hybrid Rice Research Center in Changsha County, Changsha City, Hunan Province, and stored in a -20℃ refrigerator for future use.

[0031] 1.2 Resistant material screening

[0032] The collected rice seeds were placed in a 30°C constant-temperature incubator and soaked in darkness for 12 hours. They were then transferred to petri dishes lined with sterile filter paper and placed in a lighted incubator (28°C, 12 hours of light / 25°C, 12 hours of darkness) for germination. After germination, the seedlings were transplanted into seedling pots. When the plants reached the two-leaf stage, they were foliarly sprayed with glyphosate at a dose of 2000 g ai / ha using a walking spray tower. Untreated plants served as blank controls. After application, resistant materials were screened by observing survival phenotypes and calculating plant height and fresh weight inhibition rates. Ultimately, a stable, genetically stable, glyphosate-resistant rice germplasm was obtained for subsequent experiments.

[0033] 1.3 Rice total DNA extraction

[0034] Rice leaves at the two-leaf stage were selected, quickly frozen in liquid nitrogen, and then ground. DNA was then lysed and purified using the Tiangen Biochemical Technology Co., Ltd. Plant Genomic DNA Extraction Kit. After testing for purity and concentration, the DNA was stored at -20°C.

[0035] 1.4 Rice total RNA extraction and reverse transcription

[0036] Two-leaf stage rice leaves were selected and quick-frozen in liquid nitrogen. Total RNA was extracted using the FastPureUniversal PlantTotal RNAIsolation Kit from Nanjing Novozymes Biotech according to standard procedures. Reverse transcription was then performed using a reverse transcription kit. The resulting cDNA was aliquoted into EP tubes and stored at -20°C.

[0037] 1.5 Rice EPSPS gene-specific primer design and cloning

[0038] Based on the Nipponbare EPSPS gene sequence (SEQ ID No. 2) in the NCBI database, specific primers (EPSPS-F (SEQ ID No. 3): ATGGCGGCGACCATGGCGT; EPSPS-R (SEQ ID No. 4): GTTCCTGACGAAAGTGCTTA) were designed for PCR amplification using rice DNA as a template. The amplification protocol was 95°C for 3 minutes, followed by 32 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 2 minutes, and finally 72°C for 5 minutes. The amplified product was verified by agarose gel electrophoresis, and the target band was purified using the FastPure Gel DNA Extraction Mini Kit, ligated with a D-TOPO cloning vector, and transformed into competent Escherichia coli cells. Positive clones were screened for plasmid extraction, and DNA sequencing verification was performed by Shanghai Sangon Biotechnology Co., Ltd.

[0039] 1.6 Bioinformatics Analysis

[0040] Bioinformatics analysis was performed based on the amino acid sequence of rice EPSPS protein (SEQ ID No. 1): sequence alignment was performed using DNAMAN to obtain the mutation site.

[0041] 1.7 Gene function verification

[0042] By cloning the EPSPS genomic sequence of Nipponbare and resistant rice lines, a GFP fusion expression vector driven by the EPSPS promoter was constructed ( Figure 4 ) were used to obtain transgenic plants via Agrobacterium transformation. Semi-quantitative PCR was used to detect GFP expression levels in the transgenic lines using OsACTIN1 as an internal reference (primers are shown in Table 1). Three transgenic lines with consistent exogenous GFP expression levels were selected as treatment groups, and Zhonghua 11 was used as a control group. A glyphosate spraying experiment at 2000 g ai / ha was conducted, and phenotypic parameters such as plant height and fresh weight of the transgenic plants were analyzed.

[0043] Table 1 Primer sequences

[0044]

[0045] result:

[0046] 1. Screening and phenotypic analysis of glyphosate-resistant rice materials

[0047] By applying 2000g ai / ha of glyphosate, 18 resistant rice plants were initially screened from the collected natural rice materials. These plants were propagated to obtain the second-generation natural rice materials. The natural rice materials that were not sprayed with glyphosate served as the control group, and the Nipponbare rice and second-generation rice materials sprayed with 2000g ai / ha of glyphosate served as the treatment groups. Phenotypic analysis was performed 21 days after application. The results showed that the second-generation treated group maintained a normal growth phenotype, while the Nipponbare treated group withered and died, indicating that the screened rice plants were glyphosate-resistant and that this resistance trait was stably inherited ( Figure 1 ).

[0048] 2. Cloning of EPSPS Gene

[0049] Total DNA was extracted from the above-mentioned resistant rice plants and the EPSPS gene was amplified. The sequencing results were compared and analyzed by DNAMAN. It was found that five types of amino acid mutations occurred at specific sites. The mutants were named EPSPS-W1 (V410D / A414D), EPSPS-W2 (P227Q / L509V), EPSPS-W3 (L509V), EPSPS-W4 (E54K / L509V) and EPSPS-W5 (Q251K / V401I), respectively. Figure 2 .

[0050] 3. Verification of glyphosate resistance in EPSPS-W1 and EPSPS-W2 overexpressing rice

[0051] The present invention successfully constructed six EPSPS gene overexpression vectors (pEPSPS::EPSPS:GFP, pEPSPS::EPSPS-W1:GFP, pEPSPS::EPSPS-W2:GFP, pEPSPS::EPSPS-W3:GFP, pEPSPS::EPSPS-W4:GFP, and pEPSPS::EPSPS-W5:GFP). Since the EPSPS-W3, EPSPS-W4, and EPSPS-W5 transformation lines failed to obtain stably inherited homozygous plants, the functional verification was mainly performed on the Nipponbare EPSPS, EPSPS-W1, and EPSPS-W2 overexpression lines. Phenotypic analysis revealed that after 21 days of treatment with 2000 g ai / ha of glyphosate, Zhonghua 11 plants completely withered and died, while the three transgenic lines (two overexpression lines of Nipponbare EPSPS, EPSPS-W1, and EPSPS-W2) all survived. This result indicates that compared with Nipponbare EPSPS, the genetic variation of EPSPS-W1 and EPSPS-W2 can enhance the resistance of rice to glyphosate ( Figure 3 ).

[0052] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of a point mutation of rice EPSPS protein in improving rice resistance to glyphosate, characterized in that: The point mutations are located at positions 410 and 414 of the rice EPSPS protein, with the amino acid at position 410 mutating from V to D and the amino acid at position 414 mutating from A to D; Or the point mutation is located at positions 227 and 509 of the rice EPSPS protein, the amino acid at position 227 mutates from P to Q, and the amino acid at position 509 mutates from L to V; or a point mutation at position 509 of the rice EPSPS protein, where the amino acid at position 509 is mutated from L to V; Or the point mutation is located at positions 54 and 509 of the rice EPSPS protein, the amino acid at position 54 mutates from E to K, and the amino acid at position 509 mutates from L to V; Or the point mutation is located at positions 251 and 401 of the rice EPSPS protein, the amino acid at position 251 mutates from Q to K, and the amino acid at position 401 mutates from V to I; The amino acid sequence of the rice EPSPS protein is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The gene sequence of the rice EPSPS protein is shown in SEQ ID No.

2.

3. The use according to claim 1, characterized in that The rice varieties include Nipponbare.

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