Primer group for amplifying glyphosate-resistant gene OsEPSPS molecular marker and application
By developing primer sets for amplifying the OsEPSPS molecular marker of the glyphosate-resistant gene, combined with PCR and electrophoresis analysis, the problem of existing rice varieties being sensitive to glyphosate herbicides was solved, and the rapid and accurate identification of glyphosate-resistant rice varieties were achieved, and breeding efficiency was improved.
Patent Information
- Application Number
- CN202510357728.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-04-25
AI Technical Summary
The existing rice varieties are sensitive to glyphosate herbicides, which leads to difficulties in controlling weeds and affects rice yield and quality. There is a lack of quick and accurate identification methods to breed glyphosate-resistant rice varieties.
A set of primer sets of amplified glyphosate-resistant gene OsEPSPS molecular markers were developed, and using PCR technology and electrophoretic analysis to quickly and accurately identify whether rice has glyphosate-resistant herbicide traits.
The rapid and accurate identification of glyphosate rice varieties has been achieved, which reduces the risks of field operation errors and environmental pollution, and improves the efficiency and accuracy of rice breeding.
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Figure CN119876480A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for amplifying a glyphosate-resistant gene. OsEPSPS Primer sets and applications for molecular markers. Background Art
[0002] Rice is an important food crop, and its stable and high yields play an important role in ensuring national food security. However, with the continuous advancement of industrialization, the agricultural population has continued to decline, and the small-scale rice farming form can no longer meet the current agricultural needs. The no-tillage and direct seeding methods are favored because of their advantages such as less labor and high economic efficiency. However, the extensive field management method leads to vigorous growth of weeds, which seriously affects the yield and quality of rice. According to statistics, the rice yield loss caused by weed damage in the direct seeding system exceeds 80%. With the continuous expansion of the area of direct-seeding rice, rice varieties that do not contain herbicide resistance genes have been difficult to meet market demand. Herbicide-resistant rice varieties have become the preferred varieties of farmers and are also an important research direction of rice breeding.
[0003] Glyphosate is the most widely used herbicide in the current area. It has the advantages of rapid degradation in soil, not easy to enter groundwater, and non-toxic to mammals. Glyphosate competitively inhibits the action of 5'-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimic acid synthesis pathway, leading to the accumulation of shikimic acid in the organism, the synthesis of aromatic amino acids phenylalanine, tyrosine, and tryptophan is blocked, and the photosynthesis, respiration and other physiological activities of the plant are adversely affected, eventually leading to the chlorosis, yellowing, and even death of the plant. However, due to its broad spectrum, it can also harm the growth of crops while removing weeds. Now almost all rice varieties are sensitive to glyphosate. The selection of new rice varieties with glyphosate herbicide resistance can fundamentally solve this problem. Therefore, it is of great application value to develop a primer set that can quickly and accurately identify glyphosate-resistant rice varieties with different backgrounds. Summary of the invention
[0004] In order to solve the above problems, the present invention provides a method for amplifying a glyphosate-resistant gene. OsEPSPS Molecular marker primer set and application. The invention provides amplification of glyphosate resistance gene OsEPSPS The primer set, kit and identification method of molecular markers can quickly and accurately identify new glyphosate-resistant rice varieties with different backgrounds. The primer set or kit for amplifying the glyphosate-resistant gene OsEPSPS molecular marker provided by the present invention can detect whether rice has the trait of glyphosate herbicide resistance, and can efficiently and accurately breed glyphosate-resistant rice varieties with different backgrounds. The method for identifying glyphosate-resistant rice varieties provided by the present invention has the advantages of small workload, low cost, simple operation, etc.
[0005] The first aspect of the present invention provides a method for amplifying a glyphosate-resistant gene. OsEPSPS Molecular marker primer set, the glyphosate resistance gene OsEPSPS The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; the glyphosate resistance gene OsEPSPS The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; the nucleotide sequence of the molecular marker has a G / C polymorphism at the 4th bp base, an A / G polymorphism at the 5th bp base, a C / T polymorphism at the 18th bp base, and an A / C polymorphism at the 20th bp base; the primer set includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.
[0006] A second aspect of the present invention provides a kit, which comprises the primer set described above.
[0007] Furthermore, the kit also includes dNTPs, Taq DNA polymerase and PCR reaction buffer.
[0008] The third aspect of the present invention provides an application of the primer set or kit described above in rice germplasm resource breeding.
[0009] A fourth aspect of the present invention provides an application of the primer set or kit described above in identifying rice germplasm resources.
[0010] Furthermore, the application is to identify rice varieties resistant to glyphosate.
[0011] A fifth aspect of the present invention provides a method for identifying glyphosate-resistant rice varieties, comprising the following steps: Extracting DNA from the rice to be tested; Using the DNA of the rice to be tested as a template, amplifying the DNA template by PCR using the primer set described above to obtain an amplified product; The amplified product is subjected to electrophoresis analysis. If there is a band at 605 bp, the rice to be tested is determined to be glyphosate-resistant rice.
[0012] Furthermore, each 20 μL PCR amplification reaction system includes: 2 μL~3 μL DNA template, 10 μL 2×Taq PCRMasterMix, 0.2 μL~0.5 μL upstream primer, 0.2 μL~0.5 μL downstream primer, and ddH2O is added to make up.
[0013] Furthermore, the PCR reaction procedure is: (1) initial denaturation; (2) denaturation; annealing: 58°C, 30s; extension; a total of 34 cycles; (3) terminal extension.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a set of methods for amplifying glyphosate-resistant genes OsEPSPS The present invention finds that the primer set of molecular marker OsEPSPS A molecular marker having a nucleotide sequence as shown in SEQ ID NO.1 exists on the gene, and the molecular marker is related to the glyphosate resistance trait of rice. The nucleotide sequence of the molecular marker has a G / C polymorphism at the 4th bp base, an A / G polymorphism at the 5th bp base, a C / T polymorphism at the 18th bp base, and an A / C polymorphism at the 20th bp base. The present invention utilizes the principle that the base polymorphism changes of the four SNP sites with small intervals cause the primers to be unable to bind, and develops a set of primers for amplifying the glyphosate resistance gene. OsEPSPS The molecular marker primer set can be used to quickly identify the superior rice varieties resistant to glyphosate herbicides, and can reduce the deviation of breeding material detection caused by field operation errors, environmental and water pollution, etc.; it can efficiently and accurately identify whether rice has the trait of glyphosate herbicide resistance, and provide a new idea for the selection and improvement of glyphosate-resistant rice varieties. In addition, in crop molecular breeding, SNP sites are generally detected by CAPS / dCAPS or KASP methods, but CAPS / dCAPS needs to be digested after PCR amplification, and then electrophoresis analysis is performed, which takes a long time, and the cost of using restriction endonucleases is high, while KASP primers require fluorescence detection equipment and related fluorescence kits, which are expensive. The primer set provided by the present invention can achieve the purpose of quickly and accurately identifying new glyphosate-resistant rice varieties with different backgrounds only through ordinary PCR, without the need for enzyme digestion.
[0015] The present invention also provides a method for identifying glyphosate-resistant rice varieties. This method can identify whether the variety to be tested is a glyphosate-resistant rice variety through electrophoresis results. The method has the advantages of simple operation, clear bands that are easy to judge, and low cost. The feasibility of this method is verified by electrophoresis experiments and phenotypic tests. The results are accurate and reliable, and the glyphosate resistance of rice can be easily predicted. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0017] Figure 1 for OsEPSPS Genes and glyphosate resistance OsEPSPS Differential base alignment of mutant genes; in the figure, the top row is the wild type OsEPSPS Gene bases and sequencing peaks, the bottom row is for glyphosate resistance OsEPSPS Gene bases and sequencing peaks, the red frame is the wild type OsEPSPS and glyphosate resistant OsEPSPS The difference in bases of mutant genes 。
[0018] Figure 2 The electrophoresis results of PCR products of 181 F2 populations, where Figures B, C, and D are the continuations of Figures A, B, and C, respectively. Arabic numerals represent the numbers of each plant in the F2 population, the red arrow indicates the position of the 605 bp band, and M represents DNA Marker.
[0019] Figure 3 The phenotypic results of the F2 population after spraying with glyphosate; the red arrows indicate the plants that dried up and died after spraying with glyphosate. DETAILED DESCRIPTION
[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0021] The biological materials involved in the embodiments: Zhennuo 19 was purchased from Hefei Fengle Seed Co., Ltd., and Runnong 11 was purchased from Shandong Denghai Runnong Seed Co., Ltd.
[0022] Glyphosate is the most widely used herbicide in the current area. However, due to its broad spectrum, it can harm the growth of crops while killing weeds. Almost all rice varieties are sensitive to glyphosate. The selection of new rice varieties with glyphosate herbicide resistance can fundamentally solve this problem. Therefore, it is of great application value to develop an identification method and primer set that can quickly and accurately identify glyphosate-resistant rice varieties with different backgrounds.
[0023] The present invention provides a set of methods for amplifying glyphosate-resistant genes OsEPSPS Molecular marker primer set and kit, and a method for identifying glyphosate-resistant rice varieties, wherein the glyphosate-resistant gene OsEPSPSThe nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. In the sequence shown in SEQ ID NO.1, there are polymorphic SNP sites that are close to each other. A set of amplified glyphosate resistance genes was designed with these four SNPs as the center. OsEPSPS The primer set for molecular markers was developed, and the feasibility of the identification method was verified through molecular experiments and phenotypic tests.
[0024] Example 1: A method for amplifying a glyphosate-resistant gene OsEPSPS Primer Sets for Molecular Markers The invention extracts DNA from the rice variety Zhennuo No. 19, which is resistant to glyphosate, and uses the DNA as a template to clone OsEPSPS In the CDS region of the gene, after comparing the sequencing results in the phytozome database, it was found that there were 6 SNP sites. After analyzing these 6 sites, it was found that 4 of them were close to each other. Then, the primer set was designed based on these 4 SNPs to distinguish different genotypes. The details are as follows:
[0025] 1. Sequence analysis and primer set development The genome sequence of Nipponbare rice without glyphosate herbicide resistance was used as the reference genome. The genome information was derived from the Oryza sativa v7.0 genome (Phytozome genome ID: 323) in the phytozome database. The genome sequence of Zhennuo 19 rice with glyphosate herbicide resistance was used as the reference genome. OsEPSPS The genes were sequenced and compared with the reference genome sequence, and glyphosate resistance genes were found OsEPSPS There are 4 polymorphic SNP sites in the sequence shown in molecular marker SEQ ID NO.1 that are close to each other ( Figure 1 ).
[0026] SEQ ID NO.1: , s is G / C, r is A / G, y is C / T, and m is A / C; The nucleotide sequence shown in SEQ ID No. 1 has a G / C base change at the 4th bp, an A / G base change at the 5th bp, a C / T base change at the 18th bp, and an A / C base change at the 20th bp.
[0027] The base polymorphism at the 18th and 20th bp of the nucleotide sequence shown in SEQ ID No.1 results in the amino acid encoded by the 18th to 20th bp bases of the SEQ ID No.1 sequence being proline (DNA bases are CCA or CCC) or serine (DNA bases are TCC or TCA). These two SNP sites are located at OsEPSPSThe gene encodes the catalytic region of the enzyme. Different encoded amino acids will affect the enzyme's binding activity with glyphosate, thereby changing the resistance phenotype of rice plants to glyphosate. When the amino acid encoded by the bases at 18bp~20bp changes from proline to serine, the change in amino acid leads to a decrease in the enzyme's binding activity with glyphosate, thereby making the rice plants resistant to glyphosate; the base polymorphism change at the 4bp and 5bp causes the amino acid encoded by the 3bp~5bp bases of the SEQID No.1 sequence to be glycine (DNA base is GGA or GGG) or alanine (DNA base is GCA or GCG).
[0028] The distance between the four SNP sites in the nucleotide sequence shown in SEQ ID No.1 is 16 bp, which meets the conditions for primer design. The present invention is designed to amplify the glyphosate resistance gene OsEPSPS Primer set for molecular markers: the nucleotide sequence of the upstream primer EPS-S is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer EPS-R is shown in SEQ ID No. 3; SEQ ID No.2: 5'-CTGCGACTGCAATGCGATCC-3'; SEQ ID No. 3: 5'-CAAGTACTGACTGCTGATGG-3'.
[0029] 2. Theoretical analysis of amplified fragments The genotype of glyphosate-resistant rice has the nucleotide sequence shown in SEQ ID NO.4, which can completely match with the primer, so a 605 bp band can be amplified; however, the upstream primer designed by the present invention cannot complementarily pair with the base of the wild-type gene fragment (SEQ ID NO.5), and a PCR product cannot be obtained.
[0030] SEQ ID NO.4: 。
[0031] SEQ ID NO.5: .
[0032] Example 2: A method for identifying glyphosate-resistant rice varieties (1) DNA extraction Experimental materials to be tested: Runnong 11, Zhennuo 19 and 181 F2 segregating populations of the hybrid offspring of Runnong 11 and Zhennuo 19.
[0033] Select young leaves of the rice plant to be tested and use the TPS method to extract rice genomic DNA. The specific steps are as follows: ① Take an appropriate amount of leaves and place them in a 2 mL centrifuge tube, add 600 μL of DNA extraction solution and 3 steel beads, and shake with a tissue crusher for about 60 seconds; ② Place the centrifuge tube in a 72°C water bath for 60 min, mixing by inverting it three times; ③ Centrifuge at 12000rpm for 10min, and aspirate about 300μL of supernatant into a new 1.5mL centrifuge tube; ④ Add an equal volume of pre-cooled isopropanol and mix thoroughly by inverting. Place at -20℃ for 30 minutes to allow the DNA to fully precipitate. ⑤ Centrifuge at 12000rpm for 10min, discard the supernatant, and add 500μL 75% ethanol to rinse once; ⑥ After the DNA is dry, add 200 μL ddH2O to dissolve the DNA and store it at -20℃ for later use.
[0034] (2) PCR amplification The extracted DNA was subjected to PCR amplification using the primer set of Example 1. Each 20 μL PCR amplification reaction system included: 2 μL DNA template, 10 μL 2×Taq PCR MasterMix, 0.5 μL upstream primer, 0.5 μL downstream primer, and ddH2O was added to make up.
[0035] The PCR program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, 34 cycles; extension at 72°C for 8 min, and storage at 4°C.
[0036] (3) Determination of electrophoresis results The amplified product was subjected to 1% agarose gel electrophoresis. When an obvious band was found at 605 bp in the electrophoresis result, the rice to be tested was determined to be glyphosate-resistant rice.
[0037] (4) Verification of phenotypic results The present invention verifies the accuracy of the primer set and the feasibility of the method by using the phenotypic results of rice seedlings after spraying glyphosate herbicide.
[0038] Agarose gel electrophoresis results Figure 2 As shown, some samples have obvious bands at 605bp, while some samples have no bands at 605bp. The phenotypic results of rice seedlings after spraying glyphosate herbicide are as follows Figure 3 As shown, it can be seen that some rice seedlings have chlorotic leaves, and some even have dried up and died, but some seedlings still have green leaves and grow normally. The specific experimental data are shown in Table 1. The rice traits reflected by the electrophoresis results are consistent with the phenotypic results.
[0039] Table 1 Typing results and phenotypes of experimental materials Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.
[0040] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. Amplification of glyphosate-resistant genes OsEPSPS A primer set for molecular labeling, characterized in that: The glyphosate resistance gene OsEPSPS The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; the nucleotide sequence of the molecular marker has a G / C polymorphism at the 4th bp base, an A / G polymorphism at the 5th bp base, a C / T polymorphism at the 18th bp base, and an A / C polymorphism at the 20th bp base; the primer set includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.
3.
2. A kit, characterized in that: The kit comprises the primer set according to claim 1.
3. The kit according to claim 2, characterized in that The kit also includes dNTPs, Taq DNA polymerase and PCR reaction buffer.
4. Use of the primer set according to claim 1 or the kit according to claim 2 in rice germplasm resource breeding.
5. Use of the primer set according to claim 1 or the kit according to claim 2 in identifying rice germplasm resources.
6. The use according to claim 5, characterized in that: The application is to identify rice varieties resistant to glyphosate.
7. A method for identifying glyphosate-resistant rice varieties, characterized in that: The steps include: Extracting DNA from the rice to be tested; Using the DNA of the rice to be tested as a template, amplifying the DNA template by PCR using the primer set of claim 1 to obtain an amplified product; The amplified product is subjected to electrophoresis analysis. If there is a band at 605 bp, the rice to be tested is determined to be glyphosate-resistant rice.
8. The method according to claim 7, characterized in that Each 20 μL PCR amplification reaction system includes: 2 μL~3 μL DNA template, 10 μL 2×Taq PCR MasterMix, 0.2 μL~0.5 μL upstream primer, 0.2 μL~0.5 μL downstream primer, and ddH2O.