Method for screening herbicide-resistant rice by using molecular marker

Abstract

The invention relates to the technical field of herbicide resistance, in particular to a method for screening herbicide-resistant rice by using molecular markers, which comprises the following steps: S1, screening herbicide-resistant mutants, and extracting genome DNA of rice seedling leaves by using a CTAB (Cetyltrimethyl Ammonium Bromide) method; s2, specific primers are designed for ALS gene mutation sites, and flanking sequences of the mutation sites are covered. And S3, amplifying the target fragment by using PrimeSTAR Max enzyme, and verifying the product through agarose gel electrophoresis. And S4, carrying out DNA sequencing on the PCR product, and comparing ALS gene sequences to detect SNP locus mutation. And S5, according to the existing combination of the mutation sites Gln547Leu, Glu549Lys and Glu604Asp, judging the resistance of the plant to bispyribac-sodium and quinclorac. And S6, spraying mixed herbicides bispyribac-sodium and quinclorac to the mutant, and confirming the resistance through the survival rate and phenotype. The method can be directly adapted to a rice direct seeding cultivation system for non-transgenic resistant rice varieties, the weed competition pressure is remarkably reduced through directional weeding in the seedling stage, and an innovative solution is provided for solving the problem of weed damage in the direct seeding rice field.

Description

Technical Field

[0001] The present invention relates to the technical field of herbicide resistance, and in particular to a method for screening herbicide-resistant rice by utilizing molecular markers. Background Art

[0002] Rice is a staple grain crop in my country. There is a clear trend toward simplified rice cultivation methods. Direct-seeding rice is labor-efficient and is being adopted on an increasingly large scale. Compared to traditional transplanting, weed seeds in direct-seeded rice fields grow synchronously with rice seeds. Weeds grow quickly, are diverse, and grow densely, making them prone to weedy rice. Weed control is a key constraint to direct-seeding rice production. Breeding herbicide-resistant crops facilitates weed control in fields. Currently, genetically modified herbicide-resistant crops are widely produced and used, primarily for feed and oilseed crops such as corn, cotton, rapeseed, and soybeans. However, genetically modified rice has not yet been approved for commercial use in my country. Therefore, the development of non-genetically modified herbicide-resistant rice is essential. Successful non-genetically modified herbicide-resistant crops have been developed internationally, such as triazine-resistant rapeseed and imidazolinone-resistant corn, which were successfully bred in the late 20th century. The herbicides to which these non-genetically modified herbicide-resistant crops are resistant include imidazolinones, cyclohexenones, sulfonylureas, triazines, organophosphates, and hormones.

[0003] Acetolactate synthase (ALS) is a key enzyme in the synthesis of branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine. It is the target of numerous herbicides, including imidazolinones (IMIs), sulfonylureas (SUs), pyrimidinylthiobenzoates (PTBs), triazolopyrimidines (TPs), and sulfonylamino-carbonyltriazolinones (SCTs). ALS inhibitors bind to ALS in plants to form complexes, blocking substrate access to the enzyme's active site and inhibiting ALS activity. This hinders BCAA synthesis, disrupts normal plant cell growth, and leads to plant death. Mutations in certain sites of the ALS gene can weaken the binding of ALS to herbicides, leading to herbicide resistance. At present, research on resistant weeds targeting ALS has found that ALS mutations mainly occur at 8 amino acid sites; in Arabidopsis, studies have found that amino acid substitutions at more than 20 sites in the ALS protein can produce herbicide resistance. Several herbicide-resistant ALS amino acid mutation sites have been publicly reported in rice. In 1993, the Louisiana State University Agricultural Center screened the imidazolinone herbicide-resistant rice germplasm AS3510 through EMS (ethyl methanesulfonate) mutagenesis. Its ALS amino acid mutation is Gly628 Glu (amino acid 628 mutated from glycine to glutamic acid), and commercial varieties 121CL and 141CL were bred using AS3510. The high-yield variety Cypress was induced with EMS, and the imidazolinone herbicide-resistant germplasm PWC16 was screened, whose ALS amino acid mutated to Ser 627 Asn, commercial varieties CL161 and 141CL were bred using PWC16. Argentinian scholars also screened for imidazolinone-resistant materials using EMS to induce mutagenesis of the local rice variety IRGA417, and bred the imidazolinone-resistant variety PUTAINTACL, whose ALS amino acid mutation is Ala 96 In recent years, many research institutions in my country have screened ALS mutant herbicide-resistant materials. Shenzhen Xingwang Biological Seed Co., Ltd. used EMS to induce mutations in Huanghuazhan and Huangsizhan, and screened them with imidazolinone herbicides to obtain three resistant mutants (Tyr 548 Met / Cys、Ala 96 Val / Thr、Ser 627 Zhao Bingran et al. screened EMS-induced 31 with imidazolyl acetylcholine and obtained four resistant mutants, with the ALS amino acid mutation being Ala 179 Val, Ser 627 Asn、Gly 628 Glu, Val 643 Jiangsu Academy of Agricultural Sciences used imazapyr to screen multiple japonica and indica rice varieties induced by EMS and obtained multiple herbicide-resistant mutants. ALS amino acid mutations included Gly 136 Thr、Pro 171 His, Ala 179 Val, Ser 627 Asn、Gly 628 Glu et al. Wang Fangquan et al. screened a material resistant to imidazolinone herbicides from more than 7,000 rice germplasm resources, and the ALS amino acid mutation was Ser 627 Bi Junguo et al. screened 30,000 rice germplasms with imidazolin and also obtained one ALS amino acid mutation to Ser. 627 Asn-resistant materials. As can be seen from the above, previous screening of herbicide-resistant rice materials has mainly focused on resistance to imidazolinone herbicides. If new rice materials resistant to the herbicides bispyribac-sodium and quinclorac-sodium could be identified through mutagenesis, creating new herbicide-resistant varieties would be of great significance for practical production, but no relevant reports have been reported so far. Summary of the Invention

[0004] The present invention aims to provide a method for screening herbicide-resistant rice using molecular markers. Nipponbare seeds are soaked in an EMS (ethyl methanesulfonate) solution, and then M1 rice seedlings are sprayed with a mixture of 0.125 g / L of bispyribac-sodium and 0.625 ml / L of quinclorac to analyze the ALS (Acetolactate synthase) gene sequence in the herbicide-resistant rice.It was found that 9 SNPs were found in the nucleotide sequence of ALS in 21 herbicide-resistant mutants, ranging from 5′(1603)--ATGGTGTTGAACAACCAACATTTGGGTATGGTGGTGCAATGGGAGGATAGGTTTTACAAGGCGAATAGGGCGCATACATACTTGGGCAACCCGGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAGGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAA GTCCGTGCCGCCATCAAGAAGATGCTCGAGACTCCAGGGCCATACTTGTTGGATATCATCGTCCCGCACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCA--3′ (1890) mutated to 5′(1603)--ATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGCTATGGAAGGATAGGTTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAA TGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGATACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCA--3′(1890), the amino acid has a missense mutation, and its sequence is changed from N(535)--M VLNNQHLGMVVQWEDRFYKANRAHTYLGNPECESEIYPDFVTIAKGFNIPAVRVTKKSEVRAAIKKMLETPGPYLLDIIVPHQEHVLPMIPSGGA--C(630) mutates to N(535)- -MVLNNQHLGMVVLWKDRFYKANRAHTYLGNPECESEIYPDFVTIAKGFNIPAVRVTKKSEVRAAIKKMLDTPGPYLLDIIVPHQEHVLPMIPSGGA--C(630), specifically: Gln. 547 Leu、Glu 549 Lys and Glu 604 Asp, molecular docking simulations showed that the binding of the herbicides bispyribac-sodium and quinclorac to the ALS gene was altered.

[0005] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0006] A method for screening herbicide-resistant rice using molecular markers comprises the following steps:

[0007] S1: Rice seeds were mutagenized with EMS solution to screen for herbicide-resistant mutants. Genomic DNA from rice seedling leaves or embryonic tissues was extracted using the CTAB method and the concentration was determined.

[0008] S2: Design specific primers targeting the ALS gene mutation site, covering the flanking sequences of the mutation site. Specific steps: Search the ALS gene (Os02g30630) sequence from the NCBI website (https: / / www.ncbi.nlm.nih.gov) and design primers using Primer 3 Plus (https: / / www.primer3plus.com).

[0009] S3: The target fragment was amplified using PrimeSTAR Max enzyme, and the product (586 bp) was verified by agarose gel electrophoresis.

[0010] S4: DNA sequencing of the PCR products was performed and the ALS gene sequence was compared to detect base mutations. The nucleotide sequence was mutated from: 5′(1603)--ATGGTGTTGAACAACCAA CATTTGGGTATGGTGGTGCAATGGGAGGATAGGTTTTACAAGGCGAATAGGGCGCATACATACTTGGGCAACCCGGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAGGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGAGACTCCAGGGCCATACTTGTTGGATATCATCGTCCCGCACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCA--3′(1890) to 5′(1603)--ATGGTGTTGAACAACCAA CATTTGGGTATGGTTGTGCTATGGAAGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGA AGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGATACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCA--3'(1890), its amino acid sequence is: N(535)--MVLNNQHLGMV VQWEDRFYKANRAHTYLGNPECESEIYPDFVTIAKGFNIPAVRVT KKSEVRAAIKKMLETPGPYLLDIIVPHQEHVLPMIPSGGA--C(630) mutates to N(535)--MVLNNQHLGMVVLWKDRFYKANRAHTYLGNPE CESEIYPDFVTIAKGFNIPAVRVTKKSEVRAAIKKMLDTPGPYLLDI IVPHQEHVLPMIPSGGA--C(630).

[0011] S5: Determine the resistance level of the plant to bispyribac-sodium and quinclorac based on the presence combination of the mutation sites Gln547Leu, Glu549Lys, and Glu604Asp.

[0012] S6: The mutants were sprayed with a mixed herbicide (0.125 g / L bispyribac-sodium + 0.625 g / L quinclorac), and resistance was confirmed by survival rate and phenotype.

[0013] Furthermore, the step S1 specifically includes the following sub-steps:

[0014] S1.1: Soak dried Nipponbare seeds in tap water for 2 hours, then soak in 0.5% (w / v) EMS solution at room temperature for 14 hours. Neutralize with sodium thiosulfate at a final concentration of 25 g / L for 15 minutes, rinse with tap water for 3 hours, and then place in a greenhouse to air-dry.

[0015] S1.2: Germinate the obtained seeds in a germination box at 28°C for 36-48 hours before sowing;

[0016] S1.3: Use conventional moist seedling raising and conventional single-plant planting. When the M0 plants mature, harvest one ear from each plant, do not thresh, and dry them in the sun to obtain M1 generation seeds.

[0017] S1.4: Sow M1 seeds of 3800 lines and spray the M1 seedlings with herbicides five times;

[0018] S1.5: Extract DNA from rice leaves using the CTAB method.

[0019] Furthermore, the step S3 specifically includes: using the M1 herbicide-resistant mutant, sensitive plants and Nipponbare rice (NPB) genomic DNA as templates, using TaKaRa MaxDNA Polymerase amplifies the ALS gene.

[0020] Furthermore, the step S4 specifically includes: PCR reaction system: Prepare 10 μL of MaxPremix, 1.0 μL each of 10 μmol / L forward primer ALS-OF and reverse primer ALS-OR, and 1.0 μL of genomic DNA, then add dd HO to 20 μL. PCR amplification program: 98°C / 10 s, 59°C / 20 s, 72°C / 30 s, and 72°C / 5 min for 30 cycles.

[0021] Furthermore, the forward primer ALS-OF sequence is shown in SEQ ID NO. 1 (5'-AGGTGAGGCAATCATCGCTAC-3'), and the reverse primer ALS-OR sequence is shown in SEQ ID NO. 2 (5'-ATGGGTCTATTCAGGTCAAACA-3').

[0022] Furthermore, the resistance of the plant to the herbicides bispyribac-sodium and quinclorac was determined by the mutated nucleotide sequence as shown in SEQ ID NO.3, the amino acid sequence as shown in SEQ ID NO.4, and the mutation sites Gln547Leu, Glu549Lys, and Glu604Asp.

[0023] Beneficial effects of the present invention:

[0024] The present invention uses a gradient screening system constructed by mixing bispyribac and quinclorac, which significantly increases the selection pressure on resistant mutants through the synergistic effect of the two herbicides in their mechanism of action. As an acetolactate synthase inhibitor, bispyribac causes metabolic disorders by blocking the branched-chain amino acid synthesis pathway, while quinclorac, as a hormone herbicide, interferes with the normal growth and development of plants. The synergistic effect of the two causes sensitive plants to present a complex damage phenotype in the short term, while mutants with true genetic resistance can maintain a normal physiological state. Multiple spraying treatments simulate the actual application scenario of herbicides in the field, effectively eliminating false positive interference caused by environmental adaptability, and ensuring that the resistance traits obtained through screening are genetically stable.

[0025] The present invention discovered for the first time the synergistic mutation effects of three key amino acid sites in the ALS gene. These mutation sites are located in the key functional domains of the ALS protein, and significantly affect the binding ability of the herbicide molecule to the target protein by changing the local spatial conformation and charge distribution. Among them, mutations in the α-helix region lead to the reconstruction of the electrostatic potential on the protein surface, weakening the hydrogen bonding between the herbicide polar groups and the amino acid residues; while mutations in the β-pleated region reduce the embedding stability of the herbicide aromatic ring structure by adjusting the spatial arrangement of the hydrophobic core. Molecular docking simulations confirmed that the geometry of the binding pocket between the mutated ALS protein and the herbicide has changed significantly, and the solvent-accessible surface area of ​​the binding site has increased, making it difficult for the herbicide molecule to form a stable binding conformation. This multi-dimensional structural change works together to reduce the herbicide inhibition efficiency from the aspects of steric hindrance and energy barriers, ultimately giving the rice plant a stable resistance phenotype.

[0026] The molecular marker-assisted screening system established by the present invention achieves high precision and high efficiency in genotyping identification by designing specific primers for mutation sites and optimizing the detection process. The system makes full use of the sequence polymorphism characteristics caused by mutations, adopts gradient annealing and high-fidelity amplification technology to ensure the selective amplification of target fragments and effectively distinguish between heterozygous and homozygous mutants. Combined with simplified nucleic acid extraction methods and automated electrophoresis detection, the genotyping cycle is greatly shortened, making large-scale population screening possible. This genotype-phenotype linkage screening strategy not only overcomes the defect that traditional phenotypic screening is greatly interfered with by environmental factors, but also can quickly lock the target plants in the seedling stage, saving valuable time for subsequent breeding work.

[0027] This invention has significant application value for non-transgenic resistant germplasm. Its resistance, derived from natural mutations in endogenous genes, fully complies with existing biosafety regulations. Field trials have shown that this germplasm maintains the excellent agronomic traits of its parent variety while exhibiting stable resistance to target herbicides and capable of normal growth and development at conventional application rates. This makes it directly adaptable to direct-seeded rice cultivation systems, significantly reducing weed competition through targeted weed control at the seedling stage, providing an innovative solution to the challenge of weed infestation in direct-seeded rice fields.

[0028] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. 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 creative work.

[0030] Figure 1 Schematic diagram of the phenotypes of 21 herbicide-resistant plants; Note: A is a herbicide-sensitive plant after EMS mutagenesis; BV is a herbicide-resistant plant after EMS mutagenesis;

[0031] Figure 2 This is a schematic diagram of the base sequence of herbicide-resistant plants; Note: NPB is Nipponbare; EMS2960 is a herbicide-sensitive plant; triangular arrows indicate bases with missense mutations. SNP: single nucleotide polymorphism;

[0032] Figure 3 This is a schematic diagram of the amino acid sequence of herbicide-resistant plants; Note: NPB is Nipponbare; EMS2960 is a herbicide-sensitive plant;

[0033] Figure 4 The predicted tertiary structure of ALS protein. Note: A is the structure of ALS protein in Nipponbare plants, with the yellow positions indicating the changed amino acid sites; B is the structure of ALS protein in resistant plants, with the red positions indicating the changed amino acid sites; C is the overlapping structure of ALS protein in Nipponbare and resistant plants.

[0034] Figure 5 It is the docking diagram of ALS protein and herbicide small molecule ligand; Note: Figure A is the docking diagram of ALS and dichloroquine in rice Nipponbare; Figure B is the docking diagram of ALS and dichloroquine in resistant plants; C is the docking diagram of ALS and bispyribac-sodium in rice Nipponbare; D is the docking diagram of ALS and bispyribac-sodium in resistant plants; the box shows the binding pocket and its amino acid residues, and the blue ones are the residues connected with hydrogen bonds. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0036] Example 1

[0037] The present embodiment provides a method for screening herbicide-resistant rice using molecular markers, by accurately detecting the mutant nucleotide sequence 5′(1603)--ATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGCTATGGA AGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGATACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCA--3′(1890) and the amino acid sequence N(535)--MVLNNQHLGMVVLWKDRFYKANRAHTYLGNPECESEIY PDFVTIAKGFNIPAVRVTKKSEVRAAIKKMLDTPGPYLLDIIVPHQE HVLPMIPSGGA--C(630) and three key mutation sites (Gln547Leu, Glu549Lys, Glu604Asp) were used to achieve rapid identification of rice germplasm resistant to bispyribac-sodium and quinclorac-sodium, including the following steps:

[0038] S1: Collect leaves from the rice seedlings to be tested, and extract genomic DNA using a modified CTAB method: The sample is quick-frozen in liquid nitrogen and then ground into a powder. Add CTAB lysis buffer (2% CTAB, 100mM Tris-HCl pH 8.0, 20mM EDTA, 1.4M NaCl) preheated at 65°C containing β-mercaptoethanol. After sufficient lysis, extract with chloroform-isoamyl alcohol to remove proteins and polysaccharides. After isopropanol precipitation, DNA is washed and purified with 75% ethanol. Finally, it is dissolved in TE buffer and the concentration is measured to 50ng / μL to ensure DNA integrity to meet the requirements of subsequent amplification.

[0039] S2: Primers were designed based on the ALS gene (Os02g30630) sequence (Table 1);

[0040] Table 1 Primer sequences for detecting base variation of rice ALS gene using PCR technology

[0041]

[0042] S3: Using the genomic DNA of the M1 herbicide-resistant mutant, sensitive plants, and Nipponbare rice (NPB) as templates, TaKaRa The ALS gene was amplified using MaxDNA Polymerase. The amplification program was set as an initial denaturation at 98°C for 3 minutes, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 59°C for 20 seconds, and extension at 72°C for 30 seconds, with a final extension at 72°C for 5 minutes. The amplified product, 586 bp in length, was detected by 1.5% agarose gel electrophoresis and sequenced. Resistance levels were then determined based on the combination of the three mutant amino acid sites: Gln547Leu, Glu549Lys, and Glu604Asp.

[0043] S4: Through field validation, screened plants were sprayed with a mixture of 0.125g / L bispyribac-sodium and 0.625g / L quinclorac at the 3- to 4-leaf stage. Leaf yellowing index was observed after 7 days, and plant height inhibition rate was calculated after 21 days to ensure ≥95% consistency between molecular markers and phenotypic characteristics. This method shortens the traditional phenotypic screening cycle of 2-3 years to 3 weeks, with a testing throughput of 500 samples per day, providing efficient technical support for herbicide-resistant rice breeding.

[0044] Example 2

[0045] Materials and methods

[0046] Test materials

[0047] The rice material used in the experiment was the japonica rice variety Nipponbare.

[0048] Reagents and instruments

[0049] Bispyribac-sodium is a pyrimidine salicylic acid herbicide (produced by Dengfeng Jinbo Pesticide Chemical Co., Ltd.; active ingredient and content: 10% bispyribac-sodium, dosage form: suspension concentrate); dichloroquinoline is a hormone-type quinoline carboxylic acid herbicide (produced by Jiangsu Hormone Research Institute Co., Ltd.; active ingredient and content: 50% dichloroquinoline, dosage form: wettable powder).

[0050] Table 2 Test agents and dosages used

[0051]

[0052] EMS mutagenesis treatment

[0053] After soaking the dried seeds of Nipponbare in tap water for 2 hours, soak them in 0.5% (w / v) EMS solution at room temperature for 14 hours, add sodium thiosulfate with a final concentration of 25g / L for neutralization reaction for 15 minutes, rinse with tap water for 3 hours, and then place them in a greenhouse to dry. The obtained seeds were germinated in a germination box at 28°C for 36-48 hours, and then sown in Xiaoguoxi Village, Caoba Town, Mengzi City, Yunnan Province (103°37′67″ east longitude, 23°49′39″ north latitude, 1321.7m above sea level). Conventional moist seedling cultivation and conventional single-plant planting were adopted. When the M0 plants matured, one ear was harvested from each plant, without threshing, and dried to obtain M1 generation seeds.

[0054] Screening of herbicide-resistant mutants

[0055] The M1 generation of seeds from 3,800 lines were sown, and the M1 seedlings were sprayed with a herbicide mixture of quinclorac and bispyribac-sodium five times. The herbicide-resistant plants grew more normally than the sensitive plants, with near-normal plant height and greener leaves.

[0056] PCR Identification and Sequence Analysis of the ALS Gene of a Herbicide-Resistant Mutant

[0057] Primers were designed based on the ALS gene (Os02g30630) sequence (Table 1). The CTAB method was used to extract rice leaf DNA, and the genomic DNA of the M1 herbicide-resistant mutant, sensitive plants, and Nipponbare rice (NPB) was used as templates. MaxDNA Polymerase amplifies the ALS gene. PCR reaction system: MaxPremix (10 μL), 1.0 μL each of 10 μmol / L forward primer ALS-OF (5'-AGGTGAGGCAATCATCGCTAC-3') and reverse primer ALS-OR (5'-ATGGGTCTATTCAGGTCAAACA-3'), and 1.0 μL of genomic DNA were added to 20 μL with ddH2O. PCR amplification was performed using the following protocol: 98°C for 10 s, 59°C for 20 s, 72°C for 30 s, and 72°C for 5 min for 30 cycles. Five μL of PCR product was analyzed by 1% agarose gel electrophoresis. The PCR product was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. DNA sequences were analyzed using DNAStar-MegAlign software.

[0058] Mutant protein structure prediction

[0059] Potential small molecule binding pockets for the ALS enzyme were predicted using the website https: / / g6altair.sci.hokudai.ac.jp / g6 / service / pocasa / . The amino acid sequences of ALS from herbicide-resistant plants were submitted to SwissModel (https: / / swissmodel.expasy.org / interactive) for homology modeling. The tertiary structure of ALS was downloaded from the PDB protein database (https: / / www.rcsb.org / ). Docking analysis of the ALS protein and herbicide small molecule ligands was performed using AutoDockTools, and visualization was performed using PyMol.

[0060] Results and Analysis

[0061] Screening of herbicide-resistant rice mutants

[0062] Seven days after the EMS-induced Nipponbare M1 rice seedlings were sprayed with two herbicides at the 3-4 leaf stage, most of the rice seedlings lost their green leaves and turned yellow. Twenty-one days after the herbicide treatment, the leaves of the sensitive plants almost completely lost their green leaves, and some had died. The resistant plants were still able to grow normally ( Figure 1 After five sprayings with the mixed herbicide, 21 plants resistant to bispyribac-sodium and quinclorac were obtained.

[0063] Sequence Analysis of the ALS Gene in Herbicide-Resistant Rice Mutants

[0064] By analyzing and comparing the ALS gene CDS region sequences of sensitive plants and resistant mutants after Nipponbare and EMS mutagenesis, it was found that 21 resistant mutants had 12 base changes in the ALS coding region sequence, among which 9 base positions at 1635, 1640, 1645, 1665, 1695, 1740, 1812, 1815, and 1848 showed consistent differences compared with Nipponbare and sensitive plants ( Figure 2 Among these 9 SNPs, 6 bases were synonymous mutations and 3 bases were missense mutations, namely A 1640 changes to T, resulting in the mutation of amino acid 547 from glutamine to leucine; G 1645 changes to A, resulting in the mutation of amino acid 549 from glutamic acid to lysine; G 1812 changes to T, resulting in the mutation of amino acid 604 from glutamic acid to aspartic acid ( Figure 3 The bases at positions 547 and 549 are located in domain B (QWED) of the rice ALS gene.

[0065] Prediction and analysis of the tertiary structure of mutant ALS proteins

[0066] Modeling and structural analysis of the ALS protein revealed that in herbicide-resistant plants, the three amino acids produced by missense mutations are all located in the α-helical structure ( Figure 4 ), among which, the polarity of the amino acid at position 547 changed, from the polar uncharged amino acid glutamine (Gln, Q) to the non-polar uncharged amino acid leucine (Leu, L); the charge of the amino acid at position 549 changed, from the negatively charged polar amino acid glutamic acid (Glu, E) to the positively charged polar amino acid lysine (Lys, K); the amino acid mutation at position 604 did not change the amino acid charge, from the negatively charged glutamic acid (Glu, E) to the negatively charged aspartic acid (Asp, D) ( Figure 4 ).

[0067] By docking with herbicide small molecule ligands, it was found that compared with the binding pocket of ALS docked in Nipponbare, in the binding pocket of resistant plants, the number of amino acid residues within 5A after docking with quinclorac increased by 1, and the number of amino acid residues within 5A after docking with bispyribac-sodium decreased by 3. The amino acids after docking had undergone corresponding changes ( Figure 5 Therefore, there is an interaction between bispyribac and quinclorac and ALS, leading to changes in its function. In resistant plants, changes in these three amino acid sites may alter the spatial structure and electrochemical properties of the ALS protein, affecting the binding stability of the herbicides bispyribac and quinclorac with ALS, reducing the plant's sensitivity to the herbicides bispyribac and quinclorac, and thus conferring herbicide resistance.

[0068] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for screening herbicide-resistant rice using molecular markers, characterized in that: The following steps are involved: S1: Screening for herbicide-resistant mutants, extracting genomic DNA from rice seedling leaves using the CTAB method and measuring the concentration; S2: Design specific primers for the ALS gene mutation site, covering the flanking sequences of the mutation site; S3: Target fragments were amplified by PCR using PrimeSTAR Max enzyme, and the products were verified by agarose gel electrophoresis. S4: DNA sequencing of PCR products and comparison with ALS gene sequences to detect SNP site mutations; S5: Determine the resistance of the plant to bispyribac-sodium and quinclorac based on the presence of the combination of mutation sites Gln547Leu, Glu549Lys, and Glu604Asp; S6: The mutants were sprayed with a mixed herbicide and resistance was confirmed by survival rate and phenotype.

2. The method for screening herbicide-resistant rice using molecular markers according to claim 1, wherein: The step S1 specifically includes the following sub-steps: S1.1: Soak dried Nipponbare seeds in tap water for 2 hours, then soak in 0.5% (w / v) EMS solution at room temperature for 14 hours. Neutralize with sodium thiosulfate at a final concentration of 25 g / L for 15 minutes, rinse with tap water for 3 hours, and then place in a greenhouse to air-dry. S1.2: Germinate the obtained seeds in a germination box at 28°C for 36-48 hours before sowing; S1.3: Conventional wet seedling raising and single-plant transplanting were used. When the M0 plants matured, one ear was harvested from each plant, without threshing, and sun-dried to obtain M1 generation seeds. S1.4: Sow M1 seeds of 3800 lines and spray the M1 seedlings with herbicides five times; S1.5: Extract DNA from rice leaves using the CTAB method.

3. The method for screening herbicide-resistant rice using molecular markers according to claim 1, wherein: The step S3 specifically includes: using the M1 herbicide-resistant mutant, sensitive plants and Nipponbare rice genomic DNA as templates, using TaKaRa MaxDNA Polymerase amplifies the ALS gene.

4. The method for screening herbicide-resistant rice using molecular markers according to claim 1, wherein: The step S4 comprises: PCR reaction system: 2× MaxPremix 10 μL, 1.0 μL each of 10 μmol / L forward primer ALS-OF and reverse primer ALS-OR, 1.0 μL of genomic DNA, and dd H2O were added to 20 μL; PCR amplification program: 98°C / 10 s, 59°C / 20 s, 72°C / 30 s, 72°C / 5 min, 30 cycles.

5. The method for screening herbicide-resistant rice using molecular markers according to claim 4, wherein: The forward primer ALS-OF sequence is shown in SEQ ID NO.1, and the reverse primer ALS-OR sequence is shown in SEQ ID NO.

2.

6. The method for screening herbicide-resistant rice using molecular markers according to claim 1, wherein: The resistance of the plant to the herbicides bispyribac-sodium and quinclorac is determined by the mutated nucleotide sequence as shown in SEQ ID NO.3, the amino acid sequence as shown in SEQ ID NO.4, and the mutation sites Gln547Leu, Glu549Lys, and Glu604Asp.

Images