Rice herbicide-resistant os epsps mutant gene, protein and application thereof

Abstract

The present application relates to the technical field of biological genes, in particular to a rice herbicide-resistant OsEPSPS mutant gene, protein and application thereof. The rice herbicide-resistant OsEPSPS mutant gene provided by the present application is a wild-type rice OsEPSPS gene which is mutated at the 836th nucleotide, from T to C. Plants containing the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein of the present application have significant resistance to glyphosate herbicide, and can grow normally in the herbicide without being affected, while wild-type plants grow significantly inhibited under the same conditions until death. The mutant gene and the mutant protein have great market economic prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological genes, and particularly relates to a rice herbicide-resistant OsEPSPS mutant gene, protein and application thereof. BACKGROUND

[0002] Since the agricultural production, farmers have been plagued by weeds in the field. Weeds not only directly lead to the decrease of crop yield and quality, but also are the habitat of various diseases and pests, which indirectly affect the normal growth and development of crops and yield formation. Therefore, the control of paddy field weeds is extremely important, and chemical weeding is the main measure to control paddy field weeds. However, due to the similarity in taxonomy and physiology between weeds and rice, weeds are difficult to control, and improper use of herbicides can cause significant phytotoxicity to crops. Glyphosate is an organophosphorus herbicide with high weeding efficiency, wide range, and low environmental impact. It is the most widely used post-emergence herbicide since its commercialization in 1974. It has been applied in soybean, corn, cotton, rape and sugar beet fields. However, as a non-selective herbicide, glyphosate not only has high weeding efficiency, but also harms crops without selection. Therefore, cultivating and planting glyphosate-resistant crop varieties can not only reduce the burden of manual weeding, but also improve crop yield and facilitate mechanized production of crops.

[0003] The action pathway of glyphosate is mainly to interfere with the synthesis of shikimic acid to achieve the goal of weeding. OsEPSPS is a chloroplast enzyme that catalyzes the reaction of phosphoenolpyruvate (PEP) and 3-phosphoshikimic acid (S3P) to produce 5-enolpyruvyl-3-phosphate (EPSP). Glyphosate competes with phosphoenolpyruvate to form a stable OsEPSPS-S3P-glyphosate complex in plants, thereby destroying the activity of OsEPSPS. This hinders the accumulation of shikimic acid, which is necessary for the synthesis of aromatic amino acids, leading to the inhibition of the synthesis of hormones and secondary metabolites required for plant growth. These, in turn, cause plant growth and metabolism to be disordered, ultimately leading to plant death. Currently, some variants of OsEPSPS protein have been found in glyphosate-resistant elephant grass, but it is difficult to obtain a naturally mutated glyphosate-resistant OsEPSPS gene in crops. Therefore, the mutated OsEPSPS gene can only be introduced into crops by transgenic methods to obtain glyphosate-resistant crops.

[0004] Therefore, there is an urgent need to develop a gene that does not affect plant growth and development and has glyphosate resistance, which is of great significance to the production of crops such as rice. SUMMARY

[0005] In order to solve the above technical problems, the application provides a rice herbicide-resistant OsEPSPS mutant gene, a protein and an application thereof, a plant containing the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein has significant resistance to glyphosate herbicide and can grow normally in the herbicide without being affected, while a wild-type plant grows under the same conditions is significantly inhibited until death, and the mutant gene and the mutant protein have great market economic prospects.

[0006] To this end, the application provides the following technical solutions,

[0007] In a first aspect, the application provides, in optional embodiments, a rice herbicide-resistant OsEPSPS mutant gene, wherein the rice herbicide-resistant OsEPSPS mutant gene is a wild-type rice OsEPSPS gene with a mutation at the 836th nucleotide, from T to C.

[0008] Preferably, the nucleotide sequence of the rice herbicide-resistant OsEPSPS mutant gene is shown in SEQ ID NO. 1.

[0009] In a second aspect, the application provides, in optional embodiments, a rice herbicide-resistant OsEPSPS mutant protein, wherein the rice herbicide-resistant OsEPSPS mutant protein is a wild-type rice OsEPSPS protein with a mutation at the 279th amino acid, from methionine to threonine.

[0010] Preferably, the amino acid sequence of the rice herbicide-resistant OsEPSPS mutant protein is shown in SEQ ID NO. 2.

[0011] In the application, the case where methionine is mutated into other amino acids is also within the protection scope of the application, for example, the 279th amino acid is mutated from methionine to alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, glycine, cysteine, arginine, asparagine, glutamine, lysine, histidine or glutamic acid.

[0012] In a third aspect, the application provides, in optional embodiments, a kit, a recombinant vector or a recombinant cell containing the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or the above-mentioned rice herbicide-resistant OsEPSPS mutant protein.

[0013] In the application, the above-mentioned kit, recombinant vector or recombinant cell can be used to enhance the herbicide resistance of target plants.

[0014] In a fourth aspect, the present application provides, in optional embodiments, a nucleic acid molecule expressing the above-mentioned herbicide-resistant OsEPSPS mutant protein of rice.

[0015] In a fifth aspect, the present application provides, in optional embodiments, use of the above-mentioned herbicide-resistant OsEPSPS mutant gene of rice, the above-mentioned herbicide-resistant OsEPSPS mutant protein of rice, the above-mentioned kit, the recombinant vector, the recombinant cell, or the above-mentioned nucleic acid molecule in imparting herbicide resistance to a target plant.

[0016] In the present application, the herbicide-resistant OsEPSPS mutant gene of rice, the herbicide-resistant OsEPSPS mutant protein of rice, the kit, the recombinant vector, the recombinant cell, or the nucleic acid molecule can enhance the herbicide resistance of a plant, a plant tissue, or a plant cell.

[0017] Preferably, the herbicide-resistant OsEPSPS mutant gene of rice, the herbicide-resistant OsEPSPS mutant protein of rice, the kit, the recombinant vector, the recombinant cell, or the nucleic acid molecule is introduced into a target plant to impart herbicide resistance to the target plant; and / or the herbicide is glyphosate. The method for introducing the herbicide-resistant OsEPSPS mutant gene of rice into a target plant comprises chemical mutagenesis, transgenesis, gene editing, hybridization, or backcrossing.

[0018] Further, the method for screening a rice callus with herbicide resistance comprises the following steps:

[0019] (1) Sterilize the mature, non-molded rice seeds after shelling, carefully separate the embryo with a scalpel, and place it on a callus induction medium to generate secondary calli;

[0020] (2) Transfer the secondary calli to a new callus induction medium for pre-culture;

[0021] (3) Transfer the calli obtained in step (2) to a mutagenic medium for mutagenic culture for 7-14 days;

[0022] (4) Place the calli of step (3) on a pre-screening medium for 5-7 days;

[0023] (5) Transfer the calli of step (4) to a screening medium to obtain resistant calli;

[0024] (6) Transfer the resistant calli to a differentiation and regeneration medium to differentiate into seedlings;

[0025] (7) Transfer the seedlings of step (6) to a rooting medium to root.

[0026] (8) extracting DNA of the seedling of step (7), amplifying the OsEPSPS gene, sequencing and comparing with the wild type OsEPSPS gene, testing whether the rice contains the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or whether the rice expresses the above-mentioned rice herbicide-resistant OsEPSPS mutant protein, if containing or expressing, the rice callus has herbicide resistance.

[0027] In a sixth aspect, the present application provides, in optional embodiments, a rice plant that is resistant to herbicides, wherein the rice plant expresses the above-mentioned rice OsEPSPS mutant gene.

[0028] In a seventh aspect, the present application provides, in optional embodiments, use of the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or the above-mentioned rice herbicide-resistant OsEPSPS mutant protein in plant breeding.

[0029] In an eighth aspect, the present application provides, in optional embodiments, use of the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or the above-mentioned rice herbicide-resistant OsEPSPS mutant protein in breeding transgenic or non-transgenic plants.

[0030] The nucleotide sequence shown in SEQ ID NO. 1 is shown as follows:

[0031] ATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGGCGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCGCGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGTGGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGATCGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAGTCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCTGTGGTGGCAAGTTTCCTGTTGAGAAGGATGcGAAAGAGGAAGTGCAACTCTTCTTGGGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAACGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATGATCACAGGATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA.

[0032] The amino acid sequence represented by SEQ ID NO. 2 is as follows:

[0033] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRA RGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKALGLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAAPLALGDVEIEIIDKLISIPYVETTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKKHLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0034] The present application has one or more of the following advantages over the prior art:

[0035] 1. Plants containing the rice herbicide-resistant OsEPSPS mutant gene or rice herbicide-resistant OsEPSPS mutant protein of the present application have significant resistance to glyphosate herbicide and can grow normally in the herbicide with little effect, while wild-type plants grow significantly inhibited under the same conditions until death, and the mutant gene and mutant protein have great market economic prospects. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Photos of new resistant calli and differentiated seedlings in Example 1 of the present application;

[0037] Figure 2 Comparison chart of sequencing results near the mutation site of the mutant rice and wild-type rice in Example 2 of the present application;

[0038] Figure 3Comparison of growth of wild type rice and rice containing rice herbicide-resistant OsEPSPS mutant gene in Example 3 of the present application after spraying of glyphosate herbicide at a field recommended concentration for 30 days, wherein the left side is wild type rice and the right side is rice containing rice herbicide-resistant OsEPSPS mutant gene;

[0039] Figure 4 Comparison of gene sequencing results of wild type rice OsEPSPS gene at nucleotide position 836 of the gene where T is mutated to G and T is mutated to A in Example 4 of the present application;

[0040] Figure 5 Comparison of growth of wild type rice and rice containing mutant gene at nucleotide position 836 of wild type rice OsEPSPS gene where T is mutated to C, T is mutated to G and T is mutated to A after spraying of glyphosate herbicide at a field recommended concentration for 7 days in Example 4 of the present application. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in details below in combination with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0042] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0043] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0044] Chemical mutagenesis breeding is a rapidly developing crop breeding technology, which has the characteristics of convenient use, strong specificity and stable inheritance of mutagenic offspring. Common chemical mutagens can be divided into three categories: the first category is direct mutagenesis of DNA structure, such as alkylating agents and sodium nitrite; the second category is base analog mutagens, the commonly used base analogs are thymine analogs such as 5-bromouracil (5-BU) and 5-bromodeoxyuridine (BU-dR) and adenine analog 5-amino purine (2-AP); the third category is mutagens that induce frameshift mutations, such as acridines and antibiotics. EMS belongs to one of the alkylating agents and is a commonly used chemical mutagen. Its mechanism of action is mainly to alkylate guanine during DNA replication, which makes it pair with thymine, leading to base substitution, mainly causing G / C→A / T changes. Sodium azide is a chemical mutagen that can efficiently produce point mutations. It can easily penetrate the cell membrane into the cell and affect the normal synthesis of DNA in the form of base substitution, leading to the production of point mutations. Sodium azide has a high mutagenesis rate and is safe, and has been applied to mutagenic breeding of crops such as rice and barley. Studies have shown that sodium azide mainly causes A / T→G / C changes at the molecular level. Given that most of the current glyphosate-resistant plants are produced by base mutations in the EPSPS gene, selecting the appropriate chemical mutagen, and screening for glyphosate-resistant EPSPS mutant genes for directed mutagenesis provides an effective method for creating non-transgenic or non-genetically edited glyphosate-resistant plants.

[0045] Example 1

[0046] The present embodiment provides a method for preparing a rice herbicide-resistant OsEPSPS mutant, comprising the following steps:

[0047] (1) Select mature, healthy and dry rice seeds, peel them by hand, then disinfect them with a 70% ethanol solution for 2 minutes, and then treat them with a 50% sodium hypochlorite solution containing 1 drop of Tween 20 for 40 minutes, with intermittent shaking during the treatment. After the treatment, wash the seeds with water three times to remove the sodium hypochlorite. Dry the seeds on sterile filter paper and carefully separate the seed embryos with a scalpel. Uniformly distribute the separated seed embryos on callus induction medium and place them in a light-free incubator at an ambient temperature of 30°C for 10-12 days to obtain new callus.

[0048] (2) Use the phosphate buffer solution with pH 7.0, 0.2 mol / L to prepare different NaN3 solutions, 1.0 mmol / L, 2.0 mmol / L, 3.0 mmol / L, 4.0 mmol / L and 5.0 mmol / L, respectively, and 0.2 mol / L phosphate buffer solution as the control group, respectively, into the liquid induction medium, to prepare the control group medium and NaN3 liquid medium with different concentrations. The subcultured rice callus was inoculated in the NaN3 liquid medium with different concentrations and the control group medium, and after shaking for 3 hours at 120 r / min, it was washed with sterile water for 3-4 times, and the water was absorbed with sterile filter paper. Then it was inoculated into the solid induction medium for culture. The survival rate was counted at the 14th day (callus survival rate = (the number of surviving callus pieces / the number of inoculated callus pieces) x 100%), so as to determine the lethal concentration and the semi-lethal concentration of NaN3 mutagenic callus. The results are shown in Table 1.

[0049] Table 1 Survival rate of mutagenic callus

[0050]

[0051] As can be seen from Table 1, with the increase of the concentration of NaN3, the survival rate of callus decreases. When the concentration of NaN3 is 3.0 mmol / L and 5.0 mmol / L, the survival rate of callus after mutagenic treatment for 14 days is 48% and 5%, respectively, indicating that 3.0 mmol / L and 5.0 mmol / L are the semi-lethal concentration and the lethal concentration of NaN3 mutagenic callus.

[0052] The callus with the concentration of 3.0 mmol / L of NaN3 was continuously cultured, and the survival rate of callus was only 10% at the 21st day of mutagenic treatment. Therefore, the concentration of 3.0 mmol / L of NaN3 treated for 14 days was selected as the best mutagenic concentration of rice callus.

[0053] (3) About 20,000 induced rice embryogenic callus particles were selected and inoculated in the mutagenic medium containing 3.0 mmol / L NaN3. After growing for 14 days, about 10,000 calli survived, and were transferred to the screening medium containing 5 mM glyphosate for culture for 28-42 days for herbicide screening. Only 114 calli survived and grew new resistant callus. They were transferred to the differentiation medium, and only 1 plantlet was differentiated, and the results are shown in Table 2. Figure 1 .

[0054] Example 2

[0055] This example provides a mutation site analysis of the rice herbicide-resistant OsEPSPS mutant of Example 1:

[0056] The leaf of the mutant plant with glyphosate resistance obtained in Example 1 was selected, and the genomic DNA was extracted. The primer pair 5'-ATGGCGGCGACCATGGCGTC-3' and 5'-CATTTCCAGGAGATCTGTCA-3' was used to amplify and sequence the OsEPSPS, and the results are shown in Table 1. Figure 2 .

[0057] By Figure 2 It can be seen that, compared with the wild type Nipponbare rice OsEPSPS gene, a base mutation is found at the 836th position, which is mutated from T to C, thereby causing the amino acid at the 279th position of the OsEPSPS protein to be mutated from methionine to threonine. That is, the nucleotide sequence of the rice herbicide-resistant OsEPSPS mutant gene is shown in SEQ ID NO. 1, and the amino acid sequence of the rice herbicide-resistant OsEPSPS mutant protein encoded thereby is shown in SEQ ID NO. 2.

[0058] Example 3

[0059] This example provides a resistance analysis of the rice herbicide-resistant OsEPSPS mutant of Example 1:

[0060] The mutant plant with glyphosate resistance obtained in Example 1 was transplanted to the field, and after two generations of homozygous breeding, a suitable amount of seeds (containing the rice herbicide-resistant OsEPSPS mutant gene) were obtained.

[0061] A suitable amount of seeds and wild type Nipponbare rice seeds were soaked at 28°C for 1-2 days, and then sowed in the field after germination, and transplanted. When it grew to the tillering stage, 10 mmol / L glyphosate herbicide (recommended concentration in the field) was sprayed, and after 30 days of spraying, the growth of the rice was observed, and the results are shown in Table 2. Figure 3 .

[0062] By Figure 3 It can be seen that: after 30 days of spraying, the rice containing the rice herbicide-resistant OsEPSPS mutant gene can still develop normally, while the wild type rice dies completely after 10 days of spraying 10 mmol / L glyphosate herbicide, therefore, the rice containing the rice herbicide-resistant OsEPSPS mutant gene of the present application has excellent glyphosate resistance.

[0063] Example 4

[0064] This example provides a glyphosate herbicide resistance experiment of a rice containing a wild type rice OsEPSPS gene with a T to A mutation at the 836th position in the nucleotide sequence and a T to G mutation:

[0065] The precision editing tool is used to guide editing to make directional mutation. According to the mutation of the 836th base, the corresponding pegRNA sequence is designed,

[0066] PEGRNA1 for realizing T to G:

[0067] CTCCATTCCTTACGTTGAAAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATCTCAATGTCCTTTCAACGTAAGGCCTTAAACTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0068] PEGRNA2 for realizing T to A:

[0069] CTCCATTCCTTACGTTGAAAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATCTCAATGTCTTTTCAACGTAAGGCCTTAAACTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0070] The forward oligonucleotide chain and the reverse oligonucleotide chain complementary thereto of the pegRNA are synthesized respectively, and annealed to form a double strand. The guide editing pHUC411-PE2 skeleton fragment and the sgRNA Scoffold are connected by the Goldgate method (NEB Company), and are introduced into E. coli. The positive transformants are obtained by selecting the bacterial plaque with kanamycin resistance and without spectinomycin resistance. After sequencing verification, the positive plasmid is extracted, and a guide editing vector plasmid for plant OsEPSPS gene is constructed, which are named as pHUC411-PE2-OsEPSPS1 and pHUC411-PE2-OsEPSPS2 respectively, and are introduced into EHA105 Agrobacterium. The above Agrobacterium is used for genetic transformation of rice callus, and 37 regenerated plants of pHUC411-PE2-OsEPSPS1 are obtained, of which 9 are T-to-G homozygous mutants, 13 are heterozygous mutants, and 15 are non-mutant plants; 24 regenerated plants of pHUC411-PE2-OsEPSPS2 are obtained, of which 4 are T-to-A homozygous mutants, 15 are heterozygous mutants, and 5 are non-mutant plants; the sequencing results of the mutation types are shown in Table 1. Figure 4 .

[0071] The homozygous mutant seeds of T-to-G and T-to-A were collected, and the homozygous mutant seeds of T-to-C obtained in Example 1 and wild-type Nipponbare rice seeds were used as controls. After being soaked at 28°C for 1-2 days, the seeds were placed on moist filter paper to germinate. 100 seedlings of each of the three mutant rice and wild-type rice with the same growth and size at the 3-4 leaf stage were randomly selected, 5-8 seedlings were placed in one pot, and three replicates were set for each material. The prepared 10 mM glyphosate herbicide solution was sprayed. After 7 days, it was found that the wild-type Nipponbare rice, the rice with T-to-G mutation in the OsEPSPS gene and the rice with T-to-A mutation in the OsEPSPS gene all began to wither and die, while the leaves of the rice with T-to-C mutation in the OsEPSPS gene almost grew normally. The results are shown in Table 1. Figure 5 .

[0072] Therefore, it is shown that when the base at position 836 of the nucleotide sequence of the OsEPSPS gene is mutated from T to C, the rice can be resistant to glyphosate herbicide, and the mutant genes in which the base at position 836 of the nucleotide sequence of the OsEPSPS gene is mutated from T to A and from T to G cannot make the rice resistant to glyphosate herbicide.

[0073] Although the principles of the present application have been described in detail with reference to the preferred embodiments thereof, it is understood that the above-described embodiments are merely illustrative of the present application and are not intended to limit the scope of the present application. The details in the embodiments do not constitute a limitation on the scope of the present application. Any equivalent changes, simple replacements and the like based on the technical solutions of the present application, which do not depart from the spirit and scope of the present application, are all within the scope of protection of the present application.

Claims

1. A herbicide-resistant OsEPSPS mutant gene of rice, characterized in that, The rice herbicide-resistant OsEPSPS mutant gene is a wild-type rice OsEPSPS gene with a mutation at the 836th nucleotide, from T to C, and the nucleotide sequence of the rice herbicide-resistant OsEPSPS mutant gene is shown as SEQ ID NO.

1.

2. A herbicide resistant OsEPSPS mutant protein of rice, characterized in that, The rice herbicide-resistant OsEPSPS mutant protein is a wild-type rice OsEPSPS protein with a mutation at the 279th amino acid, from methionine to threonine, and the amino acid sequence of the rice herbicide-resistant OsEPSPS mutant protein is shown as SEQ ID NO.

2.

3. A kit or a recombinant vector comprising the rice herbicide-resistant OsEPSPS mutant gene of claim 1 or the rice herbicide-resistant OsEPSPS mutant protein of claim 2.

4. A nucleic acid molecule, characterized in that, expressing the rice herbicide-resistant OsEPSPS mutant protein of claim 2.

5. Use of the rice herbicide-resistant OsEPSPS mutant gene of claim 1, the recombinant vector of claim 3, or the nucleic acid molecule of claim 4 in cultivating a herbicide-resistant plant; wherein The cultivating method is: introducing the rice herbicide-resistant OsEPSPS mutant gene, the recombinant vector, or the nucleic acid molecule into a target plant, so that the target plant has herbicide resistance, the plant is rice, and the herbicide is glyphosate.

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