Rice herbicide-resistant OsEPSPS mutant gene, protein and application thereof

Abstract

The invention relates to the technical field of biological genes, in particular to a rice herbicide-resistant OsEPSPS mutant gene, a protein and application thereof. The rice herbicide-resistant OsEPSPS mutant gene provided by the invention is characterized in that the 836th site of the nucleotide sequence of a wild rice OsEPSPS gene is mutated, and T is mutated into C. A plant containing the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein has remarkable resistance to glyphosate herbicide and can normally grow in the herbicide almost without being influenced, and under the same condition, the growth of a wild plant is remarkably inhibited until the wild plant dies, so that the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein has a good application prospect. The mutant gene and the mutant protein have huge market economic prospects.

Description

Technical Field

[0001] The invention relates to the field of biological gene technology, and in particular to a rice herbicide-resistant OsEPSPS mutant gene, protein and application thereof. Background Art

[0002] Since the beginning of agricultural production, farmers have been troubled by weeds in the fields. Weeds not only directly lead to a decrease in crop yield and quality, but are also the source of a variety of pests and diseases, indirectly affecting the normal growth and development of crops and yield formation. Therefore, weed control in rice fields is extremely important, among which chemical weed control is the main measure to control weeds in rice fields. However, since weeds are very similar to rice in taxonomy and physiology, weeds are extremely difficult to control, and improper use of herbicides can cause serious phytotoxicity to crops. Glyphosate is an organophosphorus herbicide with the advantages of high weed control efficiency, wide range, insoluble in water, low soil residue, and relatively friendly environment. It is the post-emergence herbicide with the widest weed control spectrum. Since its commercialization in 1974, it has been quickly applied in soybean, corn, cotton, rapeseed and sugar beet fields. But at the same time, as a non-selective herbicide, glyphosate also indiscriminately harms crops while efficiently killing weeds. Therefore, cultivating and planting glyphosate-resistant crop varieties can not only reduce the burden of manual weeding, but also increase crop yields and facilitate mechanized crop production.

[0003] The main action pathway of glyphosate is to achieve the goal of weed control by interfering with shikimic acid synthesis. OsEPSPS is a chloroplast enzyme that catalyzes the reaction of phosphoenolpyruvate (PEP) and 3-phosphoshikimic acid (S3P) to produce 5-enol-3-phosphate (EPSP). Glyphosate competes with phosphoenolpyruvate to form a stable OsEPSPS-S3P-glyphosate complex with OsEPSPS in plants to destroy the activity of OsEPSPS. It hinders the accumulation of shikimate, which is necessary for the production of aromatic amino acids, resulting in the obstruction of plant protein synthesis. It inhibits the synthesis of hormones and secondary metabolites required for plant growth. These in turn cause plant growth and metabolic disorders, ultimately leading to plant death. At present, some variants of OsEPSPS proteins have been found in glyphosate-resistant goosegrass, but it is difficult to obtain naturally mutated glyphosate-resistant OsEPSPS genes in crops. The only way is to transfer the mutated OsEPSPS genes into crops through transgenic methods to obtain glyphosate-resistant crops.

[0004] Therefore, there is an urgent need to develop genes that do not affect plant growth and development and that make plants resistant to glyphosate, which is of great significance to the production of crops such as rice. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a rice herbicide-resistant OsEPSPS mutant gene, protein and application thereof. Plants comprising the rice herbicide-resistant OsEPSPS mutant gene or rice herbicide-resistant OsEPSPS mutant protein of the present invention have significant resistance to glyphosate herbicide and can grow normally in the herbicide with almost no influence, while the growth of wild-type plants under the same conditions is significantly inhibited until death. The mutant gene and mutant protein have huge market economic prospects.

[0006] To this end, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a rice herbicide-resistant OsEPSPS mutant gene in an optional embodiment, wherein the rice herbicide-resistant OsEPSPS mutant gene is a wild-type rice OsEPSPS gene with a mutation at position 836 in the nucleotide sequence, from T to C.

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

[0009] In a second aspect, the present invention provides a rice herbicide-resistant OsEPSPS mutant protein in an optional embodiment, wherein the rice herbicide-resistant OsEPSPS mutant protein is a wild-type rice OsEPSPS protein with a mutation at position 279 in the amino acid sequence, 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 present invention, the mutation of methionine to other amino acids is also within the protection scope of the present invention, for example, the 279th amino acid mutates from methionine to alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, glycine, cysteine, arginine, asparagine, glutamine, lysine, histidine or glutamic acid.

[0012] The third invention, in an optional embodiment, the present invention provides a kit, a recombinant vector or a recombinant cell comprising the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or the above-mentioned rice herbicide-resistant OsEPSPS mutant protein.

[0013] In the present invention, 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 invention provides, in an optional embodiment, a nucleic acid molecule for expressing the above-mentioned rice herbicide-resistant OsEPSPS mutant protein.

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

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

[0017] Preferably, the rice herbicide-resistant OsEPSPS mutant gene, rice herbicide-resistant OsEPSPS mutant protein, kit, recombinant vector, recombinant cell or nucleic acid molecule is introduced into a target plant to make the target plant resistant to herbicides; and / or the herbicide is glyphosate. Methods for introducing the rice herbicide-resistant OsEPSPS mutant gene into a target plant include: chemical mutagenesis, transgenic, gene editing, hybridization or backcrossing.

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

[0019] (1) After the mature, mold-free rice seeds are husked and sterilized, the embryos are carefully separated with a scalpel and placed on a callus induction medium to produce secondary callus;

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

[0021] (3) transferring the callus obtained in step (2) to a mutagenesis medium for mutagenesis culture for 7-14 days;

[0022] (4) placing the callus tissue from step (3) on a pre-screening medium and culturing for 5-7 days;

[0023] (5) transferring the callus tissue of step (4) to a screening medium to obtain resistant callus tissue;

[0024] (6) transferring the resistant callus to a differentiation and regeneration medium to differentiate into shoots;

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

[0026] (8) Extracting DNA from the seedlings of step (7), amplifying the OsEPSPS gene, sequencing it and comparing it with the wild-type OsEPSPS gene, testing whether the rice contains the above-mentioned rice herbicide-resistant OsEPSPS mutant gene, or testing whether the rice expresses the above-mentioned rice herbicide-resistant OsEPSPS mutant protein; if so, the rice callus has herbicide resistance.

[0027] In a sixth aspect, the present invention provides, in an optional embodiment, a herbicide-resistant rice, wherein the herbicide-resistant rice expresses the above-mentioned rice OsEPSPS mutant gene.

[0028] In a seventh aspect, the present invention provides, in an optional embodiment, a 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 invention provides, in an optional embodiment, a use of the above-mentioned rice herbicide-resistant OsEPSPS mutant gene or rice herbicide-resistant OsEPSPS mutant protein in cultivating transgenic or non-transgenic plants.

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

[0031] ATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGGCGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCGCGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGTGGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGATCGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAGTCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCTGTGGTGGCAAGTTTCCTGTTGAGAAGGATGcGAAAGAGGAAGTGCAACTCTTCTTGGGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAACGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATGATCACAGGATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA。

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

[0033] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRA RGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKALGLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQ LFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAAPLALGDVEIEIIDKLISIPYVE TTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKKHLKAVDVNMNK MPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0034] Compared with the prior art, the present invention has one of the following beneficial effects:

[0035] 1. Plants containing the rice herbicide-resistant OsEPSPS mutant gene or rice herbicide-resistant OsEPSPS mutant protein of the present invention have significant resistance to glyphosate herbicide and can grow normally in the presence of the herbicide with almost no influence, while the growth of wild-type plants is significantly inhibited until death under the same conditions. The mutant gene and mutant protein have great market economic prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The photos are of the new resistant callus and the seedlings differentiated therefrom in Example 1 of the present invention;

[0037] Figure 2 This is a comparison chart of sequencing results near the mutation site of mutant rice and wild-type rice in Example 2 of the present invention;

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

[0039] Figure 4 It is a comparison chart of the gene sequencing results of the mutation from T to G and from T to A at the nucleotide 836 of the wild-type rice OsEPSPS gene in Example 4 of the present invention;

[0040] Figure 5 The growth of rice containing the mutant gene in Example 4 of the present invention, wherein the nucleotide 836 of the wild-type rice OsEPSPS gene is mutated from T to C, from T to G, and from T to A, is compared with the wild-type rice after being sprayed with the glyphosate herbicide at the recommended field concentration for 7 days. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0044] Chemical mutagenesis is a rapidly developing crop breeding technology with the characteristics of easy use, strong specificity and easy stable inheritance of induced offspring. Commonly used chemical mutagens can be divided into three categories: the first category is mutagens that directly mutate DNA structure, such as alkylating agents and sodium nitrite; the second category is base analogue mutagens, currently commonly used base analogues include thymine analogues such as 5-bromouracil (5-BU) and 5-bromodeoxyuridine (BU-dR) and adenine analogue 5-aminopurine (2-AP); the third category is mutagens that induce frameshift mutations, such as acridines and antibiotics. EMS is a type of alkylating agent and is a widely used chemical mutagen. Its mechanism of action is mainly to alkylate guanine during DNA replication to make it pair with thymine, resulting in base substitution, mainly causing changes in G / C→A / T. Sodium azide is a chemical mutagen that can efficiently produce point mutations. It can easily penetrate the cell membrane and enter the cell, affecting the normal synthesis of DNA by base substitution, leading to the generation of point mutations. Azide has a high mutagenesis rate and is safe, and has been used in mutagenesis breeding of crops such as rice and barley. Studies have shown that sodium azide often causes changes from A / T to G / C at the molecular level. Given that most of the current glyphosate-resistant plants are produced by base mutations in the EPSPS gene, selecting appropriate chemical mutagens, directed mutagenesis, and screening for glyphosate-resistant EPSPS mutant genes provide an effective method for creating non-transgenic or non-gene-edited glyphosate-resistant plants.

[0045] Example 1

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

[0047] (1) Select mature, healthy, dry rice seeds, manually shell them, 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 them three times with water to remove the sodium hypochlorite. Dry the seeds on sterile filter paper, carefully separate the seed embryos with a scalpel, evenly distribute the separated seed embryos on the callus induction medium, and place them in a lightless incubator at an ambient temperature of 30°C for 10-12 days to obtain new callus tissue.

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

[0049] Table 1 Survival rate of induced callus

[0050]

[0051] It can be seen from Table 1 that as the concentration of NaN3 increases, the survival rate of callus tissue decreases. When the NaN3 concentration is 3.0mmol / L and 5.0mmol / L, the survival rate of callus tissue after 14 days of mutagenesis is 48% and 5%, respectively, indicating that 3.0mmol / L and 5.0mmol / L are the semi-lethal concentration and lethal concentration of NaN3-induced callus tissue.

[0052] The callus tissue was continued to be cultured at a NaN3 concentration of 3.0mmol / L. On the 21st day of the mutagenesis treatment, the survival rate of the callus tissue was only 10%. Therefore, a NaN3 concentration of 3.0mmol / L was selected for 14 days as the optimal mutagenesis concentration for rice callus tissue.

[0053] (3) About 20,000 induced rice embryonic callus particles were selected and inoculated on a mutagenesis medium containing 3.0 mmol / L NaN3. After 14 days of growth, about 10,000 callus tissues survived and were transferred to a screening medium containing 5 mM glyphosate for 28-42 days for herbicide screening. Only 114 callus survived and grew new resistant callus tissues. When they were transferred to a differentiation medium, only one plant differentiated into a seedling. The results are shown in Figure 1 .

[0054] Example 2

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

[0056] Leaves were selected from a glyphosate-resistant mutant plant obtained in Example 1, and genomic DNA was extracted. The primer pair 5'-ATGGCGGCGACCATGGCGTC-3' and 5'-CATTTCCAGGAGATCTGTCA-3' were used to amplify and sequence OsEPSPS. The results are shown in Figure 2 .

[0057] pass Figure 2 It can be seen that the sequencing results, compared with the wild-type Nipponbare rice OsEPSPS gene, found that a base mutation occurred at position 836, from T to C, which resulted in the amino acid mutation of the OsEPSPS protein at position 279 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 by it 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 glyphosate-resistant mutant plant obtained in Example 1 was transplanted into the field, and after two generations of homozygous breeding, an appropriate amount of seeds (containing the rice herbicide-resistant OsEPSPS mutant gene) were obtained.

[0061] Soak an appropriate amount of seeds and wild-type Nipponbare rice seeds at 28°C for 1-2 days, sow them in the field after germination, and transplant them. When they grow to the tillering stage, spray them with 10mmol / L glyphosate herbicide (recommended field concentration). After 30 days of spraying, observe the growth of rice. The results are shown in Figure 3 .

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

[0063] Example 4

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

[0065] Use precision editing tools to guide editing for directed mutagenesis, and design the corresponding pegRNA sequence based on the mutation at base 836.

[0066] PEGRNA1 that changes T to G:

[0067] CTCCATCCTTACGTTGAAAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATCTCAATGTCCTTTCAACGTAAGGCCTTAAACTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0068] PEGRNA2 that converts T to A:

[0069] CTCCATCCTTACGTTGAAAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATCTCAATGTCTTTTCAACGTAAGGCCTTAAACTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0070] The forward oligonucleotide chain of pegRNA and the reverse oligonucleotide chain complementary thereto were synthesized separately and annealed to form a double strand. The guide editing pHUC411-PE2 backbone fragment and sgRNA Scoffold were connected by the Goldgate method (NEB) and transferred into Escherichia coli. Positive transformants were obtained by selecting plaques with kanamycin resistance and without spectinomycin resistance. After sequencing verification, the positive plasmid was extracted to form a guide editing vector plasmid for the plant OsEPSPS gene, which was named pHUC411-PE2-OsEPSPS1 and pHUC411-PE2-OsEPSPS2, respectively, and transferred into EHA105 Agrobacterium. The above Agrobacterium was used for genetic transformation of rice callus to obtain 37 pHUC411-PE2-OsEPSPS1 regenerated plants, including 9 T-to-G homozygous mutations, 13 heterozygous mutations, and 15 non-mutated plants; 24 pHUC411-PE2-OsEPSPS2 regenerated plants, including 4 T-to-A homozygous mutations, 15 heterozygous mutations, and 5 non-mutated plants; the mutation type sequencing results can be found in Figure 4 .

[0071] 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 of the present invention and wild-type Nipponbare rice seeds were used as controls. After soaking at 28°C for 1-2 days, they were placed on moist filter paper for germination. 100 20-day-old 3-4-leaf seedlings of the three mutant rices and wild-type rice with the same growth and size were randomly selected. 5-8 plants were in one pot, and three replicates were set for each material. The prepared 10mM glyphosate herbicide solution was used for spraying. After 7 days, it can be found that the wild-type Nipponbare rice, the rice with T-to-G mutation on the OsEPSPS gene, and the rice with T-to-A mutation on the OsEPSPS gene all began to wilt and die, while the leaves of the rice with T-to-C mutation on the OsEPSPS gene can almost grow normally. The results are shown in Figure 5 .

[0072] This indicates that when the 836th base of the nucleotide sequence of the OsEPSPS gene mutates from T to C, rice can become resistant to glyphosate herbicide, while the mutant gene in which the 836th base of the nucleotide sequence of the OsEPSPS gene mutates from T to A and from T to G cannot make rice resistant to glyphosate herbicide.

[0073] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A rice herbicide-resistant OsEPSPS mutant gene, characterized in that: The rice herbicide-resistant OsEPSPS mutant gene is a wild-type rice OsEPSPS gene with a mutation at position 836 in the nucleotide sequence, from T to C.

2. The rice herbicide-resistant OsEPSPS mutant gene according to claim 1, characterized in that: The nucleotide sequence of the rice herbicide-resistant OsEPSPS mutant gene is shown in SEQ ID NO.

1.

3. A rice herbicide-resistant OsEPSPS mutant protein, characterized in that: The rice herbicide-resistant OsEPSPS mutant protein is a wild-type rice OsEPSPS protein with a mutation at position 279 in the amino acid sequence, from methionine to threonine.

4. The rice herbicide-resistant OsEPSPS mutant protein according to claim 3, characterized in that: The amino acid sequence of the rice herbicide-resistant OsEPSPS mutant protein is shown in SEQ ID NO.

2.

5. A kit, a recombinant vector or a recombinant cell comprising the rice herbicide-resistant OsEPSPS mutant gene according to claim 1 or 2 or the rice herbicide-resistant OsEPSPS mutant protein according to claim 3 or 4.

6. A nucleic acid molecule, characterized in that Expressing the rice herbicide-resistant OsEPSPS mutant protein according to claim 3 or 4.

7. Use of the rice herbicide-resistant OsEPSPS mutant gene according to claim 1 or 2, the rice herbicide-resistant OsEPSPS mutant protein according to claim 3 or 4, the kit, the recombinant vector, the recombinant cell according to claim 5 or the nucleic acid molecule according to claim 6 in conferring herbicide resistance to a target plant.

8. The use according to claim 7, characterized in that: Introducing the rice herbicide-resistant OsEPSPS mutant gene, rice herbicide-resistant OsEPSPS mutant protein, kit, recombinant vector, recombinant cell or nucleic acid molecule into a target plant to make the target plant resistant to herbicides; and / or, The herbicide is glyphosate.

9. The use according to claim 8, characterized in that: The method of introducing the rice herbicide-resistant OsEPSPS mutant gene into the target plant includes: chemical mutagenesis, transgenic, gene editing, hybridization or backcrossing.

10. A herbicide-resistant rice, characterized in that: The herbicide-resistant rice expresses the rice herbicide-resistant OsEPSPS mutant gene according to claim 1 or 2.

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