Herbicide-resistant rice mutant gene, protein and application thereof

Abstract

The invention relates to the technical field of biological genes, in particular to a herbicide-resistant rice mutant gene, protein and application thereof. The herbicide-resistant rice mutant gene provided by the invention is characterized in that a wild rice OsEPSPS gene is mutated at the 1415th site of a nucleotide sequence, T is mutated into C, and the nucleotide sequence is shown as SEQ ID NO.1. A plant containing the herbicide-resistant rice mutant gene or the herbicide-resistant rice mutant protein provided by the invention has excellent resistance to glyphosate herbicides, and the herbicide-resistant rice mutant gene can be widely applied to the work of herbicide-resistant breeding of plants.

Description

Technical Field

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

[0002] With the promotion of direct-seeding rice technology, problems such as the increasing number of weeds in rice fields and the increased use of herbicides have gradually emerged. The diversity of weed species and dominant populations has increased the resistance of weeds to herbicides, making weed control difficult and affecting the high and stable yields of direct-seeded rice. At the same time, the production safety issues caused by the excessive use of herbicides in direct-seeded rice fields deserve attention.

[0003] Glyphosate is a non-selective herbicide that kills weeds and other plants that compete with crops and is used to protect agricultural and horticultural crops. Long-term and large-scale application of glyphosate has led to at least 48 types of weeds becoming resistant to glyphosate. Glyphosate is mainly transported throughout the plant's meristem to exert its herbicidal effect, so any mechanism that prevents glyphosate from reaching its target, enolpyruvylshikimate phosphate synthase (EPSPS), can lead to plant resistance to glyphosate.

[0004] In general, glyphosate can bind to natural EPSPS very well. Studies have found that mutated EPSPS has a reduced ability to bind to glyphosate and can tolerate glyphosate. In the 1990s, seed giant Monsanto was the first to isolate the mutant CP4 gene of the EPSPS gene in Agrobacterium, and transferred the gene into plants to make them glyphosate-resistant, thus obtaining glyphosate-resistant soybeans, corn and cotton. With the study of glyphosate-resistant weeds, new EPSPS proteins that are insensitive to glyphosate have been discovered, opening up a new path for the cultivation of glyphosate-resistant crops. For example, some variants of EPSPS proteins were found in glyphosate-resistant Goosegrass, such as the simultaneous mutation of Thr (T) at position 102 to lle (I) and Pro (P) at position 106 to Ser (S).

[0005] However, when using biotechnology methods such as gene editing to create TIPS double mutations of endogenous EPSPS in crops such as rice, it is found that the obtained plants can only survive in the heterozygous state of the EPSPS mutant gene. And according to the current crop biological breeding regulatory policy, gene editing improved varieties cannot be directly used in the production process. Therefore, it is urgent to develop genes that do not affect plant growth and development and have glyphosate resistance in crops. The development of homozygous varieties resistant to glyphosate herbicides is of great significance. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a herbicide-resistant rice mutant gene, protein and application thereof. Plants containing the herbicide-resistant rice mutant gene or herbicide-resistant rice mutant protein provided by the present invention have excellent resistance to glyphosate herbicide. The herbicide-resistant rice mutant gene can be widely used in the work of herbicide-resistant plant breeding.

[0007] To this end, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a herbicide-resistant rice mutant gene in an optional embodiment, wherein the herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene with a mutation at position 1415 in the nucleotide sequence, from T to C, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] Preferably, the herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene that undergoes mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutating from T to C and position 1416 mutating from G to T, and its nucleotide sequence is shown in SEQ ID NO.2.

[0010] Preferably, the herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene that undergoes mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutating from T to C and position 1416 mutating from G to C, and its nucleotide sequence is shown in SEQ ID NO.3.

[0011] Preferably, the herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene that undergoes mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutating from T to C and position 1416 mutating from G to A, and its nucleotide sequence is shown in SEQ ID NO.4.

[0012] In a second aspect, the present invention provides a herbicide-resistant rice mutant protein in an optional embodiment, wherein the herbicide-resistant rice mutant protein is a wild-type rice OsEPSPS protein with a mutation at position 472 in the amino acid sequence, from methionine to threonine.

[0013] Preferably, the amino acid sequence of the rice mutant protein is shown in SEQ ID NO. 5. The amino acid sequence of the wild-type rice OsEPSPS protein is shown in SEQ ID NO. 6.

[0014] In a third aspect, the present invention provides, in an optional embodiment, a kit, a recombinant vector or a recombinant cell containing the above-mentioned herbicide-resistant rice mutant gene or the above-mentioned herbicide-resistant rice mutant protein.

[0015] In a fourth aspect, the present invention provides, in an optional embodiment, a herbicide-resistant rice, comprising the above-mentioned herbicide-resistant rice mutant gene, the above-mentioned herbicide-resistant rice mutant protein or the above-mentioned kit, recombinant vector or recombinant cell.

[0016] Preferably, the herbicide is glyphosate.

[0017] In a fifth aspect, the present invention provides a method for obtaining herbicide-resistant rice in an optional embodiment, such that the rice contains the above-mentioned herbicide-resistant rice mutant gene; or, the rice expresses the above-mentioned herbicide-resistant rice mutant protein.

[0018] In a sixth aspect, the present invention provides, in an optional embodiment, the use of the above-mentioned herbicide-resistant rice mutant gene, the above-mentioned herbicide-resistant rice mutant protein, the above-mentioned kit, the recombinant vector or the recombinant cell in making plants resistant (tolerant) to glyphosate herbicide.

[0019] Preferably, the application comprises introducing the above-mentioned herbicide-resistant rice mutant gene, the above-mentioned herbicide-resistant rice mutant protein, the above-mentioned kit, the recombinant vector or the recombinant cell into a target plant, so that the target plant has resistance to (tolerance to) glyphosate herbicide.

[0020] Methods for introducing the above-mentioned herbicide-resistant rice mutant gene into the target plant include chemical mutagenesis, transgenic, gene editing, hybridization or backcrossing.

[0021] In a seventh aspect, the present invention provides, in an optional embodiment, a use of the above-mentioned herbicide-resistant rice mutant gene or the above-mentioned herbicide-resistant rice mutant protein in cultivating plant progeny or plant seeds.

[0022] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0023]

[0024] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0025] ATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGG

[0026] CGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCG

[0027] CGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGT

[0028] GGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGAT

[0029] CGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAG

[0030] TCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGT

[0031] GGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCC

[0032] CTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCT

[0033] GTGGTGGCAAGTTTCCTGTTGAGAAGGATGcGAAAGAGGAAGTGCAACTCTTCTT

[0034] GGGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGT

[0035] GGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTG

[0036] GTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGC

[0037] ACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGG

[0038] TTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCT

[0039] CCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCT

[0040] TACGTTGAAATGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTC

[0041] TGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAA

[0042] ATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCA

[0043] ATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTG

[0044] ATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGAC

[0045] ACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGA

[0046] AAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTT

[0047] GTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGT

[0048] AAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGC

[0049] ATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAAC

[0050] ATCACGGCAATCGACACCTACGATGATCACAGGACTGCCATGGCCTTCTCCCTCGC

[0051] TGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA。

[0052] The nucleotide sequence shown in SEQ ID NO.3 is as follows:

[0053] ATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGG

[0054] CGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCG

[0055] CGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGT

[0056] GGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGAT

[0057] CGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAG

[0058] TCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGT

[0059] GGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCC

[0060] CTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCT

[0061] GTGGTGGCAAGTTTCCTGTTGAGAAGGATGcGAAAGAGGAAGTGCAACTCTTCTT

[0062] GGGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGT

[0063] GGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTG

[0064] GTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGC

[0065] ACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGG

[0066] TTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCT

[0067] CCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCT

[0068] TACGTTGAAATGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTC

[0069] TGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAA

[0070] ATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCA

[0071] ATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTG

[0072] ATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGAC

[0073] ACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGA

[0074] AAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTT

[0075] GTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGT

[0076] AAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGC

[0077] ATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAAC

[0078] ATCACGGCAATCGACACCTACGATGATCACAGGACCGCCATGGCCTTCTCCCTCGC

[0079] TGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA。

[0080] The nucleotide sequence shown in SEQ ID NO.4 is as follows:

[0081] ATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGG

[0082] CGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCG

[0083] CGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGT

[0084] GGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGAT

[0085] CGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAG

[0086] TCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGT

[0087] GGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCC

[0088] CTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCT

[0089] GTGGTGGCAAGTTTCCTGTTGAGAAGGATGcGAAAGAGGAAGTGCAACTCTTCTT

[0090] GGGGAACGCTGGAACTGCAATGCGACCATTGACAGCAGCCGTGACTGCTGCTGGT

[0091] GGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTG

[0092] GTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGC

[0093] ACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGG

[0094] TTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCT

[0095] CCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCT

[0096] TACGTTGAAATGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTC

[0097] TGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAA

[0098] ATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCA

[0099] ATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTG

[0100] ATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGAC

[0101] ACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGA

[0102] AAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTT

[0103] GTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGT

[0104] AAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGC

[0105] ATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAAC

[0106] ATCACGGCAATCGACACCTACGATGATCACAGGACAGCCATGGCCTTCTCCCTCGC

[0107] TGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA。

[0108] The amino acid sequence shown in SEQ ID NO.5 is shown as follows:

[0109] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKALGLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAAPLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKKHLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRTAFAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0110] The amino acid sequence shown in SEQ ID NO.6 is shown as follows:

[0111] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKAL GLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAA PLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKK HLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0112] Compared with the prior art, the present invention has one of the following beneficial effects:

[0113] 1. The plant containing the herbicide-resistant rice mutant gene or the herbicide-resistant rice mutant protein provided by the present invention has excellent resistance to glyphosate herbicide, and the herbicide-resistant rice mutant gene can be widely used in the work of herbicide-resistant plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 This is a photo of resistant callus screened on a medium containing glyphosate herbicide in Example 2 of the present invention;

[0115] Figure 2 The results of site sequencing of the herbicide-resistant rice mutant gene in Example 2 of the present invention;

[0116] Figure 3 This is a schematic diagram of the rice growth results of four mutant rices and wild-type rice in Example 3 of the present invention when sprayed with 1 times the recommended concentration of glyphosate herbicide. DETAILED DESCRIPTION

[0117] 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.

[0118] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0119] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0120] Example 1

[0121] This embodiment provides a method for constructing a guide editing vector for a herbicide-resistant rice mutant gene, comprising the following steps:

[0122] Taking rice as an example, the nucleotide sequence cacctacgatgatcacaggat in the plant OsEPSPS gene was selected. gg , as the target site, the underlined part is the PAM sequence, and the SpCas9 protein-mediated guide editing system pHUC411-PE2 is used, which can introduce pre-designed target mutations into the genome. According to the analysis of the plant pegRNA design website PlantPegDesigner (http: / / www.plantgenomeediting.net / ), the SgRNA, RT and PBS sequences of the corresponding targets were obtained. According to the analysis of the pegRNA design and optimization tool pegLIT (https: / / peglit.liugroup.us / ), there is also a corresponding 8bplinker between the PBS sequence and evopreQ1 of each target. Therefore, the pegRNA sequences of each herbicide-resistant rice mutant gene obtained are as follows:

[0123] PEGRNA1 that converts TG to CG:

[0124] CACCTACGATGATCACAGGAGTTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGGCCATGGCCGTCCTGTGATCATCGCCCTAATTTTGACGCGGTTCTATCTAGTTACGCGTTAAAACCAACTAGAAA.

[0125] PEGRNA2 that converts TG to CA:

[0126] CACCTACGATGATCACAGGAGTTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGGCCATGGCTGTCCTGTGATCATCGCCCTAATTTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0127] PEGRNA3 that converts TG to CC:

[0128] CACCTACGATGATCACAGGAGTTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGGCCATGGCGGTCCTGTGATCATCGCCCTAATTTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0129] PEGRNA4 that converts TG to CT:

[0130] CACCTACGATGATCACAGGAGTTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGGCCATGGCAGTCCTGTGATCATCGCCCTAATTTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA.

[0131] The forward oligonucleotide chain of pegRNA and the reverse oligonucleotide chain complementary thereto are synthesized separately and annealed to form a double strand.

[0132] The pHUC411-PE2 vector was digested with BsaI endonuclease (purchased from NEB) at 37°C for 4 hours and incubated for 10 minutes at an inactivated enzyme digestion system at 65°C as the backbone fragment for constructing the recombinant vector. The backbone fragment of the recombinant vector and sgRNA Scoffold were connected end to end using the Goldgate method (NEB) and transferred into Escherichia coli. Positive transformants were obtained by selecting plaques with kanamycin resistance and no spectinomycin resistance. After sequencing verification, the positive plasmids were extracted to form the guide editing vector plasmids for plant OsEPSPS gene CRISPR / Cas9, which were named pHUC411-PE2-OsEPSPS1, pHUC411-PE2-OsEPSPS2, pHUC411-PE2-OsEPSPS3, and pHUC411-PE2-OsEPSPS4, respectively. The plant expression vector was transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw method, and positive clones were obtained by colony PCR screening to obtain Agrobacterium containing pHUC411-PE2-OsEPSPS1, pHUC411-PE2-OsEPSPS2, pHUC411-PE2-OsEPSPS3, and pHUC411-PE2-OsEPSPS4.

[0133] Example 2

[0134] This embodiment provides a method for obtaining a positive transgenic plant, comprising the following steps:

[0135] (1) After removing the husk from mature seeds, soak the seeds in 70% alcohol for 1 minute, pour out the alcohol, and then soak the seeds in 50% sodium hypochlorite solution (the effective chlorine concentration of the original solution is greater than 4%) containing 1 drop of Tween 20 for 40 minutes (150r / min), pour out the sodium hypochlorite, and wash with sterile water 5 times until the solution is clear and there is no smell of sodium hypochlorite. Soak the seeds in sterile water overnight, peel off the endosperm of the pretreated seeds with a scalpel, peel off the embryo, and inoculate the embryo on the callus induction medium. After 11 days of dark culture at 30°C, separate the callus from the endosperm and embryo to obtain primary callus tissue with good bud removal and vigorous division.

[0136] (2) Add the primary callus to a screening medium containing 5 mM glyphosate solution (add 5 mM glyphosate solution to the induction medium), screen and culture for 28-42 days, and observe irregularly until resistant callus particles that can grow normally appear. Figure 1 .

[0137] (3) The callus particles after resistance screening were transferred to a differentiation medium with a slightly lower concentration to regenerate and differentiate into seedlings. The specific differentiation medium was: N6 macronutrients, MS iron salts, B5 micronutrients, B5 vitamins, 500 mg / L proline, 1 g / L casein hydrolyzate, 30 g / L sucrose, 1.5 mg / L naphthaleneacetic acid, 1 mg / L 6-benzyladenine, 2.5 g / L phytagel, and 2 mM glyphosate. Finally, the buds or seedlings were transferred to MS rooting medium, sampled, tested, and subjected to tissue sequencing analysis.

[0138] After genetic transformation of rice, rice of pHUC411-PE2-OsEPSPS series were obtained, and genomic DNA extracted from the plants was sent to Invitrogen for genome sequencing. The sequencing results were compared with the wild-type Nipponbare OsEPSPS gene, and it was found that the nucleotide sequence of the OsEPSPS gene had mutations at positions 1415 and 1416, respectively: a mutation from T to C occurred at position 1415, and the nucleotide sequence of the mutation is shown in SEQ ID NO.1; a mutation from T to C at position 1415 and from G to T at position 1416, and the nucleotide sequence of the mutation is shown in SEQ ID NO.2; a mutation from T to C at position 1415 and from G to C at position 1416, and the nucleotide sequence of the mutation is shown in SEQ ID NO.3; a mutation from T to C at position 1415 and from G to A at position 1416, and the nucleotide sequence of the mutation is shown in SEQ ID NO.4. The results are shown in Figure 2 As a result, the amino acid sequence of the OsEPSPS protein mutated from methionine (M) to threonine (T) at position 472. The amino acid sequence of the mutation is shown in SEQ ID NO.5.

[0139] Example 3

[0140] This example provides a resistance experiment of the rice mutation site of Example 2:

[0141] The mutants OsEPSPS1, OsEPSPS2, OsEPSPS3 and OsEPSPS4 are obtained by introducing the genomic sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 into the wild type Japanese rice.

[0142] The four mutant rices and wild-type Japanese Tenqing rice were planted separately and homozygous seeds were harvested.

[0143] Prepare the herbicide dilution: select glyphosate isopropylamine salt herbicide with 41% glyphosate active ingredient, 10mM is the recommended concentration for use in the field, and further used for seedlings in the 3-4 leaf stage after germination.

[0144] Homozygous seeds of four mutant rices and wild-type rice seeds were used as materials. 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 four mutant rices and wild-type rice with the same growth and size were randomly selected, with 5-8 plants in a pot. The four mutant rices and wild-type rice were divided into five areas. The areas with the four mutant rices were used as experimental groups, and the areas with the wild-type rice were used as control groups. 50 seedlings were planted in each area.

[0145] The prepared herbicide dilution was evenly sprayed on the leaf surfaces of wild-type rice and four mutant rice plants, and the phenotypic changes of the rice plants were observed regularly. It was found that 7 days after the herbicide was sprayed, a large area of ​​wild-type rice began to wilt and die, while the leaves of the four mutant rice plants could grow almost normally. After further observation, 20 days after the spraying treatment, the mutant plants stretched to 25 cm, and the leaves grew completely normally. The results are shown in Figure 3 .

[0146] This indicates that mutations at positions 1415 and 1416 of the nucleotide sequence of the OsEPSPS gene can make rice resistant to glyphosate herbicide.

[0147] The surviving plants are further transferred to the field for growth, and the seeds are harvested when mature.

[0148] 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 herbicide-resistant rice mutant gene, characterized in that: The herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene with a mutation at position 1415 in the nucleotide sequence, from T to C, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The herbicide-resistant rice mutant gene according to claim 1, characterized in that: The herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene with mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutated from T to C and position 1416 mutated from G to T. Its nucleotide sequence is shown in SEQ ID NO.

2.

3. The herbicide-resistant rice mutant gene according to claim 1, characterized in that: The herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene with mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutated from T to C and position 1416 mutated from G to C. Its nucleotide sequence is shown in SEQ ID NO.

3.

4. The herbicide-resistant rice mutant gene according to claim 1, characterized in that: The herbicide-resistant rice mutant gene is a wild-type rice OsEPSPS gene with mutations at positions 1415 and 1416 in the nucleotide sequence, with position 1415 mutated from T to C and position 1416 mutated from G to A. Its nucleotide sequence is shown in SEQ ID NO.

4.

5. A herbicide-resistant rice mutant protein, characterized in that: The herbicide-resistant rice mutant protein is a wild-type rice OsEPSPS protein with a mutation at position 472 in the amino acid sequence, from methionine to threonine.

6. The herbicide-resistant rice mutant protein according to claim 5, characterized in that: The amino acid sequence of the rice mutant protein is shown in SEQ ID NO.

5.

7. A kit, a recombinant vector or a recombinant cell comprising the herbicide-resistant rice mutant gene according to any one of claims 1 to 4 or the herbicide-resistant rice mutant protein according to claim 5 or 6.

8. A herbicide-resistant rice, characterized in that: It comprises the herbicide-resistant rice mutant gene according to any one of claims 1 to 4, the herbicide-resistant rice mutant protein according to claim 5 or 6, or the kit, recombinant vector or recombinant cell according to claim 7.

9. The herbicide-resistant rice according to claim 8, characterized in that: The herbicide is glyphosate.

10. A method for obtaining herbicide-resistant rice, characterized in that: Making rice contain the herbicide-resistant rice mutant gene according to any one of claims 1 to 4; or, The herbicide-resistant rice mutant protein according to claim 5 or 6 is expressed in rice.

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