Rice OsEPSPS mutant gene and application thereof

Abstract

The invention relates to the technical field of biological genes, in particular to a rice OsEPSPS mutant gene and application thereof. In an optional embodiment, the invention provides a rice OsEPSPS mutant gene, the rice OsEPSPS mutant gene is obtained by mutation of a wild type OsEPSPS gene at the 1397th site of a nucleotide sequence, and C is mutated into T. According to the rice OsEPSPS mutant gene provided by the invention, mutation is generated at the 1397th site of a nucleotide sequence of a wild type OsEPSPS gene, C is mutated into T, the mutation can enable rice to have glyphosate resistance, and after the C is mutated into G or A at the 1397th site, the mutation at the site cannot enable the rice to have glyphosate resistance, so that the mutation at the site has very strong directivity, and the mutation at the site can be applied to the field of glyphosate resistance. Only a specific mutation direction can endow the rice straw with glyphosate resistance.

Description

Technical Field

[0001] The invention relates to the technical field of biological genes, and in particular to a rice OsEPSPS mutant gene and an application thereof. Background Art

[0002] During crop planting, weeds compete with crops for limited light, water and nutrients, which will seriously affect the yield of crops. Glyphosate, which was successfully developed in the 1960s, has been promoted and applied for nearly 50 years. It has the advantages of high efficiency, low cost, broad spectrum and water decomposition. It is currently the most widely used herbicide. The use of various chemical herbicides will seriously challenge the sustainable development of crops and food security. 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] Glyphosate achieves its weed control goal mainly by interfering with the shikimate synthesis pathway of plants. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway of plants. Under its catalytic action, phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) can synthesize amino acid precursors, which is an important part of plant physiological metabolism. Due to its unique chemical structure, glyphosate will competitively bind to EPSPS with PEP, resulting in EPSPS being unable to participate in important plant metabolic links, aromatic amino acids being unable to be synthesized in plant cells, and various metabolic reactions of plants being inhibited, eventually leading to plant death. V.Mohan Murli Achari et al. found that double mutants of the rice EPSPS gene can significantly improve glyphosate resistance. Ouyang Chao et al. found that a naturally evolved TIPS-EPSPS mutation was confirmed to be highly resistant to glyphosate in goosegrass, which was 180 times that of the wild type (WT) and 32 times higher than the P106S mutant studied by previous researchers. However, the EPSPS genes that are widely studied now are all from bacteria, and there are very few studies on endogenous EPSPS genes in plants.

[0004] At present, it is difficult to obtain naturally mutated EPSPS genes that are resistant to glyphosate in crops. The only way is to transfer mutated EPSPS genes into crops through genetic modification to obtain glyphosate-resistant crops. Therefore, it is urgent to develop genes that do not affect plant growth and development and 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 OsEPSPS mutant gene and an application thereof. The rice OsEPSPS mutant gene provided by the present invention produces a mutation at the 1397th position of the nucleotide sequence of the wild-type OsEPSPS gene, from C to T. This mutation can make the rice gene resistant to glyphosate, while after the mutation at the 1397th position from C to G or A, the rice gene cannot be made resistant to glyphosate, indicating that the mutation at this site has a strong directionality, and only a specific mutation direction can confer glyphosate resistance to rice.

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

[0007] In a first aspect, the present invention provides a rice OsEPSPS mutant gene in an optional embodiment, wherein the rice OsEPSPS mutant gene is a wild-type OsEPSPS gene with a mutation at position 1397 of the nucleotide sequence, from C to T.

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

[0009] In a second aspect, the present invention provides a rice OsEPSPS mutant protein in an optional embodiment, wherein the rice PsEPSPS mutant protein is a wild-type OsEPSPS protein with a mutation at position 466 of the amino acid sequence, from threonine to isoleucine.

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

[0011] In a third aspect, the present invention provides, in an optional embodiment, an expression cassette, a recombinant vector or a recombinant cell comprising the above rice OsEPSPS mutant gene.

[0012] In a fourth aspect, the present invention provides, in an optional embodiment, a use of the above rice OsEPSPS mutant gene, the above rice OsEPSPS mutant protein, the above expression cassette, the recombinant vector or the recombinant cell in cultivating herbicide-resistant rice.

[0013] Preferably, the herbicide is glyphosate herbicide. The application includes: introducing the rice OsEPSPS mutant gene, the rice OsEPSPS mutant protein, the expression cassette, the recombinant vector or the recombinant cell into rice, so that the rice has resistance or tolerance to glyphosate. The method of introducing into rice includes genetic modification, hybridization or backcrossing.

[0014] Furthermore, the application includes introducing one of the rice OsEPSPS mutant gene, the rice OsEPSPS mutant protein, the expression cassette, the recombinant vector or the recombinant cell into rice, so that the rice has herbicide resistance.

[0015] Specifically, the transgenic method comprises the following steps:

[0016] (1) constructing an expression vector containing the OsEPSPS mutant gene;

[0017] (2) genetically transforming rice with the above expression vector so that the rice contains the above rice herbicide-resistant OsEPSPS mutant gene;

[0018] (3) Make rice express rice herbicide-resistant OsEPSPS mutant protein.

[0019] Furthermore, the application comprises the following steps:

[0020] (1) After hulling and sterilizing rice seeds, the embryos are separated and placed on a callus induction medium to produce secondary callus;

[0021] (2) transferring the secondary callus to a new callus induction medium for pre-culture;

[0022] (3) contacting the callus obtained in step (2) with Agrobacterium for 15 minutes to obtain resistant callus, wherein the Agrobacterium is transferred with a recombinant vector having a rice OsEPSPS mutant gene;

[0023] (4) transferring the callus tissue of step (3) to a culture dish with three sterile filter papers on top, and culturing at 21-23° C. for 48 hours;

[0024] (5) placing the callus tissue of step (4) on a pre-screening medium and culturing for 5-7 days;

[0025] (6) transferring the callus tissue of step (5) to a screening medium to obtain resistant callus tissue;

[0026] (7) transferring the resistant callus to a differentiation and regeneration medium to differentiate into shoots;

[0027] (8) Transfer the seedlings from step (7) to a rooting medium for rooting.

[0028] In a fifth aspect, the present invention provides a host cell in an optional embodiment, comprising the above-mentioned rice OsEPSPS mutant gene; the host cell is an Escherichia coli cell.

[0029] In a sixth aspect, the present invention provides, in an optional embodiment, a use of the above-mentioned rice OsEPSPS mutant gene, the above-mentioned rice OsEPSPS mutant protein, the above-mentioned expression cassette, recombinant vector or recombinant cell in cultivating homozygous rice progeny or rice seeds.

[0030] In a seventh 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.

[0031] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0032]

[0033] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0034] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKAL GLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAA PLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKK HLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDIYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0035] Compared with the prior art, the present invention has one of the following beneficial effects:

[0036] 1. The rice OsEPSPS mutant gene provided by the present invention has a mutation at the 1397th position of the nucleotide sequence of the wild-type OsEPSPS gene, from C to T. This mutation can make the rice have a glyphosate-resistant gene, while after the mutation at the 1397th position from C to G or A, the rice cannot have a glyphosate-resistant gene, indicating that the mutation at this site has a strong directionality, and only a specific mutation direction can confer glyphosate resistance to rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The photos are of resistant callus and resistant buds produced on a medium containing glyphosate herbicide in Example 1 of the present invention;

[0038] Figure 2 This is a sequencing comparison diagram of the wild type and mutant near the OsEPSPS mutation site in Example 1 of the present invention;

[0039] Figure 3 This is a comparison diagram of the growth of rice containing the OsEPSPS mutant gene (left) and wild-type rice (right) after spraying the herbicide in Example 2 of the present invention 7 days after spraying the herbicide;

[0040] Figure 4 This is a comparison of the growth of plants with a mutation in the nucleotide at position 1397 in Example 3 of the present invention before and after spraying the herbicide, wherein the upper plant is the result of not spraying the glyphosate herbicide, and the lower plant is the result of spraying 10mmol·L -1 Results of glyphosate herbicides. 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] Example 1

[0045] This embodiment provides a method for directional screening of herbicide resistance sites by mutagenesis of rice callus, comprising the following steps:

[0046] 1. Preparation of explants: Select healthy, mature and dry seeds, remove the shells and soak them in 70% alcohol for 90 seconds, then pour out the alcohol. Next, rinse the seeds once with sterilized water and then pour out the water.

[0047] 2. Disinfection: Soak the seeds in a 50% sodium hypochlorite solution containing 1 drop of Tween 20 for 35 minutes (with intermittent shaking). Then, pour out the sodium hypochlorite and rinse several times with sterile water until the solution is clear and there is no smell of sodium hypochlorite. Then soak the seeds in sterile water overnight. Finally, inoculate the seeds on the induction medium, placing 18-20 seeds per dish.

[0048] 3. Mutagenesis culture: transfer the callus in good condition to the mutagenesis medium for 2 weeks. The formula of the mutagenesis medium is: N6 macronutrient, B5 micronutrient, MS iron source, B5 vitamin, 500mg / L proline, 500mg / L glutamine, 300mg / L enzymatic caseinate, 2mg / L2,4-D, 30g / L sucrose, 3.0g / L phytagel, KOH to adjust the pH to 5.80, and add 0.05% NaNO2 mutagen. Cultivate at 30°C, with 16 hours of light and 8 hours of darkness, for 1-2 weeks to achieve chemical mutagenesis of rice callus.

[0049] 4. Screening culture: Rice has different resistance to herbicides at different growth stages, and also shows different resistance levels during tissue culture. In order to screen resistant mutants, glyphosate was used as a simulated herbicide and added to the culture medium for directional screening. The appropriate screening concentration was determined based on the test results and added to the screening medium. Subsequently, the rice callus after 1-2 weeks of mutagenesis culture was transferred to the screening medium for culture, and the screening culture time was 30 days. The screening medium is an induction medium with 5mM glyphosate added. Observe at irregular intervals until resistant callus particles that can grow normally appear. For results, see Figure 1 .

[0050] 5. Differentiation culture: The callus particles after resistance screening are transferred to a differentiation medium with a slightly lower concentration to promote their regeneration and differentiation into seedlings. The specific components of the differentiation medium are: N6 macroelements, MS iron salts, B5 trace elements, B5 vitamins, 500mg / L proline, 1g / L enzymatic casein, 30g / L sucrose, 1.5mg / L NAA, 1mg / L 6-BA, 2.5g / L phytagel and 2mM glyphosate. The regenerated buds or seedlings will be moved to MS rooting medium for rooting culture. After completion, the samples will be taken out for tissue sequencing analysis.

[0051] 6. Repeat the above steps to obtain different mutants, and further culture the seedlings of these mutants to promote their growth. In a specific growth period, glyphosate is induced in the seedlings. Under the same concentration of glyphosate induction conditions, plants with relatively high resistance levels are selected.

[0052] 7. The genomic DNA of plants with high glyphosate resistance was extracted, amplified by PCR, and sequenced by Sanger sequencing. The sequencing results were compared with the wild-type Nipponbare OsEPSPS gene, and it was found that the 1397th C in OsEPSPS was mutated to T. The results are shown in Figure 2The nucleotide sequence of the OsEPSPS gene of the herbicide-resistant mutant is shown in SEQ ID NO. 1. The base mutation causes the amino acid at position 466 of the OsEPSPS gene to change from threonine (T) to isoleucine (I), and the amino acid sequence is shown in SEQ ID NO. 2. This mutation can significantly enhance the resistance of rice to glyphosate herbicides.

[0053] Calli and shoots of the mutant showed resistance to the herbicide in medium supplemented with 5 mM glyphosate.

[0054] Example 2

[0055] This example provides a rice resistance identification experiment containing a rice OsEPSPS mutant gene obtained in Example 1, comprising the following steps:

[0056] The rice containing the rice OsEPSPS mutant gene (hereinafter referred to as mutant) and the rice containing the wild-type Nipponbare OsEPSPS gene (hereinafter referred to as wild-type) obtained in Example 1 were used as materials, soaked at 28°C for 1-2 days, and then placed on a moist filter paper to promote germination. After the seeds germinated, a similar number of wild-type and mutant seeds were taken and placed in a rice nutrient solution for culture.

[0057] Select 20-day-old mutant and wild-type seedlings with the same growth, and plant 3-5 seedlings in a pot. Plant the wild-type and mutant in different areas as the experimental group and the control group, with 50 seedlings in one area. After 30 days of growth, select glyphosate isopropylamine salt herbicide (with an active ingredient concentration of 41%) and use 10mmol·L -1 The glyphosate isopropylamine salt herbicide (recommended concentration for rice fields) was sprayed evenly on the leaf surfaces of wild-type and mutant plants. Subsequently, the phenotypic changes of rice plants were observed regularly. Figure 3 .

[0058] The results showed that after 7 days, the wild-type rice in the experimental group began to wilt on a large scale, while the leaves of the homozygous mutant plants maintained almost normal growth.

[0059] Example 3

[0060] In order to test whether other types of mutations at nucleotide position 1397 also confer herbicide resistance, this example uses a guide editing tool to design a pegRNA for the mutation of nucleotide position 1397 from C to G:

[0061] GGCCATCCTGTGATCATCGTGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGCAATCGACAGCTACGATGATCACAGGTAAATAAATTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA;

[0062] After the nucleotide at position 1397 was mutated from C to A, the pegRNA was designed:

[0063] GGCCATCCTGTGATCATCGTGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGCAATCGACAACTACGATGATCACAGGTAAATAAATTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA;

[0064] The above-mentioned pegRNA was constructed on the guide editing vector ePE2, and rice genetic transformation was carried out to obtain homozygous mutant plants containing nucleotide mutations at position 1397 from C to G and nucleotide mutations at position 1397 from C to A. Rice plants containing nucleotide mutations at position 1397 (i.e., nucleotide mutations at position 1397 from C to T, G, and A) were sprayed with 10 mmol·L -1 glyphosate isopropylamine salt herbicide, glyphosate herbicide resistance identification, the results are shown in Figure 4 .

[0065] The results showed that when no herbicide was sprayed, rice plants with the nucleotide mutation at position 1397 (i.e., the nucleotide at position 1397 mutated from C to T, G, and A) grew normally, but when 10 mmol·L -1 After being exposed to the glyphosate isopropylamine salt herbicide, only the nucleotide at position 1397 mutated from C to T to have herbicide resistance, while the other two mutation types could not survive. Therefore, the mutant gene in which the nucleotide at position 1397 mutated from C to T has important application value in creating herbicide-resistant rice. Only such a site-directed mutation can make the plant resistant to (tolerant to) glyphosate herbicide.

[0066] 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 OsEPSPS mutant gene, characterized in that: The rice OsEPSPS mutant gene is a wild-type OsEPSPS gene that has a mutation at position 1397 of the nucleotide sequence, from C to T.

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

1.

3. A rice OsEPSPS mutant protein, characterized in that: The rice PsEPSPS mutant protein is a wild-type OsEPSPS protein with a mutation at position 466 of the amino acid sequence, from threonine to isoleucine.

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

2.

5. An expression cassette, a recombinant vector or a recombinant cell comprising the rice OsEPSPS mutant gene according to claim 1 or 2 or the rice OsEPSPS mutant protein according to claim 3 or 4.

6. Use of the rice OsEPSPS mutant gene according to claim 1 or 2, the rice OsEPSPS mutant protein according to claim 3 or 4, the expression cassette, recombinant vector or recombinant cell according to claim 5 in cultivating herbicide-resistant rice; The herbicide is glyphosate herbicide.

7. The use according to claim 6, characterized in that: The application comprises: introducing the rice OsEPSPS mutant gene according to claim 1 or 2, the rice OsEPSPS mutant protein according to claim 3 or 4, the expression cassette, recombinant vector or recombinant cell according to claim 5 into rice, so that the rice has resistance or tolerance to glyphosate; The methods of introducing rice include genetic modification, hybridization or backcrossing.

8. A host cell, characterized in that The method comprises the rice OsEPSPS mutant gene according to claim 1 or 2; The host cell is an Escherichia coli cell.

9. Use of the rice OsEPSPS mutant gene according to claim 1 or 2, the rice OsEPSPS mutant protein according to claim 3 or 4, the expression cassette, recombinant vector or recombinant cell according to claim 5 in cultivating homozygous rice progeny or rice seeds.

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

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