Plant herbicide-resistant epsp synthase mutant gene and use thereof

Abstract

The present application relates to the technical field of biological genes, and particularly relates to a plant herbicide-resistant EPSPS mutant gene and application thereof. The present application provides a plant herbicide-resistant EPSPS mutant gene, wherein the plant herbicide-resistant EPSPS mutant gene is mutated at the 742th nucleotide sequence of a wild-type EPSPS gene, and T is mutated into C. The EPSPS enzyme coded by the plant herbicide-resistant EPSPS mutant gene provided by the present application has resistance to herbicides, and can maintain normal catalytic activity even under the action of herbicides, so as to ensure that a plant can normally synthesize aromatic amino acids, maintain growth and development, and a rice plant containing the EPSPS mutant gene not only has resistance to glyphosate herbicide with 1 times of a field recommended concentration, but also has resistance to glyphosate herbicide with 3 times of the field recommended concentration, and the mutant gene has the characteristics of resistance to high-concentration herbicides.

Description

Technical Field

[0001] This invention relates to the field of biogenetics, specifically to a mutant gene for plant resistance to herbicides EPSPS and its applications. Background Technology

[0002] Weeds are the most serious biological limiting factor in agriculture, significantly impacting crop growth, development, and yield. They infest entire fields, actively competing with crops for nutrients, water, sunlight, and space. Weeds also serve as substitute hosts for major insects, pests, and diseases. Furthermore, some harmful weeds release phytotoxins into the soil, negatively affecting crop growth. Traditional weed control measures, including hand-harvesting, burning weeds before new crop planting, tilling, harrowing, and irrigation of the entire field through watering and crop rotation, are highly effective methods for small-scale farming. However, due to their labor-intensive and time-consuming nature, these methods are difficult to manage weed infestations in large-scale planting areas. On the other hand, chemical-based weed control measures are efficient and economical, offering a better option for achieving higher productivity.

[0003] 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in plants, involved in the synthesis of aromatic amino acids and crucial for plant growth and development. Glyphosate herbicides work by inhibiting EPSPS activity, thereby suppressing the synthesis of aromatic amino acids and achieving weed control. This mechanism of action often results in crop damage when treated with herbicides.

[0004] To solve this problem, a mutant EPSPS gene was successfully developed using gene editing technology. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a herbicide-resistant EPSPS mutant gene for plants and its application. This mutant gene encodes an EPSPS enzyme resistant to herbicides, maintaining its normal catalytic activity even under herbicide exposure, thus ensuring the plant's normal synthesis of aromatic amino acids and maintaining its growth and development. Specifically, the EPSPS mutant gene alters the binding site between the EPSPS enzyme and the herbicide, reducing the herbicide's affinity for the enzyme and making it difficult for herbicides to inhibit enzyme activity. The application of this mutant gene not only improves crop resistance to herbicides but also reduces herbicide damage, bringing significant economic benefits to agricultural production. Therefore, the development and application of EPSPS mutant genes are of great significance for improving agricultural production efficiency, reducing production costs, and protecting the environment. This patented technology was proposed against this background, aiming to provide a more efficient, safe, and environmentally friendly solution for agricultural production by utilizing the EPSPS mutant gene. Rice plants containing the EPSPS mutant gene of this invention are resistant not only to glyphosate herbicides at 1 times the recommended field concentration, but also to glyphosate herbicides at 3 times the recommended field concentration. This mutant gene has the characteristic of resistance to high concentrations of herbicides.

[0006] Therefore, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a plant herbicide-resistant EPSPS mutant gene in an optional embodiment, wherein the plant herbicide-resistant EPSPS mutant gene has a mutation at position 742 of the wild-type EPSPS gene, changing from T to C.

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

[0009] Secondly, in an optional embodiment, the present invention provides a plant herbicide-resistant EPSPS mutant protein, wherein the plant herbicide-resistant EPSPS mutant protein is a wild-type EPSPS protein in which a mutation occurs at position 248 of the amino acid sequence, changing from tyrosine to histidine.

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

[0011] In this invention, the mutation of the amino acid site in the EPSPS mutant protein of this invention into other amino acids will also produce glyphosate resistance. These mutated amino acids are also within the protection scope of this invention. For example, the amino acid at position 248 is mutated from tyrosine to glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, arginine, asparagine, glutamine, lysine, histidine, or glutamic acid.

[0012] Thirdly, in an optional embodiment, the present invention provides an expression cassette comprising the aforementioned plant herbicide-resistant EPSPS mutant gene or the aforementioned plant herbicide-resistant EPSPS mutant protein.

[0013] Fourthly, in an optional embodiment, the present invention provides a recombinant vector comprising the aforementioned plant herbicide-resistant EPSPS mutant gene or the aforementioned plant herbicide-resistant EPSPS mutant protein.

[0014] Fifthly, in optional embodiments, the present invention provides the use of the above-mentioned plant herbicide-resistant EPSPS mutant gene, the above-mentioned plant herbicide-resistant EPSPS mutant protein, the above-mentioned expression cassette, or the above-mentioned recombinant vector in equipping plants with glyphosate resistance or tolerance.

[0015] Preferably, the application includes: introducing the above-mentioned plant herbicide-resistant EPSPS mutant gene, the above-mentioned plant herbicide-resistant EPSPS mutant protein, the above-mentioned expression cassette, or the above-mentioned recombinant vector into a target plant, thereby terminating the target plant's resistance to or tolerance to glyphosate. The method of introducing the herbicide into the target plant includes transgenic, hybridization, or backcrossing. Further, the application includes introducing one of the above-mentioned fragments into the target plant, thereby terminating the target plant's herbicide resistance.

[0016] Specifically, the application includes the following steps:

[0017] (1) After removing the shells and sterilizing the rice seeds, the embryos were separated and placed on the callus induction medium to generate secondary callus.

[0018] (2) Transfer the secondary callus to a new callus induction medium for pre-culture;

[0019] (3) The callus obtained in step (2) was contacted with Agrobacterium for 15 minutes to obtain resistant callus, in which a recombinant expression vector containing the plant herbicide resistance EPSPS mutant gene was introduced.

[0020] (4) Transfer the callus tissue from step (3) to a culture dish with three sterile filter papers on it and incubate at 21-23℃ for 48 hours.

[0021] (5) Place the callus tissue from step (4) on a pre-screening medium and culture for 5-7 days;

[0022] (6) Transfer the callus tissue from step (5) onto a screening medium to obtain resistant callus tissue;

[0023] (7) The resistant callus tissue was transferred to a differentiation and regeneration medium to differentiate into seedlings;

[0024] (8) Transfer the seedlings from step (7) to the rooting medium to root.

[0025] In a sixth aspect, the present invention provides, in optional embodiments, the application of the above-mentioned plant herbicide-resistant EPSPS mutant gene, the above-mentioned plant herbicide-resistant EPSPS mutant protein, the above-mentioned expression cassette, or the above-mentioned recombinant vector in the cultivation of homozygous plant offspring or plant seeds.

[0026] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0027]

[0028] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0029] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKAL GLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGTAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQHLSALLMAA PLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKK HLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN.

[0030] Compared with the prior art, the present invention has one of the following beneficial effects:

[0031] 1. The EPSPS enzyme encoded by the plant herbicide-resistant EPSPS mutant gene provided by this invention is resistant to herbicides. Even under the action of herbicides, it can maintain its normal catalytic activity, thereby ensuring that the plant can synthesize aromatic amino acids normally and maintain its growth and development. Furthermore, rice plants containing the EPSPS mutant gene are resistant not only to glyphosate herbicides at 1 times the field recommended concentration, but also to glyphosate herbicides at 3 times the field recommended concentration. This mutant gene has the characteristic of resistance to high concentrations of herbicides.

[0032] 2. This invention also provides herbicide-resistant plants that express an EPSPS gene containing a herbicide-resistant mutation site. This gene sequence differs from the EPSPS gene sequence of wild-type plants; it is a sequence of the EPSPS gene containing a herbicide-resistant mutation site. It has been found that plants carrying the EPSPS mutation site are resistant to glyphosate herbicides, while wild-type plants are sensitive to glyphosate herbicides.

[0033] 3. The present invention also provides the application of the mutated nucleic acids or genes and proteins in plant breeding for cultivating herbicide-resistant plants, especially crops, and provides the application of these proteins and their encoding genes in transgenic or non-transgenic plants such as rice. Attached Figure Description

[0034] Figure 1 Photographs of resistant callus and resistant shoots produced on a herbicide-containing culture medium in Example 1;

[0035] Figure 2 This is a sequencing comparison diagram of the wild type and the mutant near the mutation site in Example 1;

[0036] Figure 3 This is a comparison of the growth of wild-type rice plants (top) and mutant rice plants (bottom) after spraying different concentrations of glyphosate herbicide in Example 2. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are merely for explaining the present invention and are not limited to the present invention.

[0041] Example 1

[0042] Screening of mutation sites and analysis of mutation sites

[0043] The method for targeted screening of herbicide resistance sites through mutagenesis of rice callus specifically includes the following steps:

[0044] 1. Preparation of explants

[0045] After removing the husks from mature seeds, soak them in 70% alcohol for 1 minute, then discard the alcohol. Soak the seeds in a 50% sodium hypochlorite solution (with an effective chlorine concentration greater than 4%) containing 1 drop of Tween 20 for 40 minutes (150 rpm). Discard the sodium hypochlorite and wash the seeds five times with sterile water until the solution is clear and has no sodium hypochlorite odor. Soak the seeds in sterile water overnight. Using a scalpel, peel the embryo off the seed along the aleurone layer and inoculate the embryo onto callus induction medium. After culturing in the dark at 30°C for 11 days, separate the callus from the endosperm and plumule, and collect the well-formed, vigorously dividing primary callus tissue.

[0046] 2. Mutagenesis culture

[0047] After induction, callus tissue in good condition was transferred to mutagenesis medium for 7-14 days. The mutagenesis medium consisted of N6 majors, B5 minors, MS iron, B5 vitamin, 500 mg / L proline, 500 mg / L glutamine, 300 mg / L casein acids enzymatic, 2 mg / L 2,4-D, 30 g / L sucrose, 3.0 g / L phytagel, pH 5.70, with an additional 0.05% NaNO2. The callus tissue was cultured at 30℃ for 16 hours of light + 8 hours of darkness for 1-2 weeks to chemically mutate the genome of rice callus tissue.

[0048] 3. Screening and Cultivation

[0049] Rice exhibits varying tolerance to herbicides at different growth and development stages. Similarly, its tolerance to abiotic stress differs at different stages under tissue culture conditions. Using glyphosate to simulate a herbicide environment, we added it to the culture medium for targeted screening of resistant mutants. After testing, we added an appropriate screening concentration to the screening medium. Rice calluses induced 1-2 weeks prior were transferred to the screening medium and cultured for 28-42 days. The screening medium consisted of induction medium + 5 mM glyphosate. Observations were made periodically until resistant callus particles capable of normal growth appeared. (See [link to relevant documentation]). Figure 1 .

[0050] 4. Differentiation culture

[0051] After resistance screening, callus particles were transferred to a differentiation medium with a slightly lower concentration for regeneration and differentiation into seedlings. The specific resistance-specific differentiation medium consisted of: N6 majors, MS iron salts, B5 minors, B5 vitamins, 500 mg / L proline, 1 g / L casein enzymatic hydrolysate, 30 g / L sucrose, 1.5 mg / L NAA, 1 mg / L 6BA, 2.5 g / L phytagel, and 2 mM Glyphosate. Once shoots or seedlings formed, they were transferred to MS rooting medium, samples were taken, and tissue sequencing analysis was performed.

[0052] 5. Mutation site analysis

[0053] Genomic DNA extracted from this plant was sent to Invitrogen for genome sequencing. The sequencing results were compared with those of the wild-type Nipponbare EPSPS gene; see [link to results]. Figure 2 A mutation was found at position 742 of the EPSPS gene, changing from T to C. This resulted in a change from tyrosine (Y) to histidine (H) at position 248 of the EPSPS gene. This type of mutation can significantly increase the resistance of rice to glyphosate herbicides. The nucleotide sequence of the EPSPS gene of the herbicide-resistant mutant is shown in SEQ ID NO:1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO:2, which is not listed as a separate sequence here.

[0054] The mutant callus and shoots showed resistance to the herbicide in a medium supplemented with 5 mM glyphosate herbicide resistance.

[0055] Example 2

[0056] Resistance identification of mutant plants

[0057] To further verify the effects of this mutant on herbicide resistance and yield, both mutant and wild plants were planted in an experimental field. The mutant plant was Nipponbare rice with the introduced mutant gene.

[0058] The homozygous mutant seeds obtained in the above experiments were planted to cultivate seedlings and then further developed into plants for experimental comparison.

[0059] The specific process is as follows:

[0060] Preparation of herbicide dilution: Use glyphosate isopropylamine salt herbicide with 30% glyphosate active ingredient. Add 90 ml of water to each milliliter of stock solution to obtain 20 mmol·L⁻¹. -1 Secondary dilution, set aside. Then dilute with water to prepare the field-recommended concentration (7 mmol·L⁻¹).-1 Glyphosate solutions at concentrations of 1, 2, and 3 times.

[0061] Rice seedling cultivation: Seeds containing homozygous lines of mutants were soaked at 28℃ for 1-2 days, germinated on moist filter paper, and after germination, seeds with roughly equivalent buds were selected from wild-type and mutant plants and cultured in rice nutrient solution.

[0062] 400 mutant and 400 wild-type seedlings of the same size and growth were randomly selected at 20 days old. 3-5 seedlings were planted in one pot. Wild-type zone 1 (no glyphosate solution sprayed), wild-type zone 2 (sprayed with glyphosate solution at 1 times the recommended concentration), wild-type zone 3 (sprayed with glyphosate solution at 2 times the recommended concentration), and wild-type zone 4 (sprayed with glyphosate solution at 3 times the recommended concentration) served as the control group. Mutant zone 1 (no glyphosate solution sprayed), mutant zone 2 (sprayed with glyphosate solution at 1 times the recommended concentration), mutant zone 2 (sprayed with glyphosate solution at 2 times the recommended concentration), and mutant zone 4 (sprayed with glyphosate solution at 3 times the recommended concentration) served as the experimental group. 50 seedlings were planted in each zone.

[0063] After 20 days of growth, wild-type zone 1 was sprayed with water, wild-type zone 2 was sprayed with glyphosate solution at 1x the recommended concentration, wild-type zone 3 was sprayed with glyphosate solution at 2x the recommended concentration, and wild-type zone 4 was sprayed with glyphosate solution at 3x the recommended concentration. Mutant zone 1 was sprayed with water, mutant zone 2 was sprayed with glyphosate solution at 1x the field recommended concentration, mutant zone 3 was sprayed with glyphosate solution at 2x the field recommended concentration, and mutant zone 4 was sprayed with glyphosate solution at 3x the recommended concentration. All spraying areas were the leaf surfaces of the plants. Phenotypic changes in the rice plants were observed regularly. Results are shown in [link to results]. Figure 3 It was observed that 7 days after the herbicide was sprayed, the wild-type rice plants in zones 2 to 4 of the experimental group began to wither over a large area, while the leaves of the mutants in zones 2 to 4 could grow almost normally.

[0064] This shows that rice plants containing the EPSPS mutant gene are resistant not only to glyphosate herbicides at 1 times the recommended field concentration, but also to glyphosate herbicides at 3 times the recommended field concentration, indicating that the mutant gene has the property of resisting high concentrations of herbicides.

[0065] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions 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 plant herbicide-resistant EPSPS mutant gene, characterized in that, The nucleotide sequence of the plant herbicide-resistant EPSPS mutant gene is shown in SEQ ID NO.

1.

2. A mutant protein of plant herbicide-resistant EPSPS, characterized in that, The amino acid sequence of the plant herbicide-resistant EPSPS mutant protein is shown in SEQ ID NO.

2.

3. An expression box, characterized in that, The expression cassette contains the plant herbicide-resistant EPSPS mutant gene as described in claim 1.

4. A recombinant vector, characterized in that, The recombinant vector contains the plant herbicide-resistant EPSPS mutant gene as described in claim 1.

5. The use of the plant herbicide-resistant EPSPS mutant gene of claim 1, the expression cassette of claim 3, or the recombinant vector of claim 4 in equipping rice with glyphosate resistance.

6. The application according to claim 5, characterized in that, The application includes: introducing the plant herbicide-resistant EPSPS mutant gene of claim 1, the expression cassette of claim 3, or the recombinant vector of claim 4 into target rice, so that the target rice has glyphosate resistance.

7. The application according to claim 6, characterized in that, Methods for introducing the target rice include genetic modification, hybridization, or backcrossing.

8. The application of the plant herbicide-resistant EPSPS mutant gene of claim 1, the expression cassette of claim 3, or the recombinant vector of claim 4 in the breeding of homozygous rice progeny with glyphosate resistance.

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