A plant herbicide-resistant EPSPS mutant protein and its application

Abstract

The present invention discloses a plant EPSPS mutant protein and its application. The present invention also provides a gene sequence encoding the EPSPS mutant protein. The mutant plants containing the mutant of the present invention have excellent performance in herbicide resistance, especially glyphosate herbicide resistance. At the same time, considering that there have been many reports on genes with herbicide resistance at present. The mutant protein of the present invention not only endows plants with herbicide resistance, but also can increase the yield of plants, which is of great significance to agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of genes, and particularly to a plant herbicide-resistant EPSPS mutant protein and its application. Background Art

[0002] With the development of social economy, the production mode of rice is undergoing a huge transformation, and direct seeding of rice is becoming more and more common in production. The weed damage and weedy rice in paddy fields caused by direct seeding have become the key bottleneck problems restricting the improvement of rice production quality and efficiency. In particular, the frequent occurrence of volunteer rice in direct seeding seriously affects the yield and quality of rice. At present, volunteer rice (also known as weedy rice) in direct seeding generally causes a reduction in production of 5% - 10%, and in severe cases, more than 50% reduction, and the rice quality drops by 1 grade. Volunteer rice is usually caused by the falling of miscellaneous rice grains in the previous year and the introduction by external machinery. In terms of genetic essence, it is very close to rice and is difficult to control by traditional chemical methods. At present, it can only be prevented by manual removal or cultivation method transformation, but the control effect is poor and the cost is high. Therefore, there is an urgent need to develop herbicide-resistant rice materials.

[0003] Glyphosate, an organophosphorus herbicide, is systemic. Due to its high herbicidal efficiency, wide range, low solubility in water, low soil residue, and relatively friendly environment, it is the post-emergence herbicide with the broadest weed control spectrum and can eradicate annual and perennial weeds. Since its commercialization in 1974, it has been quickly applied in glyphosate-resistant soybean, corn, cotton, rapeseed, and sugar beet fields and has quickly occupied a place in the pesticide market and become the most widely used herbicide. However, at the same time, as a non-selective herbicide, glyphosate also harms crops without discrimination while efficiently controlling weeds. Therefore, cultivating and planting glyphosate-resistant crop varieties can not only reduce the burden of manual weeding but also increase crop yields, which is beneficial to the mechanized production of crops.

[0004] Glyphosate mainly achieves its herbicidal goal by interfering with the shikimic acid synthesis pathway in plants. Its mechanism of action is mainly to competitively inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway. This synthase is a key enzyme in the biosynthesis of aromatic amino acids in fungi, bacteria, algae, and higher plants. Generally, glyphosate can bind well to natural EPSPS. Studies have found that the binding ability of mutant EPSPS to glyphosate decreases, and it can tolerate glyphosate. In the 1990s, the seed industry giant Monsanto was the first to isolate the mutant CP4 gene of the EPSPS gene from Agrobacterium tumefaciens and transfer this gene into plants to make them glyphosate-resistant, obtaining glyphosate-resistant transgenic soybeans, corn, and cotton. Through the study of glyphosate-resistant weeds, new EPSPS proteins insensitive to glyphosate were 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). However, currently, when using biotechnological methods such as gene editing to create the TIPS double mutation 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 regulatory policies for crop bio-breeding, gene-edited improved varieties cannot be directly applied in the production process. Therefore, there is an urgent need to develop genes that do not affect plant growth and development and confer glyphosate resistance to crops, which is of great significance for the development of homozygous varieties resistant to glyphosate herbicides. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an EPSPS mutant protein that can confer glyphosate resistance to rice, and homozygous offspring with herbicide resistance can be obtained using this mutant protein.

[0006] Another object of the present invention is to provide the application of the above-mentioned coding gene in cultivating glyphosate-resistant plants. Compared with the existing EPSPS mutant proteins, the EPSPS-P173G mutant protein of the present invention can not only improve the herbicide resistance of plants but also increase the plant yield.

[0007] The specific EPSPS-P173G mutant herbicide-resistant plant of the present invention is obtained by chemical mutagenesis.

[0008] Specifically, the present invention provides a mutant EPSPS protein for plant herbicide resistance, and the 173rd position of the mutant EPSPS protein for plant herbicide resistance is mutated.

[0009] Preferably, the amino acid sequence of the EPSPS mutant protein is as shown in SEQ ID NO: 1, 3, 4 or 5 in the sequence listing.

[0010] On the other hand, the present invention provides a plant herbicide-resistant EPSPS mutant gene, and the plant herbicide-resistant EPSPS mutant gene is a gene encoding the protein described in claim 1. Preferably, the EPSPS mutant gene comprises at least the sequence shown in SEQ ID NO: 2 in the sequence listing.

[0011] On the other hand, the present invention provides an expression cassette, and the expression cassette contains the plant herbicide-resistant EPSPS mutant protein described in claim 1 or the plant herbicide-resistant EPSPS mutant gene described in claim 3.

[0012] On the other hand, the present invention provides a recombinant vector, and the recombinant vector contains the plant herbicide-resistant EPSPS mutant protein or the plant herbicide-resistant EPSPS mutant gene.

[0013] On the other hand, the present invention provides an application of the plant herbicide-resistant EPSPS mutant protein, the plant herbicide-resistant EPSPS mutant gene, the expression cassette and the recombinant vector. The application includes introducing the gene sequence encoding the above protein into a target plant to make the target plant have glyphosate resistance / tolerance.

[0014] Preferably, the application includes introducing one of the above fragments into a target plant to make the target plant have herbicide resistance. The plant herbicide-resistant EPSPS mutant protein and the plant herbicide-resistant EPSPS mutant gene described in claim 2 are used to cultivate homozygous plant offspring or plant seeds.

[0015] Preferably, the method for introducing the gene into a recipient plant in the application includes transgenic technology, hybridization or backcross.

[0016] Preferably, the application includes:

[0017] (1) Shell the rice seeds, sterilize them, and then isolate the embryos, and place them on a callus induction medium to produce secondary callus;

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

[0019] (3) Contact the callus obtained in step (2) with Agrobacterium for 15 minutes to obtain resistant callus. The Agrobacterium has been transformed with a recombinant expression vector containing a plant herbicide-resistant EPSPS mutant gene or other genes encoding the mutant protein.

[0020] (4) Transfer the callus from step (3) to a petri dish with three sterile filter papers on the bottom, and culture at 21 - 23 °C for 48 hours;

[0021] (5) Culture the callus from step (4) on the pre - screening medium for 5 - 7 days;

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

[0023] (7) Transfer the resistant callus to the differentiation and regeneration medium to differentiate into seedlings; and

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

[0025] The present invention has the following advantages:

[0026] 1. The mutant protein of the present invention is obtained by using sodium nitrite or 5 - bromouracil as a specific mutagen, overcoming the problem that traditional mutagens such as EMS are difficult to mediate the variation of OsEPSPS - P173G, and greatly increasing the probability of generating the target mutation.

[0027] 2. Plants containing the mutant protein and gene of the present invention can not only obtain herbicide resistance, but their offspring can maintain homozygosity, and the yield of their offspring is significantly higher than that of the wild type.

[0028] The mutant protein obtained in the present invention is not obtained through transgenic and gene - editing operations, but through a chemical mutagenesis process, with wide adaptability. The newly created rice herbicide - resistant germplasm material can directly enter production. Moreover, the mutant protein of the present invention shows herbicide - resistant performance in rice, maize, wheat, and soybean, with a wider range of adaptable species. Description of the Drawings

[0029] Figure 1 Resistant callus produced on the herbicide - containing medium after induction by the chemical mutagen sodium nitrite.

[0030] Figure 2 Rice mutant plants growing in a herbicide environment.

[0031] Figure 3 Maize mutant plants growing in a herbicide environment.

[0032] Figure 4 Soybean mutant plants growing in a herbicide environment.

[0033] Figure 5 Wheat mutant plants growing in a herbicide environment.

[0034] Figure 6Is a wild-type rice plant grown in a herbicide environment.

[0035] Figure 7 Is a wild-type corn plant grown in a herbicide environment.

[0036] Figure 8 Is a wild-type soybean plant grown in a herbicide environment.

[0037] Figure 9 Is a wild-type wheat plant grown in a herbicide environment. Detailed implementation mode

[0038] The present invention will be described in detail below in conjunction with the accompanying drawings and its embodiments, but the protection scope of the present invention is not limited to the scope described in the embodiments.

[0039] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation mode of the present invention is not limited thereto. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following embodiments are all commercially available products unless otherwise specified.

[0040] The following combines the attached Figure 1 And the detailed implementation mode to further describe the technical solution of the present invention in detail. The specific embodiments described herein are only used to explain the present invention and are not limited to the present invention.

[0041] The method for mutagenesis and directional screening of herbicide-resistant sites in rice callus of the present invention specifically includes the following steps:

[0042] 1. Preparation of explants: After removing the glumes from mature seeds, soak the seeds in 70% alcohol for 1 min and pour out the alcohol. Soak the seeds in a 50% sodium hypochlorite solution (the effective chlorine concentration of the stock solution is greater than 4%) containing 1 drop of Tween 20 for 40 min (150 r / 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. Use a scalpel to peel the embryo along the aleurone layer of the seeds and inoculate the embryo on the callus induction medium. After dark culture at 30 °C for 11 days, separate the callus from the endosperm and germ, and select the primary callus with good state and strong division after removing the buds.

[0043] 2. Mutagenic culture: Transfer the callus with good state after induction to the mutagenic culture medium for 7 - 14 days of mutagenic culture. The mutagenic culture medium is 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% NaNO 2 ; Culture at 30°C with a 16 - hour light / 8 - hour dark cycle for 1 - 2 weeks to obtain the genome of chemically mutagenized rice callus;

[0044] 3. Screening culture: The tolerance of rice to herbicides varies at different growth and development stages. Similarly, the tolerance to stress at each stage under tissue culture conditions also differs. Use glyphosate to simulate the herbicide environment and add it to the culture medium to screen for resistant mutants directionally. We add the appropriate screening concentration after testing to the screening medium, and transfer the rice callus after 1 - 2 weeks of mutagenesis to the screening medium for 28 - 42 days of screening culture. The screening medium is the induction medium + 5 mM Glyphosate. Observe irregularly until resistant callus particles that can grow normally appear.

[0045] 4. Differentiation culture: Transfer the resistant callus particles after screening to a differentiation medium with a slightly lower concentration to regenerate and differentiate into seedlings. The specific resistant differentiation medium is: 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 6 - BA, 2.5 g / L phytagel, 2 mM Glyphosate. After budding or growing into seedlings, transfer them to the MS rooting medium, take samples for detection, and conduct tissue sequencing analysis.

[0046] 5. Repeat the above steps to obtain different mutants, and further culture the seedlings of different mutants to make them grow. And at a specific growth stage, conduct drought induction, and select the plants with the relatively highest drought tolerance under the same degree of drought induction conditions.

[0047] 6. The genomic DNA extracted from the plant was sent to Invitrogen for genomic sequencing. By comparing the sequencing results with the EPSPS gene of wild-type Nipponbare, it was found that a C-G mutation occurred at the 517th position and a C-G mutation occurred at the 518th position in OsEPSPS, resulting in the amino acid at the 173rd position of the OsEPSPS gene changing from proline (P) to glycine (G). This type of mutation can significantly improve the resistance of rice to glyphosate herbicides. The nucleotide sequence of the EPSPS gene of the herbicide-resistant mutant and the amino acid sequence of its encoded protein are shown in SEQ ID NO:1. The 173rd position of the original gene sequence of OsEPSPS is proline (P), that is, modifying the 173rd position to proline (P) in SEQ ID NO:1 is its original sequence, which is not listed separately as a sequence here.

[0048] The callus and young buds of this mutant showed resistance to the herbicide in the medium supplemented with 5 mM glyphosate herbicide.

[0049] In this example, the gene and amino acid sequences of the mutant obtained by induction are as follows (the same as the corresponding sequences in SEQ ID NO:1 and 2), and the mutation positions are marked.

[0050]

[0051] Example 2: Application process of EPSPS gene

[0052] To further verify the effects of this mutant on herbicide resistance and yield, the applicant planted the mutant plants and wild plants in the experimental field respectively. The mutant plants are Nipponbare rice into which the mutant gene has been introduced.

[0053] The homozygous seeds of the mutant obtained in Example 1 were planted, and the obtained seeds were cultivated into seedlings and further grown into plants for experimental comparison.

[0054] The specific process is as follows:

[0055] Prepare the herbicide dilution: Select the glyphosate isopropylammonium salt herbicide with 30% glyphosate active ingredient, add 90 ml of water to each milliliter of the stock solution, and obtain a 20 mmol·L -1 secondary dilution for standby.

[0056] Cultivation of rice seedlings: The seeds of the homozygous line containing the mutant were soaked at 28 °C for 1-2 days, germinated on moist filter paper, and after germination, seeds with roughly equivalent young buds were selected from wild-type and mutant plants and cultured in rice nutrient solution.

[0057] Randomly select 100 20-day-old seedlings each of the mutant type and the wild type with the same growth vigor and size. Pot 3 - 5 seedlings in one pot. Divide the wild type and the mutant into two regions respectively, which serve as the experimental group and the control group, and plant 50 seedlings in each region.

[0058] After growing for 20 days, take an appropriate amount of the previously prepared herbicide solution and further dilute it to 7 mmol·L-1, and evenly spray it on the leaf surfaces of the plants in the experimental group (wild type and mutant) regions. Regularly observe the phenotypic changes of the rice plants. It can be found that 2 - 5 days after spraying the herbicide, the rice plants in the wild type region of the experimental group began to show large areas of withering, while the leaves of the homozygous mutant plants could grow almost normally. And after growing for 2 months, they could flower and set seeds normally, and the yield per plant was about 10.81 g.

[0059] In addition, without spraying the herbicide, the number of grains per plant of the mutant increased, but the grain weight did not change significantly, resulting in the yield of the mutant being about 17.3% higher than that of the wild type.

[0060]

[0061] The above results show that the application of the herbicide will significantly inhibit the wild type rice plants, and a large number of plants wither, while the mutant plants are hardly affected. Moreover, under normal growth conditions, the yield is higher than that of the wild type rice.

[0062] Example 3

[0063] In addition, the applicant found that the mutation site of the present invention is also effective in corn and soybeans. Therefore, in a manner similar to the above-mentioned rice, the applicant induced mutations in corn and soybeans.

[0064] Prepare corn and soybean seeds respectively. Soak the seeds in 70% alcohol for 1 min and then pour out the alcohol. Soak the seeds in a 50% sodium hypochlorite solution (the available chlorine concentration of the stock solution is greater than 4%) containing 1 drop of Tween 20 for 40 min (150 r / 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. Use a scalpel to peel off the embryos and inoculate the embryos on the callus induction medium. After dark culture at 30°C for 11 - 15 days, separate the callus from the endosperm and germ, and obtain the primary callus with good state, strong division and without buds.

[0065] Mutagenic culture: Transfer the callus with better state after induction to the mutagenic medium for mutagenic culture for 7 - 14 days; culture at 30°C under 16 hours of light / 8 hours of darkness for 1 - 2 weeks.

[0066] Considering that the tolerance of corn and soybeans to herbicides varies during different growth and development stages, and the tolerance to stress at each stage under tissue culture conditions also differs. Using glyphosate to simulate the herbicide environment and adding it to the culture medium for directional screening of resistant mutants, the inventor added the appropriate screening concentration after testing to the screening medium, transferred the calli of corn and soybeans after 1 - 2 weeks of mutagenesis to the screening medium for screening culture for 28 - 42 days. The screening medium was the induction medium + 5 mM Glyphosate. Observe irregularly until resistant callus particles that can grow normally appear. Transfer the callus particles after resistant screening to a differentiation medium with a slightly lower concentration to regenerate and differentiate into seedlings.

[0067] Take leaves from the grown corn and soybean plants for protein sequencing, and screen for sites from the obtained mutants. This mutant has the same locus as the rice mutant, and its sequences are as shown in SEQ ID No.3 and 4 in the sequence listing (the same as the following sequences).

[0068] Protein sequence at the corresponding mutant position of corn EPSPS:

[0069]

[0070] Protein sequence at the corresponding mutant position of soybean EPSPS:

[0071]

[0072]

[0073] Spray the mutant and wild - type plants after 20 days of growth period with the same concentration of glyphosate herbicide as in Example 1.

[0074] The mutant corn plants growing in the herbicide environment are as shown in Figure 3 shown. The mutant soybean plants growing in the herbicide environment are as shown in Figure 4 shown. It can be seen that the herbicide environment has little impact on the growth of the above - mentioned plants. The wild - type corn plants growing in the glyphosate herbicide environment are as shown in Figure 7 shown. The wild - type soybean plants growing in the glyphosate herbicide environment are as shown in Figure 8 shown.

[0075] Example 4

[0076] Using a similar method as above, the applicant conducted induced mutation and screening in wheat and obtained the corresponding mutant proteins.

[0077] Protein sequence at the corresponding mutant position of wheat EPSPS:

[0078]

[0079]

[0080] The mutant type and the wild type after 20 days of growth period are respectively sprayed with glyphosate herbicide at the same concentration as in Example 1.

[0081] The wheat mutant plants growing in the herbicide environment are as Figure 5 shown, and the wild wheat plants growing in the herbicide environment are as Figure 9 shown.

[0082] Although the principles of the present invention have been described in detail above in connection with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are only explanations of the illustrative implementation manners of the present invention and do not limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention all fall within the protection scope of the present invention.

Claims

1. A rice herbicide-resistant EPSPS mutant gene, characterized in that: The rice herbicide-resistant EPSPS mutant gene is shown in SEQ ID NO:

2.

2. An expression cassette, characterized in that The expression cassette comprises the rice herbicide-resistant EPSPS mutant gene according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector comprises the rice herbicide-resistant EPSPS mutant gene according to claim 2.

4. An application of the rice herbicide-resistant EPSPS mutant gene according to claim 1, characterized in that: The application comprises introducing the rice herbicide-resistant EPSPS mutant gene into target rice, so that the target rice has resistance to / tolerance to glyphosate.

5. The use according to claim 4, characterized in that: The application comprises introducing the rice herbicide-resistant EPSPS mutant gene of claim 1 into target rice, so that the target rice has herbicide resistance, and is used for cultivating homozygous rice progeny.

6. The use according to claim 5, characterized in that: Methods for introducing the gene into recipient rice include transgenic, hybridization or backcrossing.

7. The use according to claim 6, characterized in that: The applications include: (1) After hulling and sterilizing rice seeds, the embryos are separated and placed on a callus induction medium to produce secondary callus; (2) transferring the secondary callus to a new callus induction medium for pre-culture; (3) contacting the callus obtained in step (2) with Agrobacterium for 15 minutes to obtain resistant callus, into which the recombinant expression vector having the rice herbicide-resistant EPSPS mutant gene is transferred; (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; (5) transferring the callus tissue of step (4) to a screening medium to obtain resistant callus tissue; (6) transferring the resistant callus to a differentiation and regeneration medium to differentiate into shoots; and (7) transferring the shoots of step (6) to a rooting medium for rooting.

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