Rice EPSPS mutant protein and application thereof

Abstract

The invention relates to the technical field of biological genes, in particular to a rice EPSPS mutant protein and application thereof. The rice EPSPS mutant protein is characterized in that wild EPSPS protein is mutated on the 169th site of an amino acid sequence, and threonine is mutated into serine. The rice containing the EPSPS-T169S mutant protein shows resistance to glyphosate herbicides, the offspring of the rice can be kept homozygous, and the yield of the rice is remarkably higher than that of a wild type. More importantly, the mutant protein is not obtained through a transgenosis or gene editing technology, and can be directly used for germplasm improvement and production.

Description

Technical Field

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

[0002] Weeds are one of the important factors affecting crop yields. Chemical weed control is the main measure to control weeds in rice fields. Conventional herbicides have different weed control spectra due to their different mechanisms of action. However, weed control has always been a difficult point. The main reason is that weeds are very similar to rice in terms of taxonomy and physiology. Improper use of herbicides can cause serious damage to crops. In recent years, a control strategy of using special herbicides and herbicide-resistant rice varieties to control weeds in rice fields has been proposed. Therefore, herbicide-resistant rice has received more and more attention and has become an important direction of genetic breeding.

[0003] Glyphosate is one of the most important, widely used and excellent herbicides to date. However, due to the widespread commercialization of glyphosate-resistant genetically modified crops, the use of glyphosate has increased rapidly and weed resistance has occurred, which has not only seriously affected the efficacy of glyphosate and its future sustainable application, but also posed a threat to the safety of modern agricultural production. Glyphosate is one of the most widely used herbicides in the world and has been used for nearly 40 years.

[0004] The mechanism of action of glyphosate 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. In general, glyphosate can bind to natural EPSPS very well. Studies have found that individual EPSPS mutants have reduced binding ability with glyphosate and can tolerate glyphosate. The current method for breeding glyphosate-resistant varieties is to use genetic engineering methods to introduce glyphosate-resistant genes from bacteria into crops, thereby breeding new varieties of transgenic glyphosate-resistant crops. Since the promotion began in 1996, the planting area has increased rapidly. By 2015, the global planting area of ​​glyphosate-resistant transgenic crops has reached 150 million hectares, accounting for 83% of the total planting area of ​​transgenic crops, bringing huge benefits to agricultural production and the environment. However, the most widely used glyphosate-resistant gene in agriculture is CP4EPSPS derived from the CP4 strain of Agrobacterium tumefaciens. Although many glyphosate-resistant EPSPS genes have been found in microorganisms, these genes have not yet been widely used in crops. The application of these microbial glyphosate-resistant genes in crops is to express these genes, such as CP4EPSPS, in crops by transgenic methods. At present, some variants of EPSPS proteins have been found in glyphosate-resistant cattle grass, but it is currently difficult to obtain naturally mutated glyphosate-resistant EPSPS genes in crops. Mutated EPSPS genes can only be transferred into crops by transgenic methods to obtain glyphosate-resistant crops. However, due to the regulatory policies of genetically modified crops and the acceptance of genetically modified crops around the world, their large-scale promotion and use are still subject to many restrictions. Therefore, it is urgent to develop genes that do not affect plant growth and development and have glyphosate resistance in plants, 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 EPSPS mutant protein and its application. The rice EPSPS mutant protein provided by the present invention can not only improve the herbicide resistance of rice, but also maintain the stability of agronomic traits such as rice yield and plant height, and thus can be used to cultivate glyphosate-resistant crops.

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

[0007] In a first aspect, the present invention provides a rice EPSPS mutant protein in an optional embodiment, wherein the rice EPSPS mutant protein is a wild-type EPSPS protein with a mutation at position 169 in the amino acid sequence, from threonine to serine.

[0008] Preferably, the amino acid sequence of the rice EPSPS mutant protein is shown as SEQ ID NO.2.

[0009] In a second aspect, the present invention provides a rice EPSPS mutant gene in an optional embodiment, wherein the rice EPSPS mutant gene is a wild-type EPSPS gene with a mutation at position 506 in the nucleotide sequence, from C to G.

[0010] Preferably, the nucleotide sequence of the rice EPSPS mutant gene is shown as SEQ ID NO.1.

[0011] The mutation of C at position 506 to G caused the mutation of amino acid 169 of the EPSPS gene encoding from threonine (T) to serine (S). The rice EPSPS mutant was resistant to glyphosate while maintaining its own biological enzyme catalytic activity. Plants transformed with the rice EPSPS mutant could grow normally, and the rice EPSPS mutant had broad application prospects.

[0012] In a third aspect, the present invention provides, in an optional embodiment, a nucleic acid molecule encoding the above rice EPSPS mutant protein.

[0013] In a fourth aspect, the present invention provides an expression cassette in an optional embodiment, wherein the expression cassette contains the above rice EPSPS mutant protein, the above rice EPSPS mutant gene or the above nucleic acid molecule.

[0014] In a fifth aspect, the present invention provides a recombinant vector in an optional embodiment, wherein the recombinant vector contains the above-mentioned rice EPSPS mutant protein, the above-mentioned rice EPSPS mutant gene or the above-mentioned nucleic acid molecule.

[0015] In a sixth aspect, the present invention provides, in an optional embodiment, a use of the above-mentioned rice EPSPS mutant protein, the above-mentioned rice EPSPS mutant gene, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or the above-mentioned recombinant vector in making a target plant resistant / tolerant to glyphosate.

[0016] Preferably, the method for making the target plant resistant / tolerant to glyphosate comprises:

[0017] (1) introducing the sequence encoding the rice EPSPS mutant protein or the rice EPSPS mutant gene into the target plant; or,

[0018] (2) Cultivating homozygous plant progeny or plant seeds with the rice EPSPS mutant gene or the rice EPSPS mutant protein.

[0019] Furthermore, the method of introducing the rice EPSPS mutant gene into the target plant includes hybridization, backcrossing, transgenic or gene editing technology.

[0020] Specifically, the application includes the following steps:

[0021] (1) After hulling and sterilizing rice seeds, the embryos are separated and placed on a callus induction medium to produce secondary callus;

[0022] (2) transferring the secondary callus to a new callus induction medium for pre-culture;

[0023] (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 expression vector having a plant herbicide-resistant EPSPS mutant gene or other genes encoding the mutant protein;

[0024] (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;

[0025] (5) placing the callus tissue of step (4) on a pre-screening medium and culturing for 5-7 days;

[0026] (6) transferring the callus tissue of step (5) to a screening medium to obtain resistant callus tissue;

[0027] (7) transferring the resistant callus to a differentiation and regeneration medium to differentiate into shoots; and

[0028] (8) Transfer the seedlings from step (7) to a rooting medium for rooting.

[0029] The nucleotide sequence of SEQ ID NO.1 is shown below.

[0030]

[0031] The amino acid sequence of SEQ ID NO.2 is shown below.

[0032] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKAL GLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGSAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAA PLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKK HLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a photo of the resistant callus produced on the herbicide-containing medium after induction by the chemical mutagen sodium nitrite in Example 1 of the present invention;

[0034] Figure 2 This is a schematic diagram of the comparison results of the sequencing images of the wild type and the mutant near the mutation site in Example 1 of the present invention;

[0035] Figure 3 This is a schematic diagram of the growth comparison results of the wild type and EPSPS mutant strains after spraying the herbicide in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1

[0040] Directed screening of herbicide resistance loci by rice callus mutagenesis

[0041] (1) After the mature, mildew-free rice seeds are husked and sterilized, the embryos are separated with a scalpel and placed on a callus induction medium to produce secondary callus;

[0042] (2) transferring the secondary callus obtained in step (1) to a new callus induction medium for pre-culture;

[0043] (3) transferring the callus obtained in step (2) to a mutagenesis medium for mutagenesis culture for 10 days;

[0044] (4) placing the callus tissue from step (3) on a pre-screening medium and culturing for 8 days;

[0045] (5) The callus tissue of step (4) is transferred to the screening medium to obtain resistant callus tissue. The results are shown in Figure 1 ;

[0046] (6) transferring the resistant callus from step (5) to a differentiation and regeneration medium to differentiate into seedlings;

[0047] (7) Transfer the seedlings from step (6) to a rooting medium for rooting.

[0048] The seeds in step (1) are mature seeds, the rice is japonica rice Nipponbare, and the culture medium in each step is shown in Table 1.

[0049] Table 1 Culture medium in each step

[0050]

[0051] About 20,000 calli were induced and transferred to a medium containing 5mM glyphosate for herbicide resistance. Ten resistant calli were obtained and differentiated into seedlings. The genomic DNA of one of the calli was extracted. The EPSPS gene was amplified and sent to Shanghai Biotech for genome sequencing. The sequencing results were compared with the wild-type Nipponbare EPSPS gene. The results are shown in Figure 2It was found that a mutation from C to G occurred at position 506 of EPSPS, resulting in a change from threonine (T) to serine (S) at position 169 of the EPSPS gene. 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 the EPSPS protein of the herbicide-resistant mutant is shown in SEQ ID NO.2.

[0052] Example 2

[0053] Application of mutant EPSPS in herbicide resistance

[0054] Mutant plants (denoted as T169S) and wild plants (rice containing the wild-type Nipponbare EPSPS gene, denoted as WT) were planted in the test field. The mutant plants were Nipponbare rice introduced with the mutant gene of Example 1. The homozygous mutant seeds obtained in Example 1 were used for planting and cultivated into seedlings, which were then allowed to grow further into plants. The specific process is as follows:

[0055] Prepare herbicide dilution: Choose glyphosate isopropylamine salt herbicide, whose active ingredient concentration is 41%. Mix each ml of stock solution with 90 ml of water to obtain 20mmol·L -1 The secondary dilution is set aside.

[0056] Cultivation of rice seedlings: The homozygous mutant seeds obtained in Example 1 were placed in a constant temperature incubator at 37°C to absorb water for 48 hours, and then germinated on moist filter paper. After germination, seeds with similar sprout sizes were selected from the wild type and mutant plants, respectively, and placed in rice nutrient solution for cultivation.

[0057] Planting and growth: In a hydroponic environment, after 2 weeks, select rice seedlings with uniform growth, plant mutant and wild-type rice seedlings in different areas, plant 50 seedlings in each area, and repeat the planting in 3 areas. The rice nutrient solution used is Hogland nutrient solution.

[0058] After 30 days of growth, spray 10mmol·L -1 About 5 days after spraying, the wild-type rice plants in the experimental group began to wilt on a large scale, while the leaves of the homozygous mutant plants could grow almost normally (see the results). Figure 3 ). After 2 months of growth, the mutant plants were able to bloom and bear fruit normally.

[0059] The above results show that the application of herbicides has a significant inhibitory effect on wild-type rice plants, causing a large number of plants to wilt, while the mutant plants are almost unaffected. In addition, under normal growth conditions, the agronomic traits such as yield and plant height of the mutant plants remain relatively stable (see Table 2 for the results).

[0060] Table 2 Comparison of agronomic traits between EPSPS mutants and controls

[0061]

[0062] 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 EPSPS mutant protein, characterized in that: The rice EPSPS mutant protein is a wild-type EPSPS protein with a mutation at position 169 in the amino acid sequence, from threonine to serine.

2. The rice EPSPS mutant protein according to claim 1, characterized in that The amino acid sequence of the rice EPSPS mutant protein is shown in SEQ ID NO.

2.

3. A rice EPSPS mutant gene, characterized in that: The rice EPSPS mutant gene is a wild-type EPSPS gene with a mutation at position 506 in the nucleotide sequence, from C to G.

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

1.

5. A nucleic acid molecule, characterized in that Encoding the rice EPSPS mutant protein according to claim 1 or 2.

6. An expression cassette, characterized in that The expression cassette contains the rice EPSPS mutant protein according to claim 1 or 2, the rice EPSPS mutant gene according to claim 3 or 4, or the nucleic acid molecule according to claim 5.

7. A recombinant vector, characterized in that: The recombinant vector contains the rice EPSPS mutant protein according to claim 1 or 2, the rice EPSPS mutant gene according to claim 3 or 4, or the nucleic acid molecule according to claim 5.

8. Use of the rice EPSPS mutant protein according to claim 1 or 2, the rice EPSPS mutant gene according to claim 3 or 4, the nucleic acid molecule according to claim 5, the expression cassette according to claim 6 or the recombinant vector according to claim 7 in making a target plant resistant / tolerant to glyphosate.

9. The use according to claim 8, characterized in that: Methods for making target plants resistant / tolerant to glyphosate include: (1) introducing the sequence encoding the rice EPSPS mutant protein or the rice EPSPS mutant gene into the target plant; or, (2) Cultivating homozygous plant progeny or plant seeds with the rice EPSPS mutant gene or the rice EPSPS mutant protein.

10. The use according to claim 9, characterized in that: The method of introducing the rice EPSPS mutant gene into the target plant includes hybridization, backcrossing, transgenic or gene editing technology.

Images