Rice epsps mutant protein and application thereof

Abstract

The application relates to the field of biological gene technology, and particularly relates to a rice EPSPS mutant protein and application thereof. The rice EPSPS mutant protein is wild-type EPSPS protein which is mutated at the 169th amino acid in the amino acid sequence, and is mutated from threonine to serine. The rice containing the EPSPS-T169S mutant protein in the application shows resistance to glyphosate herbicide, the offspring can also be kept homozygous, and the yield is significantly higher than that of the wild type. More importantly, the mutant protein is not obtained through transgenic or gene editing technology, and can be directly used for germplasm improvement and production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological genes, and particularly relates to a rice EPSPS mutant protein and application thereof. BACKGROUND

[0002] Weeds are one of the important factors affecting crop yield, and chemical weeding is the main measure to control weeds in rice fields. Conventional herbicides have different weed control spectrum due to different action mechanisms, but the control of weeds has always been a difficult point. The main reason is that weeds are very similar to rice in taxonomy and physiology, and improper use of herbicides can cause significant phytotoxicity 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, so herbicide-resistant rice has attracted more and more attention and become an important direction of genetic breeding.

[0003] Glyphosate is one of the most important, widely used and best herbicides so far. However, due to the wide commercialization of glyphosate-resistant transgenic crops, the use of glyphosate has rapidly increased, and weeds have developed resistance, which not only has a serious impact on the efficacy of glyphosate and its future sustainable application, but also poses 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 four decades.

[0004] The mechanism of action of glyphosate is mainly competitive inhibition of the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway. The 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, and research has found that individual EPSPS mutants have reduced binding capacity to glyphosate and can tolerate glyphosate. At present, the method for cultivating glyphosate-resistant varieties is to introduce an anti-glyphosate gene from bacteria into crops to cultivate new transgenic glyphosate-resistant crop varieties. Since the promotion in 1996, the planting area has increased rapidly, and by 2015, the global glyphosate-resistant transgenic crop planting area has reached 150 million hectares, accounting for 83% of the total transgenic crop planting area, which has brought great benefits to agricultural production and the environment. However, the most widely used glyphosate-resistant gene in agriculture is CP4EPSPS from Agrobacterium tumefaciens CP4 strain. Although many glyphosate-resistant EPSPS genes have been found in microorganisms, these genes have not been widely used in crops. The application of these microorganism glyphosate-resistant genes in crops is to express these genes such as CP4EPSPS in crops by transgenic methods. At present, some EPSPS protein variants have been found in glyphosate-resistant elephant grass, but it is difficult to obtain naturally mutated glyphosate-resistant EPSPS genes in crops, and only by transgenic methods can the mutated EPSPS gene be transferred into crops to obtain glyphosate-resistant crops. However, due to the regulatory policy of transgenic crops and the acceptance of transgenic crops in the world, the large-scale promotion and use of transgenic crops are still subject to many restrictions. Therefore, it is urgent to develop a gene that does not affect plant growth and development and has glyphosate resistance to crops such as rice, which is of great significance to crop production. SUMMARY

[0005] To solve the above technical problems, the present application provides a rice EPSPS mutant protein and its application. The rice EPSPS mutant protein provided by the present application not only can improve the glyphosate resistance of rice, but also can maintain the stability of the yield and plant height of rice, and therefore can be used for cultivating glyphosate-resistant crops.

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

[0007] In an optional embodiment, the present application provides a rice EPSPS mutant protein, wherein the rice EPSPS mutant protein is a wild-type EPSPS protein with a mutation at the 169th amino acid, which is threonine mutated to serine.

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

[0009] In an alternative embodiment, the present application provides a rice EPSPS mutant gene, which is mutated at position 506 from C to G in the nucleotide sequence of the wild-type EPSPS gene.

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

[0011] The mutation of C to G at position 506 results in the mutation of threonine (T) to serine (S) at position 169 of the amino acid encoded by the EPSPS gene. The rice EPSPS mutant has glyphosate resistance while maintaining its biological enzyme catalytic activity. Plants transformed with the rice EPSPS mutant can grow normally and have broad application prospects.

[0012] In an alternative embodiment, the present application provides a nucleic acid molecule encoding the rice EPSPS mutant protein described above.

[0013] In an alternative embodiment, the present application provides an expression cassette containing the rice EPSPS mutant protein, the rice EPSPS mutant gene or the nucleic acid molecule described above.

[0014] In an alternative embodiment, the present application provides a recombinant vector containing the rice EPSPS mutant protein, the rice EPSPS mutant gene or the nucleic acid molecule described above.

[0015] In an alternative embodiment, the present application provides the use of the rice EPSPS mutant protein, the rice EPSPS mutant gene, the nucleic acid molecule, the expression cassette or the recombinant vector described above in conferring glyphosate resistance on target plants.

[0016] Preferably, the method for conferring glyphosate resistance on target plants comprises:

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

[0018] (2) cultivating homozygous plant offspring or plant seeds of the rice EPSPS mutant gene or the rice EPSPS mutant protein.

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

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

[0021] (1) After shelling and sterilizing the rice seeds, the embryo is separated and placed on a callus induction medium to generate secondary callus;

[0022] (2) The secondary callus is transferred to a new callus induction medium for pre-culture;

[0023] (3) The callus obtained in step (2) is contacted with Agrobacterium for 15 minutes to obtain resistant callus, wherein the Agrobacterium has a recombinant expression vector with a plant herbicide-resistant EPSPS mutant gene or other gene encoding the mutant protein;

[0024] (4) The callus of step (3) is transferred to a culture dish with three sterile filter papers, and cultured at 21-23°C for 48 hours;

[0025] (5) The callus of step (4) is placed on a pre-selection medium and cultured for 5-7 days;

[0026] (6) The callus of step (5) is transferred to a selection medium to obtain resistant callus;

[0027] (7) The resistant callus is transferred to a differentiation and regeneration medium to differentiate into seedlings; and

[0028] (8) The seedlings of step (7) are transferred to a rooting medium for rooting.

[0029] The nucleotide sequence of SEQ ID NO. 1 is as follows.

[0030]

[0031] The amino acid sequence of SEQ ID NO. 2 is as follows.

[0032] MASNAAAAAAVSLDQAVAASAAFSSRKQLRLPAAARGGMRVRVRARGRREAVVVASASSSSVAAPAAKAEEIVLQPIREISGAVQLPGSKSLSNRILLLSALSEGTTVVDNLLNSEDVHYMLEALKALGLSVEADKVAKRAVVVGCGGKFPVEKDAKEEVQLFLGNAGSAMRPLTAAVTAAGGNATYVLDGVPRMRERPIGDLVVGLKQLGADVDCFLGTECPPVRVKGIGGLPGGKVKLSGSISSQYLSALLMAAPLALGDVEIEIIDKLISIPYVEMTLRLMERFGVKAEHSDSWDRFYIKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTVTVQGCGTTSLQGDVKFAEVLEMMGAKVTWTDTSVTVTGPPREPYGKKHLKAVDVNMNKMPDVAMTLAVVALFADGPTAIRDVASWRVKETERMVAIRTELTKLGASVEEGPDYCIITPPEKLNITAIDTYDDHRMAMAFSLAACADVPVTIRDPGCTRKTFPNYFDVLSTFVRN. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a photograph of the resistant callus produced on herbicide-containing medium after chemical mutagenesis with sodium nitrite in Example 1 of the present application;

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

[0035] Figure 3 Figure 3 is a comparison of the growth of the wild type and the EPSPS mutant strain after spraying herbicide in Example 2 of the present application. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0038] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0039] Example 1

[0040] Rice callus mutagenesis and directional screening of herbicide resistance sites

[0041] (1) Sterilize the mature, non-molded rice seeds after shelling, separate the embryos with a scalpel, and place them on callus induction medium to produce secondary calli;

[0042] (2) Transfer the secondary calli from step (1) to a new callus induction medium for pre-culture;

[0043] (3) Transfer the calli obtained in step (2) to a mutagenic medium for mutagenic culture for 10 days;

[0044] (4) Place the calli from step (3) on the pre-screening medium for 8 days;

[0045] (5) Transfer the calli from step (4) to the screening medium to obtain resistant calli, see Figure 1 for results;

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

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

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

[0049] Table 1 Medium in each step

[0050]

[0051] Using about 20,000 calli after mutagenesis, transfer to a medium containing 5 mM glyphosate herbicide resistance for screening, and finally 10 resistant calli are obtained, which are differentiated into seedlings. The genomic DNA of one of them is extracted. The EPSPS gene is amplified and sent to Shanghai Biosciences Company for genomic sequencing. The sequencing results are compared with the wild-type Nipponbare EPSPS gene, see Figure 2, a mutation from C to G was found at position 506 in EPSPS, which resulted in the change of the amino acid at position 169 in EPSPS gene from threonine (T) to serine (S). 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] The mutant plants (denoted as T169S) and wild-type plants (rice containing wild-type EPSPS gene of Nipponbare, denoted as WT) were planted in a test field. The mutant plants were Nipponbare rice into which the mutant gene of Example 1 was introduced, and the homozygous mutant seeds obtained in Example 1 were used for planting and growing into seedlings, and then further growing into plants. The specific process is as follows:

[0055] Preparation of herbicide dilution solution: glyphosate isopropylamine salt herbicide was selected, with an effective component concentration of 41%. Each milliliter of the original solution was mixed with 90 milliliters of water to obtain a secondary dilution solution of 20 mmol·L -1 , which was ready for use.

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

[0057] Planting and growth: after 2 weeks in the water culture environment, rice seedlings with consistent growth were selected, and mutant and wild-type rice seedlings were planted in different areas, with 50 seedlings planted in each area, and 3 areas were repeated. The rice nutrient solution used was Hoagland nutrient solution.

[0058] After 30 days of growth, 10 mmol·L -1 of glyphosate herbicide was uniformly sprayed, and the phenotypic changes were observed. About 5 days after spraying, the wild-type area of the experimental group began to wither in large areas, while the leaves of the homozygous mutant plants grew almost normally (see Figure 3 for results). After 2 months of growth, the mutant plants could normally flower and bear fruit.

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

[0060] Table 2 Agronomic trait comparison of EPSPS mutants and controls

[0061]

[0062] Although the principles of the present application have been described in relation to particular embodiments thereof, it will be appreciated by those skilled in the art that modifications can be made to the disclosed embodiments without departing from the principles of the application. For example, alternative configurations can be utilized. Other modifications and changes will be apparent to those skilled in the art. The scope of the application is not to be limited by the embodiments disclosed herein but is only to be defined by the claims and the equivalents thereof.

Claims

1. A mutant EPSPS protein from rice, characterized in that, The rice EPSPS mutant protein is a wild-type EPSPS protein with a mutation at position 169 of the amino acid sequence, changing from threonine to serine. The amino acid sequence of the rice EPSPS mutant protein is shown in SEQ ID NO.

2.

2. 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 of the nucleotide sequence, changing from C to G. The nucleotide sequence of the rice EPSPS mutant gene is shown in SEQ ID NO.

1.

3. An expression cassette containing the rice EPSPS mutant gene as described in claim 2.

4. A recombinant vector containing the rice EPSPS mutant gene as described in claim 2.

5. The application of the rice EPSPS mutant protein of claim 1, the rice EPSPS mutant gene of claim 2, the expression cassette of claim 3, or the recombinant vector of claim 4 in the cultivation of glyphosate-resistant rice.

6. The application of the rice EPSPS mutant protein of claim 1, the rice EPSPS mutant gene of claim 2, the expression cassette of claim 3, or the recombinant vector of claim 4 in the cultivation of glyphosate-resistant rice progeny or rice seeds.

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