Rice mutant protein, mutant gene and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a rice mutant protein, a mutant gene and application of the rice mutant protein and the mutant gene. The amino acid sequence of the rice mutant protein provided by the invention is as shown in SEQ ID NO. 1. The method comprises the following steps: carrying out large-scale mutagenesis on calluses of Nipponbare rice by using ethyl methanesulfonate, adding a herbicide for screening during culture, carrying out differentiation, rooting and planting on the obtained resistant calluses, selecting a rice variety with high thousand seed weight, and sequencing to obtain the rice mutant protein. According to the rice mutant protein, the thousand seed weight of rice can be remarkably increased, the grain length of seeds is increased, the yield of the rice is increased, and the rice can have the weeding resistance function.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to a rice mutant protein and mutant gene and their applications. Background Art

[0002] As one of the most important food crops globally, the yield of rice is directly related to food security and social stability. The yield of rice is determined by three major factors: the number of effective panicles, the number of grains per panicle, and the 1000-grain weight. Among them, the 1000-grain weight reflects the plumpness of the grains and is a key target trait for increasing yield.

[0003] However, traditional breeding methods have problems such as long cycle and low efficiency in increasing the 1000-grain weight, and some materials obtained through gene editing or genetic improvement are often accompanied by negative impacts on other agronomic traits, restricting their practical applications. In particular, it is difficult to balance the yield and resistance of rice. Therefore, exploring key genes that regulate rice yield and can improve resistance is of great significance for high-yield rice breeding. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a rice mutant protein and mutant gene and their applications. The present invention uses ethyl methanesulfonate to mutagenize the callus of Nipponbare rice on a large scale, adds a herbicide for screening during cultivation, differentiates, roots, and plants the obtained resistant callus, and selects a rice variety with a high 1000-grain weight from them. By sequencing, a rice mutant protein is obtained. This rice mutant protein can not only significantly increase the 1000-grain weight of rice, increase the grain length of the seeds, and improve the yield of rice, but also endow rice with the function of herbicide resistance.

[0005] Therefore, the present invention provides the following technical solutions.

[0006] In a first aspect, in an optional embodiment, the present invention provides a rice mutant protein, and the amino acid sequence of the rice mutant protein is as shown in SEQ ID NO.1.

[0007] In a second aspect, in an optional embodiment, the present invention provides a rice mutant gene, and the nucleotide sequence of the rice mutant gene is as shown in SEQ ID NO.2 or SEQ ID NO.3.

[0008] In a third aspect, in an optional embodiment, the present invention provides a plasmid containing the above rice mutant gene.

[0009] In a fourth aspect, in an optional embodiment, the present invention provides a plant expression vector containing the above rice mutant gene.

[0010] Fifth aspect, in an optional embodiment, the present invention provides a recombinant cell comprising the above-mentioned rice mutant gene.

[0011] Sixth aspect, in an optional embodiment, the present invention provides a recombinant protein comprising the above-mentioned rice mutant gene.

[0012] Seventh aspect, in an optional embodiment, the present invention provides an application of the above-mentioned rice mutant protein or the above-mentioned rice mutant gene in increasing the 1000-grain weight of rice.

[0013] Eighth aspect, in an optional embodiment, the present invention provides an application of the above-mentioned rice mutant protein or the above-mentioned rice mutant gene in increasing the yield of rice.

[0014] Ninth aspect, in an optional embodiment, the present invention provides an application of the above-mentioned rice mutant protein or the above-mentioned rice mutant gene in conferring herbicide resistance to plants.

[0015] Tenth aspect, in an optional embodiment, the present invention provides a method for obtaining a rice plant with increased rice yield and herbicide resistance, comprising the following steps:

[0016] By means of gene editing, hybridization, backcrossing, self-crossing or asexual propagation, making the plant contain the above-mentioned rice mutant protein or the above-mentioned rice mutant gene, thereby obtaining a plant with increased rice yield and herbicide resistance;

[0017] The herbicide is one or more of metamifop, cyhalofop-butyl and pinoxaden.

[0018] The amino acid sequence of SEQ ID NO.1 is shown as follows:

[0019]

[0020] The nucleotide sequence of SEQ ID NO.2 is shown as follows:

[0021]

[0022] The nucleotide sequence of SEQ ID NO.3 is shown as follows:

[0023]

[0024] Compared with the prior art, the present invention has one of the following beneficial effects:

[0025] 1. The present invention uses ethyl methyl sulfonate to mutagenize calli of Nipponbare rice on a large scale, adds a herbicide for screening during cultivation, differentiates, roots and plants the obtained resistant calli, selects a rice variety with a high 1000-grain weight therefrom, sequences to obtain a rice mutant protein. This rice mutant protein can not only significantly increase the 1000-grain weight of rice, increase the grain length of seeds, and improve the yield of rice, but also endow rice with the function of herbicide resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the resistant callus grown on the screening medium containing herbicide in Example 1 of the present invention;

[0027] Figure 2 This is the sequencing diagram of mutants MT1 and MT2 obtained in Example 2 of the present invention;

[0028] Figure 3 This is the effect diagram of spraying different herbicides on mutants MT1 and MT2 obtained in Example 3 of the present invention;

[0029] Figure 4 This is the phenotype diagram of mutants MT1 and MT2 obtained in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be 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.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0033] Example 1

[0034] Obtaining of Rice Mutant Plants

[0035] The method for mutagenizing rice calli and directionally screening herbicide-resistant sites includes the following steps:

[0036] 1. Preparation of explants: After removing the glumes from mature rice seeds, soak the seeds in 70% alcohol for 1 minute, pour out the alcohol, and then soak the seeds in a solution of sodium hypochlorite (original solution with available chlorine concentration greater than 4%) with a mass fraction of 50% containing 1 drop of Tween 20 for 40 minutes (while shaking at a rate of 150 r / min). Then pour out the sodium hypochlorite solution, wash with sterile water 5 times until the solution is clear and there is no smell of sodium hypochlorite, and then soak the seeds in sterile water overnight. The next day, use a scalpel to peel the embryo along the aleurone layer of the seeds, inoculate the embryo on the callus induction medium, and finally, after culturing in the dark at 30°C for 11 days, separate the callus from the endosperm and germ, and obtain primary callus with good state, vigorous division and without buds.

[0037] 2. Mutagenic culture: Dissolve ethyl methanesulfonate (EMS) in phosphate-buffered saline (PBS, pH 7.2), filter and sterilize it to prepare a treatment solution containing 40 μmol / L EMS, and treat the callus for 6 hours. After mutagenic treatment, the callus is rinsed 3 times with PBS buffer (pH 7.2), placed on sterilized filter paper, and the surface moisture is blotted dry in a laminar flow hood, and then inoculated into a solid callus induction medium. It is cultured in the dark at 27±1°C for 7 days to obtain about 10,000 calli. The specific composition of the induction medium is: N6 macroelements, MS iron salts, B5 microelements, B5 vitamins, 500 mg / L proline, 500 mg / L glutamine, 300 mg / L enzymatically hydrolyzed casein, 2 mg / L 2,4-D, 30 g / L sucrose, and 3 g / L phytagel, with a pH of 5.7.

[0038] 3. Screening by adding herbicide: Transfer the callus after the above mutagenic treatment to a screening medium containing cyhalofop-butyl. The medium is the induction medium added with 1 mg / L cyhalofop-butyl, and it is cultured at 30°C with 16 h light + 8 h dark for 30 days. Observe irregularly until resistant callus granules that can grow normally appear (see Figure 1 ), and 100 resistant callus granules are obtained.

[0039] 4. Differentiation culture: Transfer the callus granules after spraying the herbicide to the differentiation medium, regenerate and differentiate into seedlings, and transfer the buds or seedlings to the MS rooting medium for culture. A total of 80 seedlings are successfully differentiated. The specific composition of the differentiation medium is: N6 macroelements, MS iron salts, B5 microelements, B5 vitamins, 500 mg / L proline, 1 g / L enzymatically hydrolyzed casein, 30 g / L sucrose, 1.5 mg / L NAA, 1 mg / L 6-BA, 2.5 g / L phytagel, and 1 mg / L cyhalofop-butyl.

[0040] 5. Transplant these 80 seedlings into the field separately for self-crossing, harvest the progeny F1, and count the average 1000-grain weight of the progeny F1. It is found that the average 1000-grain weight of the progeny F1 of 15 seedlings increases compared with the average 1000-grain weight of wild rice seeds.

[0041] Example 2

[0042] Analysis of mutant sites of mutants

[0043] Plant the seeds obtained from the above progeny F1 separately and cultivate them into seedlings. Select leaves from the obtained rice mutant plants and extract genomic DNA. Use a pair of primers to amplify the upstream and downstream sequences of the target sequence.

[0044] FP: TAATCTGTCATTAGCACATG.

[0045] RP: TCCAATATACACTCACCAAC.

[0046] Send the amplification product to Invitrogen for genomic sequencing. The sequencing results are shown in SEQ ID NO.2 and SEQ ID NO.3, which are respectively named MT1 and MT2 in the present invention. See Figure 2 .

[0047] The amino acid sequences of mutant plants MT1 and MT2 are both shown in SEQ ID NO.1.

[0048] Example 3

[0049] Spraying experiment of rice mutants against herbicides (metamifop, cyhalofop-butyl and pinoxaden)

[0050] Transplant the obtained rice materials MT1 and MT2 containing mutant genes into the field respectively to harvest T1 generation seeds. After the plants mature, the seeds are mixed, harvested, dried and stored in a storage box. To verify the resistance of the mutants to herbicides, when the seedlings grow to the 3-4 leaf stage, after spraying 20 g / mu of cyhalofop-butyl (8 times the recommended concentration) herbicide, the plants still grow and develop normally, while the wild-type rice shows whole-plant death 5 days after spraying 2.5 g / mu of cyhalofop-butyl. In addition, after spraying metamifop (100 g / mu, 8 times the recommended concentration) and pinoxaden (1 g / mu, 8 times the recommended concentration) herbicides, the plants still grow and develop normally, while the wild-type rice shows whole-plant death 5 days after spraying 12.5 g / mu of metamifop and 0.125 g / mu of pinoxaden respectively (see Figure 3 ), indicating that the mutant rice can resist herbicides at 8 times the recommended concentration.

[0051] Example 4

[0052] The phenotypes of the homozygous mutant progenies of the rice materials MT1 and MT2 in the field were observed. Compared with the control material (i.e., the wild-type rice without herbicide spraying), there were no significant differences in plant height and tiller number. However, the grain length of the two mutant materials increased from 6.9 mm of the control material to 7.6 mm, and the 1000-grain weight also increased from 24.4 g of the control material to 25.6 g (see Figure 4 ).

[0053] In summary, the rice containing the two mutant genes of the present invention (i.e., containing the mutant protein) can not only improve the herbicide resistance of rice, but also increase the grain weight, which has important application value for cultivating high-yield and herbicide-resistant rice.

[0054] Although the principle of the present invention has been 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 only explanations of the illustrative implementation modes 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 transformation and simple substitution based on the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A rice mutant protein, characterized in that, The amino acid sequence of the rice mutant protein is shown in SEQ ID NO.

1.

2. A rice mutant gene, characterized in that, The nucleotide sequence of the rice mutant gene is shown in SEQ ID NO. 2 or SEQ ID NO.

3.

3. A plasmid containing the rice mutant gene according to claim 2.

4. A plant expression vector containing the rice mutant gene according to claim 2.

5. A recombinant cell containing the rice mutant gene according to claim 2.

6. A recombinant protein containing the rice mutant gene according to claim 2.

7. Use of the rice mutant protein according to claim 1 or the rice mutant gene according to claim 2 in increasing the 1000-grain weight of rice.

8. Use of the rice mutant protein according to claim 1 or the rice mutant gene according to claim 2 in increasing the yield of rice.

9. Use of the rice mutant protein according to claim 1 or the rice mutant gene according to claim 2 in conferring herbicide resistance to plants.

10. A method for obtaining rice plants with increased rice yield and herbicide resistance, characterized in that, Comprising the following steps: By means of gene editing, hybridization, backcrossing, self-crossing or asexual reproduction, making a plant contain the rice mutant protein according to claim 1 or the rice mutant gene according to claim 2, thereby obtaining a plant with increased rice yield and herbicide resistance; The herbicide is one or more of mefenacet, cyhalofop-butyl and pinoxaden.

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