Method for reducing rice seed shattering by regulating expression of SH3.1 gene and application of method

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for regulating expression of an SH3.1 gene to reduce rice seed shattering and application of the method. The method for reducing the seed shattering of the rice comprises the following steps: inhibiting the expression of an SH3.1 gene in receptor rice to obtain target rice of which the seed shattering is lower than that of the receptor rice. The inventor adopts a CRISPR / Cas9 technology to edit an SH3.1 gene at a fixed point, and knocks out the SH3.1 gene of an introgression line GIL291 of an African cultivated rice IRGC104038 segment by causing frameshift mutation, so that a new rice germplasm with obviously reduced seed shattering is obtained. High seed shattering is an important factor restricting yield increase of African cultivated rice, the rice seed shattering is reduced, high-yield germplasm resources of rice are innovated, and the method has important significance on new variety cultivation, environmental sanitation and grain safety. The method has great application and popularization values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to regulating SH3.1 A method for reducing rice grain shattering through gene expression and its application. Background Art

[0002] Oryza Oryza L.) has a total of 22 species, including two cultivated species, namely Asiatic cultivated rice ( O. sativa L.) and African cultivated rice ( O. glaberrima Steud.), from common wild rice ( O. rufipogon ) and short-tongued wild rice ( O. barthii ) were domesticated.

[0003] The loss of seed shattering is one of the most critical events in rice domestication. However, due to its relatively recent domestication history and lack of systematic modern breeding and improvement, African cultivated rice retains a strong seed shattering trait. This significantly reduces harvest efficiency and is a major constraint on increasing African cultivated rice yields. Over the past few decades, scientists have conducted in-depth research on the regulatory mechanisms of seed shattering in Asian cultivated rice and the cloning of major genes responsible for seed shattering. However, research on genes associated with seed shattering domestication in African cultivated rice has remained limited to the cloning of major genes.

[0004] However, African cultivated rice, with its characteristics of early maturity, disease resistance, and drought tolerance, has certain production advantages in specific ecological environments and is a valuable genetic resource. Therefore, improving the harvest efficiency of African cultivated rice has certain application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to reduce the grain shattering property of rice, especially the grain shattering property of African cultivated rice, so as to increase the harvest yield.

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a method for reducing rice grain shattering, comprising the following steps: inhibiting the growth of SH3.1 The expression of the gene is used to obtain the target rice with lower grain shattering than the recipient rice; SH3.1 The gene is a gene encoding SH3.1 protein; the SH3.1 protein is the following protein A1), A2) or A3): A1) The amino acid sequence is the protein of SEQ ID No: 2 in the sequence listing; A2) a protein having a similar function to the protein of A1 and having a homology of more than 80% to the protein of A1 obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID No: 2 in the sequence listing; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0007] In the above methods, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0008] In the above method, the above 80% or greater identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0009] In the above method, the SH3.1 The gene may specifically be the gene shown in E1, E2 or E3 below: E1, the coding sequence of the coding strand is a nucleic acid molecule of SEQ ID No: 1; E2, a nucleotide sequence comprising the nucleic acid molecule of positions 1946-2421 of SEQ ID No: 3 in the sequence listing; E3. The nucleotide sequence is the nucleic acid molecule of SEQ ID No: 3 in the sequence listing.

[0010] In the above method, the recipient rice can be selected from African cultivated rice or its derivative plants.

[0011] In the above method, the inhibitory receptor rice SH3.1 Gene expression is achieved by SH3.1 The gene editing is achieved by using the CRISPR / Cas9 system.

[0012] In the above method, the CRISPR / Cas9 system includes a plasmid expressing Cas9 and sgRNA, and the target sequence of the sgRNA can be SEQ ID No: 3 positions 2128-2151 and SEQ ID No: 3 positions 2194-2216 in the sequence listing.

[0013] In the above method, the target rice is rice that meets the following conditions: SH3.1One or two target regions of the gene corresponding to positions 2128-2151 of SEQ ID No: 3 and positions 2194-2216 of SEQ ID No: 3 are mutated.

[0014] The present invention also provides the SH3.1 protein.

[0015] The present invention also provides a biological material related to the SH3.1 protein, wherein the biological material is any one of the following B1 to B5: B1, nucleic acid molecule encoding SH3.1 protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

[0016] In the above-mentioned biological material, SH3.1 The gene may specifically be the gene shown in E1, E2 or E3 below: E1, the coding sequence of the coding strand is a nucleic acid molecule of SEQ ID No: 1; E2, a nucleotide sequence comprising the nucleic acid molecule of positions 1946-2421 of SEQ ID No: 3 in the sequence listing; E3. The nucleotide sequence is the nucleic acid molecule of SEQ ID No: 3 in the sequence listing.

[0017] In order to solve the above technical problems, the present invention also provides an agent for reducing grain shattering, wherein the active ingredient of the agent is a substance that inhibits the expression of the gene encoding the SH3.1 protein, reduces the abundance of the SH3.1 protein, and / or knocks out the gene encoding the SH3.1 protein.

[0018] In the above-mentioned agent for reducing the particle falling property, the substance contains the following F1), F2) or F3): F1) Targeting SH3.1 sgRNA, siRNA, shRNA, miRNA, or antisense RNA of a gene; F2) produces the target SH3.1 Gene-targeting sgRNA DNA molecules are generated SH3.1 Gene-targeting siRNA DNA molecules SH3.1Gene-targeting shRNA DNA molecules SH3.1 Gene or miRNA DNA molecules targeting the SH3.1 a DNA molecule that is the antisense RNA of a gene; F3) produces the target SH3.1 Gene expression vectors targeting the sgRNA SH3.1 Gene expression vectors, producing siRNA targeting the SH3.1 Gene expression vectors, producing shRNA targeting the SH3.1 Gene expression vectors or production of miRNA targeting the SH3.1 Gene antisense RNA expression vector.

[0019] In the above-mentioned reagent for reducing grain shedding, the target sequence of the sgRNA is SEQ ID No: 3, positions 2128-2151 and SEQ ID No: 3, positions 2194-2215 in the sequence listing.

[0020] The active ingredients of the above-mentioned agent for reducing grain shattering may also contain other biological components and / or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned agent based on the effect of reducing grain shattering in plants.

[0021] The present invention also provides the method for reducing rice grain shattering, and the actual use of the protein, the biological material or the plant in cultivating high-yield rice.

[0022] The inventors of the present invention isolated and cloned the gene related to grain shattering in African cultivated rice from rice. SH3.1 , after expression in rice, it showed an effect of increasing grain shattering. The inventors used CRISPR / Cas9 technology to edit the SH3.1 The gene was knocked out in the introgression line GIL291 of the African cultivated rice IRGC104038 fragment by causing a frameshift mutation. SH3.1 Genes have been developed to produce new rice germplasm with significantly reduced grain shattering. Due to its relatively recent domestication and lack of systematic modern breeding improvements, African cultivated rice still retains a strong tendency to shatter, significantly reducing its harvest efficiency and becoming a significant factor restricting its harvest efficiency. This invention reduces rice grain shattering, creates new high-yield rice germplasm resources, and is of great significance to the development of new varieties, environmental hygiene, and food security. It has significant application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the grain shattering properties of the strain GIL291 in Example 1 of the present invention and the Asian cultivated rice Taichung65 (labeled as T65).

[0024] Figure 2 In Example 1 of the present invention SH3.1 Schematic diagram of gene location.

[0025] Figure 3 For example 2, transfer to GC- SH3.1 Electrophoresis diagrams showing the identification of transgenic lines carrying the vector and a photograph comparing their seed shattering with that of the Asian cultivated rice variety Taichung65 (labeled T65). In the electrophoresis diagram, lanes 2, 3, and 5-11 represent transgenic plants screened for hygromycin resistance, lane + represents a positive control containing no pCAMBIA1300 plasmid, and lane - represents a negative control containing deionized water. In the photograph, GC-SH3.1-2 represents transgenic lines identified by PCR into GC- SH3.1 Vector-transgenic lines.

[0026] Figure 4 For example 3, Cas9- SH3.1 Electrophoresis of gene knockout plants identified by PCR, mutation identification, and comparison of seed shattering with the introgression line GIL291 of the African cultivated rice IRGC104038. In the figure, lanes 1-11 in the electrophoresis are gene knockout-positive plants selected for hygromycin resistance, lane + is a positive control with an empty CRISPR-Cas9 vector plasmid, lane - is a negative control with deionized water, and lane Cas9- SH3.1 -3 is the Cas9-transferred protein identified by PCR SH3.1 Vector and gene-edited knockout plants. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0028] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The African rice variety IRGC104038 in the following examples is described in the non-patent document "Ning J, He W, Wu L, Chang L, Hu M, Fu Y, Liu F, Sun H, Gu P, Ndjiondjop MN, Sun C, Zhu Z. The MYB transcription factor Seed Shattering 11 controls seed shattering byrepressing lignin synthesis in African rice. Plant Biotechnol J. 2023 21(5):931-942. doi: 10.1111 / pbi.14004.", which is available to the public from the applicant to repeat the experiments of the present invention.

[0032] The Asian cultivated rice Taichung65 in the following examples is described in the non-patent document "Ning J, He W, Wu L, Chang L, Hu M, Fu Y, Liu F, Sun H, Gu P, Ndjiondjop MN, Sun C, Zhu Z. The MYB transcription factor Seed Shattering 11 controls seed shattering byrepressing lignin synthesis in African rice. Plant Biotechnol J. 2023 21(5):931-942. doi: 10.1111 / pbi.14004.", which is available to the public from the applicant to repeat the experiments of the present invention.

[0033] In the following examples, the expression vector pCAMBIA1300 is a product of Beijing Huayueyang Biological Company, with the product number VECY0070.

[0034] The CRISPR-Cas9 vector system in the following examples is described in the non-patent document "Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocotand Dicot Plants. Mol Plant. 2015 8(8):1274-84. doi: 10.1016 / j.molp.2015.04.007.", which is available to the public from the applicant to repeat the experiments of the present invention.

[0035] Example 1 Localization of Genes Regulating Seed Shattering in African Rice The African rice variety IRGC104038 was hybridized with the Asian cultivated rice Taichung65, and Taichung65 was used as the recurrent parent. After multiple backcrosses, a family GIL291 with easy grain shedding at maturity was obtained (see Figure 1 ), and it was crossed with Taichung65 to construct an F2 segregating population containing 200 individuals. In this F2 population, seed shattering was closely linked to markers Id3166 and RM15661, and SH3.1 The gene was located between these two molecular markers. The segregation population was then expanded to 8200 individual plants, and primers were designed based on the sequence between markers Id3166 and RM15661. SH3.1 The gene was located between markers Id3184 and Id3197 (see Figure 2 ), the size of this interval is about 17 kb, containing two annotated genes. Gene expression analysis was performed using specific tissues related to seed shattering, and it was found that ORF2 Higher expression levels were detected in the abscission layer, so it is believed that the gene ORF2 It regulates seed shattering SH3.1 The strain GIL291 and the African cultivated rice IRGC104038 SH3.1 The gene sequence of strain GIL291 is identical to that of Asian cultivated rice Taichung65. SH3.1 The gene sequence is different. Taichung65 SH3.1 The sequence of the gene and its upstream promoter and downstream regulatory region is shown in SEQ ID No: 4.

[0036] The sequences of the positioning primers used are shown in Table 1:

[0037] Example 2 SH3.1 Acquisition and functional verification of transgenic African rice 1. Construction of expression vector According to the African cultivated rice IRGC104038 SH3.1 Design primers based on the gene and promoter sequences, and introduce restriction endonucleases at both ends of the primers EcoR I and BamH I recognition sequence and pCAMBIA1300 homology arm sequence, the primer sequences are as follows: GC-SH3.1-1F: 5'-acagctatgaccatgattacgaattcTCTGTAGGTTAGATGTGTGT -3' (positions 1-26 are the homology arm sequence of pCAMBIA1300, positions 21-26 are the restriction endonuclease EcoR I recognition sequence, positions 27-46 are identical to positions 1-20 of SEQ ID No: 3).

[0038] GC-SH3.1-1R:5'- gcctgcaggtcgactctagaggatcc ATGCAAATCTTGTCTCCCTA -3' (positions 1-26 are the homology arm sequence of pCAMBIA1300, positions 21-26 are the restriction endonuclease BamH I recognition sequence, positions 27-46 are reverse complementary to positions 5077-5096 of SEQ ID No: 3).

[0039] The whole genome DNA of the African cultivated rice IRGC104038 was extracted using the CTAB method. Using this DNA as a template, a 5.1 kb DNA fragment (shown in SEQ ID No: 3) was amplified. Positions 1-1945 are the promoter; positions 1946-2421 are SH3.1 The genomic sequence, of which positions 1946-2032 are the first exon, positions 2125-2421 are the second exon; positions 2422-5400 are the 3'-regulatory region sequence), was recovered using a kit and cloned into the restriction site of the plant expression vector pCAMBIA1300. EcoR I and BamH Between 1 and 2, we obtained the SH3.1 Gene plant expression vector, named GC- SH3.1 .

[0040] Carrier GC- SH3.1The restriction endonuclease of the pCAMBIA1300 vector is replaced by the sequence fragment of nucleotide sequence as shown in SEQ ID No: 3. EcoR I and BamH I recognition site, keeping other sequences of the pCAMBIA1300 vector unchanged, to obtain a plant expression vector expressing SH3.1 protein.

[0041] SH3.1 The cDNA sequence of the gene is shown in SEQ ID No: 1, and the amino acid sequence of the SH3.1 protein encoded by it is shown in SEQ ID No: 2.

[0042] SEQ ID No: 1 ATGGCGGGCCATCTGCTCCTGCTCCTCCTGCTCGCTCTCCTCCTCGCCTCCTCCGTCCTTGCCAAAGCTGCCGCCGGCTTCACCGAGGAGAAGGGCGTTGCTGGGATCAGAGGCGTGATAGGGTCGAGGCCGCCGAGCTGCGAGGGGAGGTGCAGGTCGTGTGGCCACTGCGAGGCGGTGCAGGTGCCCATA ACCCCACAGCAGCTGCAGAAGAAGAGAGGCCAAGGCGACGACAGGAGGAGGAAGAAGAAGCAGCTGCTCGGCCATGGCGACAGAGCTGCTGCTGCCGGTGGGAGGGCAATGCCTGATTCCTACGACGACCACTCCAACTACAAGCCGTTGAGCTGGAGGTGCAAGTGTGGGGGAATGATCCTGAACCCATGA SEQ ID No: 2 MAGHLLLLLLLLALLLASSVLAKAAAGFTEEKGVAGIRGVIGSRPPSCEGRCRSCGHCEAVQVPITPQQLQKKRGQGDDRRRKKKQLLGHGDRAAAAGGRAMPDSYDDHSNYKPLSWRCKCGGMILNP SEQ ID No: 3 SEQ ID No:4 II. Transformation of Asian cultivated rice Taichung65 and PCR identification The vector GC- SH3.1 Transgenic plants were obtained by transformation with Agrobacterium tumefaciens (Qingke Biotechnology, TSC-A03) into the Asiatic rice variety Taichung 65, which is susceptible to grain shattering. Two rounds of selection were performed using NB medium containing 800 μg / mL hygromycin. Transgenic plants were then differentiated through pre-differentiation. A control experiment was also conducted by introducing the empty pCAMBIA1300 vector into the Asiatic rice variety Taichung 65.

[0043] The transgenic plants were identified by PCR. The specific operation was as follows: the Asian cultivated rice Taichung65 was extracted by CTAB method and transferred into GC- SH3.1 The whole genome DNA of the transgenic plants containing the vector was used as a template to amplify the approximately 500 bp hygromycin protein sequence. Deionized water was used as a negative control, and an empty pCAMBIA1300 plasmid was used as a positive control. The primer sequences are as follows: Hpt-F: AAGTTCGACAGCGTCTCCGAC; Hpt-R: TCTACACAGCCATCGGTCCAG.

[0044] The results showed that after hygromycin resistance screening and PCR identification, a total of 11 transgenic plants were obtained (part of the electrophoresis results are shown in Figure 3 The transgenic plants were self-pollinated and propagated to obtain the corresponding transgenic lines, which were numbered as GC- SH3.1 -1, GC- SH3.1 -2, GC- SH3.1 -3.GC- SH3.1 -4, GC- SH3.1 -5, GC- SH3.1 -6, GC- SH3.1 -7, GC- SH3.1 -8, GC- SH3.1 -9, GC- SH3.1 -10 and GC- SH3.1 -11.

[0045] 3. Functional identification of transgenic rice The 9 transgenic positive lines (GC- SH3.1 -2, GC- SH3.1 -3.GC- SH3.1 -5, GC- SH3.1 -6, GC- SH3.1 -7, GC- SH3.1 -8, GC-SH3.1 -9, GC- SH3.1 -10 and GC- SH3.1 -11), empty vector control lines obtained by self-pollination of the pCAMBIA1300 empty vector, and untransformed wild-type Asian cultivated rice variety Taichung 65 were used as experimental materials. Conventional planting (15 cm plant spacing, 25 cm row spacing, two rows per line) was performed. Seed shattering was observed and analyzed for each plant material after seed maturity. At least five plants of each genetic material were analyzed.

[0046] The results showed that compared with the empty control and the Asian cultivated rice variety Taichung65 without genetic modification, the 9 transgenic positive lines (GC- SH3.1 -2, GC- SH3.1 -3.GC- SH3.1 -5, GC- SH3.1 -6, GC- SH3.1 -7 and GC- SH3.1 -8, GC- SH3.1 -9, GC- SH3.1 -10 and GC- SH3.1 -11) all showed strong granulation (see some comparative photos for details). Figure 3 ). Therefore, the SH3.1 protein shown in SEQ ID No: 2 (shown in SEQ ID No: 3 SH3.1 genes) can be used to regulate seed shattering.

[0047] Example 3: Gene Editing SH3.1 Can reduce seed shattering 1. Knockout target selection and vector construction The website CRISPR-GE (http: / / skl.scau.edu.cn / ) was used to select the knockout target of SH3.1. The target site was ligated to the CRISPR / Cas9 empty vector by homologous recombination using the method proposed by Ma et al. (2015, Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, LiH, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, DongZ, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-EfficiencyMultiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015 8(8):1274-84. doi: 10.1016 / j.molp.2015.04.007.) to construct the knockout vector Cas9- SH3.1 , the target and amplification primers are as follows: Target 1: CGAGGAGGAGAGCGAGCAGG (the reverse complementary sequence is positions 1972-1991 of SEQ ID No: 3); Target 2: CACCTCCCCTCGCAGCTCGG (the reverse complementary sequence is positions 2170-2189 of SEQ ID No: 3).

[0048] First round amplification primers: UF CTCCGTTTTACCTGTGGAATCG gR-R CGGAGGAAAATTCCATCCAC (universal primer) gRT1: CGAGGAGGAGAGCGAGCAGGgttttagagctagaaat OsU6aT1:CCTGCTCGCTCTCCTCCTCGCggcagccaagccagca (amplification target 1) gRT2: CACCTCCCCTCGCAGCTCGGgttttagagctagaaat OsU6bT2:CCGAGCTGCGAGGGGAGGTGCaacacaagcggcagc (amplification target 2) Second round amplification primers (all universal primers): U-GAL: ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGGCAGCAAAGG Pgs-GA2: CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG U-GA2: GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG Pgs-GAR: TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGTCTTG Knockout vector Cas9- SH3.1 The construction steps are: 1) Target amplification: The reaction system for target 1 is shown in Table 2: Table 2

[0049] The target 2 reaction system is shown in Table 3: Table 3

[0050] The amplification program is shown in Table 4: Table 4

[0051] 2) Adapter amplification: The reaction system for target 1 is shown in Table 5: Table 5

[0052] The target 2 reaction system is shown in Table 6: Table 6

[0053] The amplification program is shown in Table 7: Table 7

[0054] 3) The reaction system for CRISPR / Cas9 empty enzyme digestion is shown in Table 8: Table 8

[0055] 37℃ 1h, 65℃ 20min.

[0056] 4) Fragment recycling: The products of the second round of amplification were recovered using a DNA gel recovery kit (Jinsha Biotechnology, GE706).

[0057] 5) Vector ligation, reaction system see Table 9: Table 9

[0058] 50℃ 20min.

[0059] 2. Transformation of the African cultivated rice introgression line GIL291 and detection of editing results The knockout vector Cas9- SH3.1 The gene was introduced into the introgression line GIL291 with the Taichung65 background and the African cultivated rice IRGC104038 fragment using the soil Agrobacterium transformation method (Qingke Bio, TSC-A03). Two rounds of screening were performed using NB medium containing hygromycin at a concentration of 800 μg / mL, and then pre-differentiation was performed to differentiate gene knockout-positive plants.

[0060] Gene knockout-positive plants were identified by PCR. Specifically, whole-genome DNA from the introgressed GIL291 wild-type and knockout-positive plants was extracted using the CTAB method. This DNA was used as a template to amplify a hygromycin protein sequence approximately 500 bp long. Deionized water was used as a negative control, and an empty CRISPR / Cas9 vector plasmid was used as a positive control. The primer sequences are as follows: Hpt-F:AAGTTCGACAGCGTCTCCGAC Hpt-R:TCTACACAGCCATCGGTCCAG After PCR identification, 11 gene knockout plants were obtained using the Cas9-SH3.1 vector, numbered as Cas9- SH3.1 -1、Cas9- SH3.1 -2.Cas9- SH3.1 -3.Cas9- SH3.1 -4、Cas9- SH3.1 -5、Cas9- SH3.1 6. Cas9 SH3.1 -7、Cas9- SH3.1 -8、Cas9- SH3.1 -9、Cas9- SH3.1 -10 and Cas9- SH3.1 -11.

[0061] The Cas9-knockout plants SH3.1 -1、Cas9- SH3.1 -2.Cas9- SH3.1 -3.Cas9- SH3.1-4、Cas9- SH3.1 -5、Cas9- SH3.1 6. Cas9 SH3.1 -7、Cas9- SH3.1 -8、Cas9- SH3.1 -9、Cas9- SH3.1 -10 and Cas9- SH3.1 -11 Using primers containing the target site and its surrounding sequences SH3.1 -F and se- SH3.1 -R was used for amplification and sequencing. The sequencing results were analyzed using the website CRISPR-GE (http: / / skl.scau.edu.cn / ) to obtain the specific method of gene editing. The primer sequences are as follows: se- SH3.1 -F:GTCTCTCTCCGGTGTCTTCT se- SH3.1 -R:ATAAACGGCAGCAAGTTGGA Among them, Cas9- SH3.1 -3 was edited by deleting 5 deoxynucleotides, CTGGG, at positions 2139-2144 of SEQ ID No: 3.

[0062] 3. Functional identification of transgenic rice The knockout lines obtained were self-pollinated and propagated to obtain the corresponding knockout lines in the T2 generation. These knockout lines were used as materials, and the unedited wild-type introgression line GIL291 was used as a control. Conventional planting was performed (field planting, with 25 cm row spacing, 12 cm plant spacing, and two rows per family). Seed shattering characteristics of each genetic material after maturity were observed and analyzed. At least five plants of each genetic material were analyzed.

[0063] The results showed that compared with the introgression line GIL291 without gene editing, the gene knockout plants screened for hygromycin resistance and identified by PCR showed a phenotype of more difficult grain shattering ( Figure 4 Therefore, SH3.1 protein and related biomaterials can be used to regulate seed shattering, and knocking out the gene encoding SH3.1 protein by gene editing can reduce seed shattering.

[0064] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for reducing rice grain shattering, characterized by: The steps include: inhibiting the receptor rice SH3.1 The expression of the gene is used to obtain the target rice with lower grain shattering than the recipient rice; SH3.1 The gene is a gene encoding SH3.1 protein; the SH3.1 protein is the following protein A1), A2) or A3): A1) The amino acid sequence is the protein of SEQ ID No: 2 in the sequence listing; A2) a protein having a similar function to the protein of A1 and having a homology of more than 80% to the protein of A1 obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID No: 2 in the sequence listing; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

2. The method according to claim 1, wherein: described SH3.1 The gene is specifically the gene shown as E1, E2 or E3 below: E1, the coding sequence of the coding strand is a nucleic acid molecule of SEQ ID No: 1; E2, a nucleotide sequence comprising the nucleic acid molecule of positions 1946-2421 of SEQ ID No: 3 in the sequence listing; E3. The nucleotide sequence is the nucleic acid molecule of SEQ ID No: 3 in the sequence listing.

3. The method according to claim 1 or 2, characterized in that: The recipient rice is selected from African cultivated rice or its derivative plants.

4. The method according to claim 1, wherein: The inhibitory receptor in rice SH3.1 Gene expression is achieved by SH3.1 The gene editing is achieved by gene editing; the gene editing is achieved with the help of the CRISPR / Cas9 system.

5. The method according to claim 4, characterized in that: The CRISPR / Cas9 system includes a plasmid expressing Cas9 and sgRNA, and the target sequence of the sgRNA is SEQ ID No: 3 positions 2128-2151 and SEQ ID No: 3 positions 2194-2215 in the sequence list.

6. The SH3.1 protein according to claim 1.

7. The biomaterial related to the SH3.1 protein according to claim 1, characterized in that: The biological material is any one of the following B1 to B5: B1, a nucleic acid molecule encoding the SH3.1 protein according to claim 1; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

8. A plant agent for reducing grain shattering, characterized in that: The active ingredient of the reagent is a substance that inhibits the expression of the gene encoding the SH3.1 protein described in claim 1, reduces the abundance of the SH3.1 protein described in claim 1, and / or knocks out the gene encoding the SH3.1 protein described in claim 1; the substance contains the following F1), F2) or F3): F1) Targeting as claimed in claim 1 SH3.1 sgRNA, siRNA, shRNA, miRNA, or antisense RNA of a gene; F2) produces the target compound described in claim 1 SH3.1 Gene targeting sgRNA DNA molecule, producing the target gene of claim 1 SH3.1 Gene-targeting siRNA DNA molecules, producing the target gene described in claim 1 SH3.1 Gene shRNA DNA molecules, producing the target gene described in claim 1 SH3.1 A DNA molecule or a miRNA targeting gene as claimed in claim 1 SH3.1 DNA molecules that are antisense RNA to a gene; F3) produces the target compound described in claim 1 SH3.1 Gene expression vector targeting sgRNA of claim 1 SH3.1 Gene siRNA expression vector, producing the target gene described in claim 1 SH3.1 Gene shRNA expression vector, producing the target gene described in claim 1 SH3.1 Gene expression vector or production of miRNA targeting the gene described in claim 1 SH3.1 Gene antisense RNA expression vector.

9. The plant agent according to claim 8, characterized in that: The target sequences of the sgRNA are SEQ ID No: 3, positions 2128-2151 and SEQ ID No: 3, positions 2194-2216 in the sequence listing.

10. Use of the method according to any one of claims 1 to 5, the protein according to claim 6, the biological material according to claim 7, or the plant agent according to claim 8 or 9 in cultivating high-yield rice.

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