Application of GSN4 gene in simultaneous regulation of 1000-grain weight and number of grains per ear in rice

Abstract

The present invention discloses the application of the GSN4 gene in simultaneously regulating the thousand-grain weight and the number of grains per panicle of rice, and relates to the technical fields of genetic engineering and genetic breeding. The nucleotide sequence of the GSN4 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2. The present invention uses the CRISPR / Cas9 system to edit GSN4 to obtain a knockout mutant of GSN4, which is characterized by reduced grain width, significantly reduced thousand-grain weight and number of grains per panicle, and clarifies the biological function of the gene in regulating the thousand-grain weight and number of grains per panicle of rice, providing new gene resources for basic research on the thousand-grain weight and number of grains per panicle of rice and breeding utilization, and effectively solving the problem that the thousand-grain weight and number of grains per panicle genes are difficult to improve simultaneously.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and genetic breeding, and in particular to the application of a GSN4 gene in simultaneously regulating the thousand-grain weight and the number of grains per ear of rice. Background Art

[0002] Rice yield is mainly composed of three factors: the number of effective panicles per plant, 1000-grain weight and the number of grains per panicle. 1000-grain weight is mainly determined by grain shape, which includes grain length, grain width, grain thickness and length-width ratio. At present, many genes related to grain shape and 1000-grain weight have been cloned, and they are involved in different signal pathways, including G protein signaling pathway, ubiquitin-proteasome pathway, mitogen-activated protein kinase pathway, transcription factor regulation pathway, plant hormone pathway, etc. The G protein signaling pathway includes one α subunit (D1 / RGA1), one β subunit (RGB1) and five γ subunits (RGG1, RGG2, GS3, DEP1 and OsGGC2), all of which are involved in regulating grain shape. Genes regulating grain shape in the ubiquitin-proteasome pathway include SMG3 / OsUBC32 and OsUBC13 encoding E2 ubiquitin conjugating enzymes, GW2, TUD1, CLG1 and DGS1 encoding E3 ubiquitin ligases, and WTG1 and OsUBP15 encoding deubiquitinating enzymes. The genes regulating granule shape by mitogen-activated protein kinase pathway mainly include OsMKKK10, OsMKK4, OsMAPK6 and GSN1 / OsMKP1. Several transcription factor family genes were reported to be directly involved in regulating granule shape, including OsSPL12, GLW7 / OsSPL13, GW8 / OsSPL16 and OsSPL18 of SPL family, GS2 / GL2 / OsGRF4 of growth regulatory factor GRF family, MYB transcription factors GL4 and GL12, etc. Plant hormones play an important role in regulating growth and development. Auxin, cytokinin and brassinolide are all involved in regulating rice grain shape and 1000-grain weight. For example, TGW6, BG1 and qGL5 regulate 1000-grain weight through auxin, BG3 / OsPUP4 and OsPUP1 regulate grain shape and 1000-grain weight through cytokinin, and OsBAK1 / 08SG2, OsBSK1-1, OsBIN2 / GSK2, DLT, GSE5 / GW5, GS9 and GW10 are reported to regulate grain shape through the BR pathway.

[0003] The number of grains per ear is mainly determined by ear length, the number of primary branches and the number of secondary branches. At present, many genes related to the number of grains per ear have been cloned. Among them, Gn1a, DST, IPA1, TAW1, FZP, RGN1, LOG and NOG1 mainly affect the number of grains per ear by regulating the number of branches, and DEP1, DEP2, DEP3, APO1, APO2, SPED1, SP1 and SP3 mainly affect the panicle shape. Existing studies have shown that 1000-grain weight and number of grains per ear are usually antagonistic and difficult to increase at the same time. For example, after GSN1 mutation, the grain shape and 1000-grain weight are significantly increased, but the number of grains per ear is significantly reduced, while after OsER1, MPK6 and MKK4 mutation, the number of grains per ear is significantly increased, but the grain shape and 1000-grain weight are significantly reduced. Therefore, it is urgent to further explore the genes related to 1000-grain weight and number of grains per ear. Breaking the balance between 1000-grain weight and number of grains per ear is an important way to further increase yield. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides the application of the GSN4 gene in simultaneously regulating the thousand-grain weight and the number of grains per ear of rice, provides new gene resources for the basic research on the thousand-grain weight and the number of grains per ear of rice and for breeding utilization, and effectively solves the problem that the genes for the thousand-grain weight and the number of grains per ear are difficult to improve simultaneously.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: to provide the application of GSN4 gene in simultaneously regulating the thousand-grain weight and the number of grains per ear of rice.

[0006] Furthermore, the nucleotide sequence of the GSN4 gene is shown in SEQ ID No.1.

[0007] Furthermore, the amino acid sequence of the protein encoded by the GSN4 gene is shown in SEQ ID NO.2.

[0008] Furthermore, the GSN4 gene is used in the simultaneous regulation of rice grain width, 1000-grain weight and number of grains per panicle.

[0009] A method for simultaneously regulating the thousand-grain weight and the number of grains per ear of rice using the above-mentioned GSN4 gene comprises the following steps:

[0010] S1. Extract the GSN4 gene, design a knockout target in the exon of the GSN4 gene, construct a knockout vector of the GSN4 gene, and then use the intermediate vector to construct the final vector;

[0011] S2, transforming competent cells with the final vector obtained in step S1, and screening the plasmid with correct sequencing;

[0012] S3. Use the plasmid obtained in step S2 to transform competent cells, screen out positive clones, and transform rice seeds to obtain knockout mutants of GSN4.

[0013] Furthermore, in step S1, the GSN4 gene is derived from a mutant related to grain shape and grain number per ear in an ethyl methanesulfonate-induced mutant library of indica rice Shuhui 498.

[0014] Furthermore, in step S1, a knockout vector of the GSN4 gene is constructed using the CRISPR / Cas9 system.

[0015] Furthermore, the intermediate vector is pYLsgRNA-OsU6a.

[0016] Further, in step S2, the competent cells are Escherichia coli DH5α.

[0017] Furthermore, dedicated sequencing primers were constructed using CRISPR / Cas9 vectors for sequencing.

[0018] Furthermore, the nucleotide sequence of the dedicated sequencing primer for CRISPR / Cas9 vector construction is 5'-GCGGTGTCATCTATGTTACTA-3' (SEQ ID No. 3).

[0019] Further, in step S3, the competent cells are Agrobacterium competent cells GV3101.

[0020] Furthermore, in step S3, the rice seeds are seeds of japonica rice Nipponbare and indica rice Shuhui 498.

[0021] Furthermore, the sequence of the editing target site in the japonica rice Nipponbare background is 5'-GGACTTGTGCGTTCGCAAGACGG-3' (SEQ ID No. 4).

[0022] Furthermore, the sequence of the editing target site in the background of indica rice Shuhui 498 is 5'-AAGACTCAACGTGCAGCGAG-3' (SEQ ID No. 5).

[0023] The present invention has the following beneficial effects:

[0024] 1. The novel 1000-grain weight and grain number per panicle gene GSN4 provided by the present invention provides a new gene resource for studying the regulatory mechanism of 1000-grain weight and grain number per panicle of rice.

[0025] 2. The gene GSN4 provided by the present invention positively regulates both the thousand-grain weight and the number of grains per ear, and can be applied to improve the thousand-grain weight and the number of grains per ear of rice, thereby increasing rice yield. It provides an excellent gene resource for simultaneously increasing the thousand-grain weight and the number of grains per ear of rice, and has important breeding application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an analysis diagram of the expression pattern of the rice GSN4 gene in Example 1 of the present invention;

[0027] Figure 2 This is a schematic diagram of the GSN4 gene editing vector in Example 2 of the present invention;

[0028] Figure 3 This is the DNA sequencing result of the japonica rice Nipponbare GSN4 gene knockout mutant;

[0029] Figure 4 This is a comparison of plant heights between the wild-type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0030] Figure 5 This is a comparison of grain length between the wild type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0031] Figure 6 This is a comparison of grain width between the wild type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0032] Figure 7 This is a comparison of panicle shapes between the wild type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0033] Figure 8 This is a comparison of the number of grains per panicle between the wild type and the GSN4 gene knockout mutant of japonica rice Nipponbare;

[0034] Fig. 9 The statistical graph of average grain length of wild-type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0035] Fig.10 The statistical graph of average grain width of wild-type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0036] Fig.11 This is a statistical chart of the thousand-grain weight of the wild-type japonica rice Nipponbare and the GSN4 gene knockout mutant;

[0037] Fig.12 This is a statistical chart of the number of grains per panicle of the wild type and GSN4 gene knockout mutant of japonica rice Nipponbare;

[0038] Fig.13 This is the result of DNA sequencing of the GSN4 gene knockout mutant of indica rice Shuhui 498;

[0039] Fig.14 This is a comparison of plant heights between the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0040] Fig.15 This is a comparison of grain length between the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0041] Fig.16 This is a comparison of grain widths between the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0042] Fig.17 This is a comparison of panicle shapes between the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0043] Fig.18 This is a comparison of the number of grains per panicle of the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0044] Fig.19 The statistical graph of average grain length of wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0045] Fig. 20 The statistical graph of average grain width of wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0046] Fig.21 This is a statistical chart of thousand-grain weight of the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498;

[0047] Fig. 22 The graph shows the statistical graph of the number of grains per panicle of the wild type and GSN4 gene knockout mutant of indica rice Shuhui 498. DETAILED DESCRIPTION

[0048] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0049] Example 1 Analysis of the expression pattern of the GSN4 gene in rice

[0050] (1) Quantitative PCR primer design

[0051] The present invention uses the quantitative PCR primer online design website (https: / / quantprime.mpimp-golm.mpg.de / ) to design a pair of quantitative PCR primers spanning exons 9 and 10 in the coding region of GSN4. The specific sequences are as follows:

[0052] Y1965-F:5'-ATAAACCGCTCACTTTCACAAC-3'(SEQ ID No.6)

[0053] Y1964-R:5'-ACCCAGTGATTCTTGCAATAGA-3'(SEQ ID No.7)

[0054] (2) RNA extraction and reverse transcription

[0055] In order to analyze the expression pattern of GSN4, materials from different tissues and different developmental stages of Shuhui 498 were collected and stored at -80°C for later use. RNA from different tissues was extracted using the Quanshijin Plant RNA Extraction Kit according to the instructions, and cDNA was obtained by reverse transcription using the RT EasyTM II (With gDNase) Reverse Transcription Kit from Fuji Biotechnology Co., Ltd. for quantitative PCR analysis.

[0056] (3) Expression profile analysis

[0057] Quantitative PCR reaction was performed using the quantitative PCR Mix of QuantiGold according to the instruction manual. Three biological replicates were performed for each sample. Actin-F: 5'-GACTCTGGTGATGGTGTCAGC-3' (SEQ ID No. 8) and Actin-R: 5'-GGCTGGAAGAGGACCTCAGG-3' (SEQ ID No. 9) primers were used as internal references. The results were analyzed as follows. Figure 1 shown. Figure 1 In the figure, R represents roots at the seedling stage, LB represents leaves at the seedling stage, FL and LS represent flag leaves and leaf sheaths at the booting stage, respectively, C represents stems at the booting stage, YPI-YP15 represent spikelets of different lengths (cm), H9-H15 represent glumes on spikelets of different lengths, and E5-E15 represent caryopsis at different days after fertilization.

[0058] Depend on Figure 1 It can be seen that GSN4 is highly expressed mainly in young panicles and glumes, which is consistent with its biological function of regulating grain shape, 1000-grain weight and grain number per spike.

[0059] Example 2 Construction of CRISPR / Cas9 knockout vector and genetic transformation of GSN4 gene

[0060] (1) Design of GSN4 gene editing target sites

[0061] In order to study whether GSN4 is involved in regulating rice thousand-grain weight and number of grains per panicle, the targetDesign online tool at the website http: / / skl.scau.edu.cn / ?tdsourcetag=s_pcqq_aiomsg was used to design knockout targets in the exons of GSN4, and knockout was performed in the backgrounds of japonica rice Nipponbare (Nip) and indica rice Shuhui 498 (R498), respectively, in the hope of obtaining loss-of-function transgenic plants to clarify the function of the GSN4 gene. The editing target sites in the backgrounds of Nipponbare and Shuhui 498 are Target 1 and Target 2, respectively, and the sequences are as follows:

[0062] Target 1: 5'-GGACTTGTGCGTTCGCAAGACGG-3' (SEQ ID No.4)

[0063] Target 2: 5'-AAGACTCAACGTGCAGCGAG-3' (SEQ ID No.5)

[0064] (2) CRISPR / Cas9 vector construction

[0065] Take 5 μL of 10 μM Target 1 target site front primer Y1982-F and rear primer Y1983-R, 10 μM Target 2 target site front primer Y3837-F and rear primer Y3838-R, mix well and anneal at 99℃ for 5 minutes to obtain primer dimers. Dilute the products 10 times for later use. The primer sequences are as follows:

[0066] Y1982-F:5'-gccgGACTTGTGCGTTCGCAAGA-3'(SEQ ID No.10)

[0067] Y1983-R:5'-aaacTCTTGCGAACGCACAAGTC-3'(SEQ ID No.11)

[0068] Y3837-F: 5'-gccgAAGACTCAACGTGCAGCGAG-3' (SEQ ID No. 12)

[0069] Y3838-R:5'-aaacCTCGCTGCACGTTGAGTCTT-3'(SEQ ID No.13)

[0070] Then, Bas1 (NEB) was used for simultaneous restriction digestion and ligation (37°C). The 10 μL restriction digestion system was as shown in Table 1 below, and the target site sequence was connected to the intermediate vector pYLsgRNA-OsU6a.

[0071] Table 1 10μL enzyme digestion system

[0072]

[0073]

[0074] Then, using universal amplification primers

[0075] Y3149-F: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No. 14) and Y3150-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No. 15) were used in combination with the target site rear primer and front primer, respectively, and the MAX enzyme (Novagen) amplification system (2x Phanta Max Buffer 10 μL; 10mM dNTP Mix 0.5 μL; 5xPCR Enhancer 4 μL; Phanta Max Super-Fidelity DNA Polymerase 0.5 μL; 40x Primer 2 μL; Template 3μL) was amplified by PCR to obtain two fragments containing the target site, one large and one small, from the intermediate vector pYLsgRNA-OsU6a. The two fragments were then fused and recovered by overlapping. Finally, Bas1 (NEB) was used for simultaneous restriction digestion and ligation (37°C) for 3h to connect the fusion fragment to the final vector pylcrispr / Cas9P35S-N (15011bp). The final vector was successfully constructed as shown in Figure 2 shown.

[0076] (3) CRISPR / Cas9 vector E. coli transformation

[0077] Take out the competent E. coli DH5α (Kangti Life) from the -80℃ refrigerator, thaw on ice, and quickly add the ligation product in the previous step after the cells are dissolved. Mix gently with a pipette and let stand on ice for 30 minutes, then heat shock at 42℃ for 1 minute, and then let stand on ice for 2 minutes. Then add 1mL of anti-antibody LB culture medium to the transformation product, put it on a 37℃ shaker, and take it out after culturing at 200rpm for 1h. After centrifugation at 4000rpm, remove most of the supernatant, and resuspend the remaining liquid (about 100μL) and apply it to the plate (LB+kanamycin resistance). After static culture at 37℃ overnight, pick a monoclonal colony and expand the culture of 3mL (LB+kanamycin resistance), then use CRISPR / Cas9 vector to construct a special sequencing primer Y3160: 5'-GCGGTGTCATCTATGTTACTA-3' (SEQ IDNo.3) for sequencing confirmation, and save the plasmid and bacterial solution with correct sequencing for use.

[0078] (4) CRISPR / Cas9 vector Agrobacterium transformation

[0079] Take out the competent Agrobacterium GV3101 (Qingke Biotechnology) from -80℃ and put it on ice to melt, add 2μL of the above positive plasmid, mix gently and let it stand on ice for 5min, then put it in liquid nitrogen for 5min, then put it in a 37℃ metal bath for 5min, and then put it on ice for 5min. Then add 1mL of anti-antibiotic LB culture medium to the transformation product, put it on a 28℃ shaker at 250rpm for 2-3h, then take it out, centrifuge at 5000rpm and remove most of the supernatant, resuspend the remaining liquid (about 100μL) and apply it to the plate (LB+Rifampicin+Kanamycin), and then stand at 28℃ overnight to pick the monoclonal colony for bacterial liquid PCR detection, and save the bacterial liquid of the detected positive clone for later use.

[0080] (5) CRISPR / Cas9 vector rice genetic transformation

[0081] Take about 500 seeds of Nip and R498, remove the seed shells and set aside. After 1 minute of treatment with 75% alcohol, wash with sterile water and 50% sodium hypochlorite respectively, then use filter paper to absorb the water, and then evenly spread the treated seeds on NMB medium to induce callus; then expand the positive clone bacterial solution to 50mL, collect the bacteria by centrifugation at 5000rpm, and resuspend the bacteria in AAM liquid medium with AS (acetosyringone), soak the well-growing callus in the resuspended bacteria environment for about 30 minutes, then remove the bacteria, and continue to culture the callus on the culture medium for 2 days. The cultured callus tissue was washed with sterile water and sterile water with cephalosporin, and placed on the selection medium for about three weeks; finally, the callus tissue was induced to root with differentiation medium, and when the seedling height was about 10cm, it could be hardened indoors for subsequent testing and transplanting.

[0082] Example 3 Phenotypic analysis of GSN4 knockout mutants in Nip background

[0083] (1) Knockout transgenic seedling detection

[0084] After the above-mentioned Agrobacterium infection and transformation, a total of 28 seedlings were obtained in the Nip background. After DNA was extracted from each plant, the vector transformation was first detected using hygromycin detection primers Y214-F: 5'-TACACAGCCATCGGTCCAGA-3' (SEQ ID No. 16) and Y215-R: 5'-TAGGAGGGCGTGGATATGTC-3' (SEQ ID No. 17). Subsequently, the cross-target site primers Y3021-F: 5'-ACAAACCTGTAGAGCCCATAGC-3' (SEQ ID No. 18) and Y3022-R: 5'-AACTCAACAAACTTAAACACCCAC-3' (SEQ ID No. 19) were used to amplify the positive plants and then sequenced. The results are shown in FIG. Figure 3As shown, two transgenic plants with different mutation patterns, GSN4-KO1 and GSN4-KO2, were obtained under the Nip background, which deleted 4 bases and 58 bases in the GSN4 coding region, respectively. Both mutation patterns led to the premature termination of GSN4 protein translation.

[0085] (2) Phenotypic analysis of knockout mutants

[0086] The phenotypes of GSN4-KO1, GSN4-KO2 and wild-type Nip were compared and analyzed. Figure 4-12 shown.

[0087] Depend on Figure 4 It can be seen that the plant heights of GSN4-KO1 and GSN4-KO2 were reduced compared with the wild-type Nip.

[0088] Depend on Figure 5 and Fig. 9 It can be seen that there is no significant difference in the grain length between GSN4-KO1, GSN4-KO2 and wild-type Nip.

[0089] Depend on Figure 6 and Fig.10 It can be seen that the grain width of GSN4-KO1 and GSN4-KO2 was significantly reduced by 8% and 7.4%, respectively.

[0090] Depend on Figure 7 It can be seen that GSN4-KO1 and GSN4-KO2 showed sparse panicle shape.

[0091] Depend on Figure 8 and Fig.12 It can be seen that the number of grains per ear decreased significantly by 22.2% and 19.6% respectively.

[0092] Depend on Fig.11 It can be seen that compared with the wild-type R498, the thousand-grain weight of GSN4-KO1 and GSN4-KO2 was significantly reduced by 9.6% and 13%, respectively.

[0093] The above results all indicate that GSN4 positively regulates rice grain width, 1000-grain weight and number of grains per panicle.

[0094] Example 4: Phenotypic analysis of GSN4 knockout mutants in R498 background

[0095] (1) Knockout transgenic seedling detection

[0096] After the above Agrobacterium infection and transformation, a total of 30 seedlings were obtained in the R498 background. After DNA was extracted from each plant, the vector transformation was first detected using the above hygromycin detection primers Y214-F and Y215-R. Then, the cross-target primers Y4829-F: 5'-ATTATTACCTTAAACCAAACATTTA-3' (SEQ ID No. 20) and Y4830-R: 5'-ATGGGTCTAGCAAATCAGTA-3' (SEQ ID No. 21) were used to amplify the positive plants and then sequenced. The results are shown in Figure 2. Fig.13 As shown, two transgenic plants with different mutation patterns, GSN4-KO3 and GSN4-KO4, were obtained under the R498 background, with one base inserted and four bases deleted in the GSN4 coding region, respectively, both of which led to the premature termination of GSN4 protein translation.

[0097] (2) Phenotypic analysis of knockout mutants

[0098] The phenotypes of GSN4-KO3 and GSN4-KO4 were analyzed and compared with those of wild-type R498. Figure 14-22 shown.

[0099] Depend on Fig.14 It can be seen that the plant heights of GSN4-KO3 and GSN4-KO4 were reduced compared with the wild-type R498.

[0100] Depend on Fig.15 and Fig.19 It can be seen that there is no significant difference in the grain length between GSN4-KO3 and GSN4-KO4 compared with the wild-type R498.

[0101] Depend on Fig.16 and Fig. 20 It can be seen that compared with the wild-type R498, the grain widths of GSN4-KO3 and GSN4-KO4 were significantly reduced by 12.7% and 10.7%, respectively.

[0102] Depend on Fig.17 It can be seen that compared with the wild type R498, GSN4-KO3 and GSN4-KO4 showed sparse panicle shape.

[0103] Depend on Fig.18 and Fig. 22 It can be seen that compared with the wild-type R498, the number of grains per ear in GSN4-KO3 and GSN4-KO4 was significantly reduced by 15.7% and 18.7%, respectively.

[0104] Depend on Fig.21 It can be seen that compared with the wild-type R498, the thousand-grain weight of GSN4-KO3 and GSN4-KO4 was significantly reduced by 22.5% and 23.2%, respectively.

[0105] The above results all indicate that GSN4 positively regulates rice grain width, 1000-grain weight and number of grains per panicle, and this phenotype is functionally conserved in indica and japonica rice.

[0106] In summary, the GSN4 gene can positively regulate both thousand-grain weight and grain number per panicle at the same time, and can be used to improve the thousand-grain weight and grain number per panicle of rice, thereby increasing rice yield.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of the GSN4 gene in simultaneously regulating the thousand-grain weight and the number of grains per ear of rice, the nucleotide sequence of the GSN4 gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the GSN4 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The GSN4 gene is used for simultaneously regulating rice grain width, thousand-grain weight and number of grains per ear.

4. A method for simultaneously regulating the thousand-grain weight and the number of grains per ear of rice using the GSN4 gene described in claim 1, characterized in that: The following steps are involved: S1. Extract the GSN4 gene, design a knockout target in the exon of the GSN4 gene, construct a knockout vector of the GSN4 gene, and then use the intermediate vector to construct the final vector; S2, transforming competent cells with the final vector obtained in step S1, and screening the plasmid with correct sequencing; S3. Use the plasmid obtained in step S2 to transform competent cells, screen out positive clones, and transform rice seeds to obtain knockout mutants of GSN4.

5. The method for simultaneously regulating rice thousand-grain weight and number of grains per ear using the GSN4 gene as claimed in claim 4, characterized in that: In step S1, the GSN4 gene is derived from mutants related to grain shape and number of grains per ear in a mutant library induced by ethyl methanesulfonate of indica rice Shuhui 498.

6. The method for simultaneously regulating rice thousand-grain weight and number of grains per ear using the GSN4 gene as claimed in claim 4, characterized in that: In step S1, a knockout vector of the GSN4 gene is constructed using the CRISPR / Cas9 system, and the intermediate vector is pYLsgRNA-OsU6a.

7. The method for simultaneously regulating rice thousand-grain weight and number of grains per ear using the GSN4 gene as claimed in claim 4, characterized in that: In step S2, the competent cells are Escherichia coli DH5α, and the CRISPR / Cas9 vector is used to construct dedicated sequencing primers for sequencing.

8. The method for simultaneously regulating rice thousand-grain weight and number of grains per ear using the GSN4 gene as claimed in claim 4, characterized in that: In step S3, the competent cells are Agrobacterium competent cells GV3101.

9. The method for simultaneously regulating rice thousand-grain weight and number of grains per ear using the GSN4 gene as claimed in claim 4, characterized in that: In step S3, the rice seeds are seeds of japonica rice Nipponbare and indica rice Shuhui 498.

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