Rice multiple grain gene qsp1 and application thereof

Abstract

The present application relates to a kind of rice multiple grain gene qSP1 and its application, belong to the field of biotechnology.Rice multiple grain gene qSP1 The promoter nucleotide sequence as shown in SEQ ID NO:2 and CDS nucleotide sequence as shown in SEQ ID NO:3, the protein encoded has the amino acid sequence as shown in SEQ ID NO:4 with rice transcriptional co-repressor LEUNIG.The qSP1 can be used to regulate rice grain number, increase rice yield.The present application increases the grain number and yield of rice by molecular marker assisted selection technology or gene editing technology, provides new gene resources and technical approach for rice genetic improvement and high-yield breeding, has important application value and social benefits.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a rice multiple-grain gene qSP1 and application thereof BACKGROUND

[0002] As one of the most important food crops in the world, rice is the staple food of more than half of the world's population, and improving rice yield is of great significance to solving the problem of food shortage caused by global climate change, continuous population growth and the year-by-year reduction of available arable land (Zhang, Strategies for developing green super rice, Proceedings of the National Academy of Sciences of the United States of America. 2007, 104, 16402-16409). With the development of urbanization in China and the year-by-year reduction of arable land, in order to make up for the loss of rice yield due to the reduction of land, improving rice yield per unit remains the primary goal of rice breeding. However, rice yield traits are complex quantitative traits controlled by multiple genes, mainly determined by three factors, i.e., effective tiller number per plant, grain number per panicle and grain weight. The three internal elements complement each other while being mutually restricted. How to break through the mutual restriction between the elements, find the node of mutual interaction and balance between the elements, has become a challenge faced by current rice scientific research and molecular design breeding (Guo et al, GRAIN SIZE AND NUMBER 1 negatively regulates the OsMKKK10-OsMKK4-OsMPK6 cascade to coordinate the trade-off between grain number per panicle and grain size in rice. The Plant Cell. 30, (2018) 871-888). Rice "grain number per panicle" is one of the three elements of yield, which largely determines the final yield, and identifying the genes controlling grain number and studying the molecular regulation mechanism thereof are of great significance to high-yield design breeding of rice. SUMMARY

[0003] Therefore, the application aims to provide a rice multiple-grain gene qSP1 and a recombinant plasmid prepared by using the gene and application thereof in cultivating multiple-grain rice varieties.

[0004] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0005] The application provides a rice multiple grain gene qSP1, a gene promoter sequence of which is shown in SEQ ID NO: 2, and a gene CDS nucleotide sequence of which is shown in SEQ ID NO: 3.

[0006] The gene expresses an amino acid sequence shown in SEQ ID NO: 4.

[0007] Further, the application provides a recombinant vector, an expression cassette or a recombinant bacterium containing the rice multiple grain gene qSP1

[0008] Further, the application provides a recombinant vector pCAMBIA1301-qSP1, wherein the recombinant vector is a qSP1 whole genome DNA sequence of SEQ ID NO: 1.

[0009] The application also provides an application of the qSP1 gene in cultivating a multiple grain rice variety, and the steps are as follows:

[0010] S1: using CRISPR / Cas9 technology to knock out the qSP1 gene;

[0011] S2: transferring the knocked-out qSP1 plasmid into agrobacterium;

[0012] S3: transforming different rice few grain varieties with positive agrobacterium;

[0013] S4: a homozygous knockout transgenic strain is a multiple grain type rice plant.

[0014] The application has the following beneficial effects:

[0015] The application provides a powerful tool for rice transgenic research and molecular marker assisted selection breeding, promotes high yield breeding research of hybrid rice, effectively increases the grain number and yield of rice through genetic engineering technology, provides a new gene resource and technical approach for genetic improvement and high yield breeding of rice, and has important application value and social benefits.

[0016] Other advantages, objects, and features of the application will be set forth in part in the following specification taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art from a consideration of the following specification and drawings and from the practice of the application. The objects and other advantages of the application will be realized and attained by means of the instrumentalities and combinations pointed out in the following specification. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the objects, technical solutions and advantages of the application clearer, the preferred embodiments of the application will be described in detail below with reference to the drawings, in which:

[0018] Figure 1For the analysis of spike-related traits and QTL identification in the single-segment substitution line Z499 containing qSP1 gene, a: panicle type of Nip and Z499. Scale bar, 5 cm; b-e: statistical analysis of spike length (b), primary branch number (c), secondary branch number (d), total grain number per panicle (e) of Nip and Z499. Data are shown as mean ± SD (n = 10). μ represents the mean value and ai represents the additive effect of QTL. P value < 0.05 represents significant difference between Nip and Z499 in t-test.

[0019] Figure 2 For the alignment of qSP1 mutant gene and wild-type gene sequence (Nip).

[0020] Figure 3 For the complementation phenotype analysis of qSP1, a: panicle type of Nip, Z499 and LOC_Os01g08190 complemented plants at the mature stage. qSP1-COM-1 and qSP1-COM-2 represent two independent LOC_Os01g08190 complemented transgenic lines, respectively. Scale bar, 5 cm; b: sequencing results of the mutant site of LOC_Os01g08190 complemented plants; c: qRT-PCR analysis of Nip, Z499 and LOC_Os01g08190 complemented plants. ACTIN was used as the internal control for qRT-PCR. Mean ± SD (n = 3); d-h: statistical analysis of spike length (d), primary branch number (e), secondary branch number (f), total grain number per panicle (g) and grain yield per plant (h) of Nip, Z499 and LOC_Os01g08190 complemented plants. Data are shown as mean ± SD (n = 10). In (c), P value < 0.05 indicates significant difference in t-test. In (d-h), different lowercase letters indicate significant difference (P < 0.05) determined by one-way ANOVA and Duncan’s multiple comparison.

[0021] Figure 4a 、 4b , 4c: protein sequence alignment of qSP1 and its predicted family proteins. The conserved sequences are shown in red and black boxes.

[0022] Figure 5 For the phylogenetic tree analysis of the conserved motifs and domains contained in the protein structure of qSP1 and its predicted family proteins, a: schematic diagram of the protein structure of qSP1; b: phylogenetic tree analysis containing conserved motifs and conserved domains. At: Arabidopsis thaliana; Os: Oryza sativa; Zm: Zea mays; FCD: Ficus carica.

[0023] Figure 6 For the spatiotemporal expression pattern analysis of qSP1, P 0.5 : 0.5 cm young panicle; P1: 1 cm young panicle; P1.5 : 1.5 cm young panicles; P2: 2 cm young panicles; P4: 4 cm young panicles; P 10 : 10 cm young panicles.

[0024] Figure 7 Figure 2 is a diagram of the interaction analysis of qSP1 and OsSEUs in yeast. SD-LT represents a SD / -Leu-Trp deficient medium; SD-LTHA represents a SD / -Leu-Trp-His-Ade deficient medium; X-a-gal represents a SD / -Leu-Trp-His-Ade deficient medium containing X-a-gal.

[0025] Figure 8 Figure 3 is an electrophoretogram in the process of constructing the complementary vector plasmid, a: target fragment amplification electrophoretogram (3 segments); b: pCAMBIA1301 vector enzyme digestion electrophoretogram; c: bacterial liquid detection electrophoretogram.

[0026] Figure 9 Figure 4 is a construction map of the complementary vector. DETAILED DESCRIPTION

[0027] The present application is described herein with reference to specific embodiments thereof which are illustrated in the attached drawings. These are presented solely for illustration of the present application and are not intended as limitations thereof. Other embodiments of the present application can be employed and modifications can be made without departing from the spirit of the present application. The following examples are provided to further illustrate the present application and are not intended to limit the scope of the application. The figures provided herein are merely schematic and are not intended to limit the present application. Certain components in the figures can be omitted, enlarged or reduced in size for better illustration of the embodiments of the present application, and do not represent the actual size of the product. It is understood by those skilled in the art that certain well-known structures and their descriptions can be omitted in the figures.

[0028] The figures are merely schematic and not actual views, and should not be construed as limiting the present application. Certain components in the figures can be omitted, enlarged or reduced in size for better illustration of the embodiments of the present application, and do not represent the actual size of the product. It is understood by those skilled in the art that certain well-known structures and their descriptions can be omitted in the figures.

[0029] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it is understood that if the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "front", "back" and the like are based on the orientations or positional relationships shown in the drawings, they are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the terms describing the positional relationships in the drawings are only used for exemplary illustration, and cannot be understood as a limitation on the present application, for those skilled in the art, the specific meanings of the above terms can be understood according to the specific circumstances.

[0030] Embodiment one

[0031] The experimental methods in the embodiments not specified with specific conditions are generally according to the conditions described in the molecular cloning experiment guide (third edition, J. Sambrook et al., Huang Peitang et al., Science Press, 2002) or according to the conditions recommended by the manufacturer.

[0032] Materials used in the embodiments: rice recipient parent material Nipponbare and multiple grain single segment substitution line Z499, both of which are cultivated by the laboratory of the inventor; M-MLV reverse transcriptase, high-fidelity DNA polymerase PFU, Taq DNA polymerase, T4 DNA ligase, restriction endonuclease, pMD19-T vector, Trizol kit, DNA gel recovery kit, plasmid extraction kit, λ-Hind III DNA marker and DL5,000 DNA marker are purchased from TaKaRa company; DNA marker III is purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd.; ampicillin (Ampicillin, Amp) and kanamycin (Kanamycin, Kan) are products of Sigma company; primer synthesis and DNA sequencing are completed by Shanghai Yingjun Biotechnology Co., Ltd.; other chemical reagents are purchased from Beijing Dingguo Biotechnology Co., Ltd.; Escherichia coli DH5α and Agrobacterium LBA4404 are preserved in the laboratory of the inventor.

[0033] A multi-grain three-segment substitution line Z1364 (Ma et al, Identification of QTL for kernel number-related traits in a rice chromosome segment substitution line and fine mapping of qSP1. The Crop Journal. 7, (2019) 494-503) was created by natural variation with japonica rice Nipponbare as the recipient parent and indica rice restorer line Xihui 18 as the donor, on the basis of which, the present application uses the F2 population generated by crossing Nipponbare and Z1364 to breed a multi-grain single-segment substitution line Z499 by QTL positioning and MAS. The present application further detects the grain number QTL qSP1 in Z499, which is located in the interval of RM10198--RM1329-RM10224--RM6777 on chromosome 1, with a shortest substitution length of 50Kb, an estimated length of 0.20Mb, and a maximum substitution length of 0.35Mb. Compared with Nipponbare, the panicle length, primary branch number, secondary branch number, and total grain number per panicle of Z499 are significantly increased Figure 1 a-e). Then, qSP1 (LOC_Os01g08190) located on chromosome 1 is cloned from Z499 by map-based cloning, with the nucleotide sequence shown as SEQ ID NO: 1, and the gene encodes a transcriptional co-repressor LEUNIG protein. Compared with Nipponbare, there is a SNP difference (replaced by G instead of A) in the fourth exon of LOC_Os01g08190 in Z499, which causes the encoded amino acid to be replaced from Ala in Nipponbare to Thr in Z499 Figure 2 ). The encoded LEUNIG protein and OsSEU1, OsSEU2, or OsSEU3 form a qSP1-OsSEUs complex to regulate the number of rice grains Figure 7 ). Therefore, determining the nucleotide sequence of the qSP1 gene, including the promoter (nucleotide sequence shown as SEQ ID NO: 2) and CDS sequence (nucleotide sequence shown as SEQ ID NO: 3) and the encoded protein sequence (amino acid sequence shown as SEQ ID NO: 4), will be beneficial to the research on the molecular mechanism of rice grain number development and hybrid rice breeding, and promote hybrid rice yield to a new level.

[0034] Based on gene localization, this invention first preliminarily identified the rice multigrain gene qSP1 as LOC_Os01g08190 through gene prediction, homology search, and gene sequence difference comparison. Subsequently, using the rice multigrain single-fragment substitution line Z499 as material, the full-length CDS of the mutant gene qSP1 was cloned. Sequencing results showed that the full-length CDS sequence of the mutant gene qSP1 remained 2634 bp, consisting of 18 exons and encoding 877 amino acids. However, compared to the receptor Nipponbare, it exhibited a base substitution in the fourth exon, leading to a change in the encoded amino acid sequence. Figure 2 Through genetic complementation, the whole-genome DNA of the recipient parent, Nipponbare (LOC_Os01g08190), was introduced into the Z499 background, and sequencing and phenotypic analysis were performed. The results showed that the T0 generation complementary transgenic plants exhibited a bimodal distribution at the mutation site, and the expression level of the complementary transgenic plants was significantly higher than that of Nipponbare and Z499. Figure 3 bc), indicating that the target plasmid was successfully introduced into Z499. Furthermore, the number of secondary branches and the total number of grains per spike in its complementary transgenic lines were significantly reduced compared to Z499, but not significantly different from Nipponbare. Figure 3 The results indicate that the LOC_Os01g08190 gene is the target gene for controlling multiple grains. qSP1 bioinformatics analysis shows that the protein contains a conserved LisH sequence at its N-terminus and seven repeating WD40 domains at its C-terminus, and is evolutionarily closely related to ZmLUG10 in maize (Figures 4-5). qRT-PCR shows that the qSP1 gene is expressed at different developmental stages of the ear, with the highest expression in ears at 1.5 cm in diameter, but very low expression in roots, stems, leaves, and sheaths. Figure 6 Compared to Nipponbare, qSP1 expression levels were significantly downregulated in Z499. Figure 3 c).

[0035] Furthermore, library screening and yeast two-hybrid experiments revealed an interaction between qSP1 and OsSEUs family proteins. Figure 7). OSHI1 / SB1 directly inhibits the expression of DEP1 and IPA1 by interacting with OsSEU to affect the development of spikelet number (Duan et al, OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 in rice. The Plant Cell 31, (2019) 1026-1042; Zeng et al, SB1 encoding RING-like zinc-finger protein regulates branch development as a transcription repressor. Rice Science. 28, (2021) 243-256). In the present application, qSP1 regulates rice spikelet number by interacting with OsSEUs family proteins.

[0036] The present application is based on the cultivated single fragment substitution line Z499 and fine mapping (Ma et al, Identification of QTL for kernel number-related traits in a rice chromosome segment substitution line and fine mapping of qSP1. The Crop Journal. 7, (2019) 494-503). First, through online gene prediction (http: / / mendel.cs.rhul.ac.uk), BLAST online alignment ( http: / / blast.ncbi.nlm.nih.gov / ) and gene function complement analysis ( Figure 2 , Figure 3), it was determined that the rice multiple grain variant gene qSP1 is the transcriptional co-repressor LEUNIG protein (LOC_Os01g08190). LEUNIG protein is widely present in plants and belongs to a subclass of plant transcriptional co-repressor family (Liu et al, Groucho / Tup1 family co-repressors in plant development. Trends in Plant Science. 13, (2008) 137-144). In Arabidopsis, LEUNIG / LUH protein co-repressor factors regulate the expression of AGAMOUS through indirect interaction with transcription factors via SEUSS, and ectopic AGAMOUS expression causes a reduction in the number of floral organs in Arabidopsis (Franks et al, SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development. 129, (2002) 253-263; Sridhar et al, Transcriptional repression of target genes by LEUNIG and SEEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proceedings of the National Academy of Sciences of the United States of America. 101, (2004) 11494-11499). LEUNIG protein can also directly interact with transcription factor YABBY to form a LEUNIG-YABBY complex to promote the initiation of adaxial cells in Arabidopsis leaves and shoot apical meristems (SAM) and the maintenance of post-embryonic SAM (Stahle et al, YABBYs and the transcriptional co-repressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarity and meristem activity in Arabidopsis. The Plant Cell. 21, (2009) 3105-3118). However, the research on LUG protein in rice is relatively less. The present application cloned the multiple grain gene qSP1 located on chromosome 1 by map-based cloning of the multiple grain rice single segment substitution line Z499.qSP1 encodes the transcriptional co-repressor LEUNIG protein. The variation of the gene coding sequence (G to A) in Z499 changes the encoded amino acid, which leads to the increase of rice grain number and the negative regulation of the development of spike grain number.

[0037] Subsequently, the present application cloned the promoter and CDS sequence of the variation gene qSP1 using rice multi-grain single fragment substitution line Z499 as the material, studied the nucleotide sequence and the structure of the encoded protein of the receptor wild type gene qSP1 by means of bioinformatics, and constructed a recombinant plant complementary vector of the wild type gene qSP1 for the transgenic research of rice.

[0038] I. Cloning, sequencing and functional complementation analysis of the variation gene qSP1

[0039] According to the sequence of rice Nipponbare gene LOC_Os01g08190 (Os01g0177100) registered in GeneBank, specific primers were designed by using VectorNTI software:

[0040] The first segment upstream primer qSP1-COM-1F (F):

[0041] 5'-CCATGATTACGAATTCGAGCTCAGACGAATGGTCAAACACGTACTAA-3' (SEQ ID NO: 4);

[0042] The first segment downstream primer qSP1-1R:

[0043] 5'-GACAGACCACCACTCCAAGAGGAAACC-3'

[0044] The second segment upstream primer qSP1-2F:

[0045] 5'-GGTTTCCTCTTGGAGTGGTGGTCTGTC-3'

[0046] The second segment downstream primer qSP1-2R:

[0047] 5'-ACCATAGCCTGAACCGCACCAACTG-3'

[0048] The third segment upstream primer qSP1-3F:

[0049] 5'-CAGTTGGTGCGGTTCAGGCTATGGT-3'

[0050] The third segment upstream primer qSP1-COM-3R (R):

[0051] 5'-GCTTGCATGCCTGCAGGTCGACTTGCAAAGTCTGTGTTCTTCCC-3' (SEQ ID NO: 5).

[0052] qSP1-detection-F:

[0053] 5'-AGCGTAGTGAAAGCAGTCATTTGC-3'

[0054] qSP1-detection-R:

[0055] 5'-TTAGTCAAACAGCTACTACATGCTGGA-3'

[0056] The whole seedlings of rice recipient parent Nipponbare and multiple grain single segment substitution line Z499 were respectively cultured for two weeks, quickly put into liquid nitrogen and grinded into powder. The total DNA was extracted according to the CTAB method. The electrophoresis results of the obtained rice recipient Nipponbare and multiple grain single segment substitution line Z499 DNA showed that the main band was clear and complete, indicating that the concentration and purity of the DNA met the experimental requirements and could be used for amplifying the whole genome DNA sequence.

[0057] The obtained Nipponbare DNA was used as a template, and specific primers qSP1-COM-F / qSP1-COM-R and high-fidelity DNA polymerase PFU were used for PCR amplification. The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 5 minutes; then denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 1 minute, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes. The RT-PCR product was detected by 1.0% (g / mL) agarose gel electrophoresis, and then gel recovery and purification were performed according to the DNA gel recovery kit instructions; the purified DNA fragment was ligated with the digested pCAMBIA1301 vector, and the enzyme digestion site was Sac I and Sal I. The ligation product was transformed into E. coli DH5α competent cells under the action of recombinase at 50℃ for 30 min. The positive clones were selected on LB plates containing kanamycin, and the plasmid was extracted, PCR identified and sequenced to obtain the recombinant vector pCAMBIA1301-qSP1. The electrophoretogram is shown in Figure 7 , and the recombinant vector is shown in Figure 9 .

[0058] The above obtained recombinant vector pCAMBIA1301-qSP1 plasmid was transformed into EHA105 Agrobacterium to obtain positive Agrobacterium colonies containing the target fragment, and was transformed into single segment substitution line Z499. The spike grain number of the positive transgenic plants was reduced and restored to similar spike grain number as Nipponbare Figure 3 a-h). It was determined that LOC_Os01g08190 gene was a multiple grain variation gene.

[0059] Multi-grain rice plants were obtained by knocking out the qSP1 gene using CRISPR / Cas9 technology. The specific steps are as follows: qSP1 knockout target primers were designed on a CDS vector → pE-U3t and gRNA were amplified by first-round PCR → sgRNA expression cassette was constructed by second-round PCR → the sgRNA expression cassette was recombined into a CRISPR / Cas9 vector → E. coli was transformed → correct positive clone transformants were obtained → plasmids were extracted → Agrobacterium was transformed → positive Agrobacterium was transformed into different low-grain rice varieties → homozygous knockout transgenic lines were the multi-grain rice plants.

[0060] II. Bioinformatics Analysis of the Mutant Gene qSP1

[0061] Using the ORF Finder in NCBI http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html Open reading frame recognition; using ( https: / / smart.embl.de / smart / show_motifs.pl Protein structure analysis was performed using MEME (conserved motif) http: / / meme-suite.org / index.html Protein conserved motif analysis was performed, and protein conserved domain analysis was conducted using CDD (conserved domain database) (http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). Amino acid sequence alignment and phylogenetic tree generation were performed using Jalview and MEGA11 software, respectively.

[0062] ORF Finder software analysis showed that the variant gene qSP1 consists of a complete and continuous open reading frame.

[0063] The amino acid sequence alignment results of the qSP1 encoded protein are shown in Figure 4. qSP1 shows high homology with the N-terminal and C-terminal sequences of proteins encoded by other genes in the LEUNIG family in rice, Arabidopsis thaliana, and maize.

[0064] The phylogenetic tree and CDD analysis results of qSP1 are as follows: Figure 5 As shown, qSP1 contains one LisH conserved domain and seven WD40 conserved domains. qSP1 and ZmLUG10 are in the same branch and are closely related.

[0065] III. Rice qSP1 regulates panicle grain number development through interactions with OsSEUs family proteins.

[0066] The interaction analysis between qSP1 and OsSEUs yielded the following results: Figure 7 As shown, qSP1 interacts with OsSEUs family members OsSEU1, OsSEU2 and OsSEU3.

[0067] IV. Application of rice multi-grain variation gene qSP1

[0068] The variation of qSP1 can increase the number of grains per panicle of rice, and the additive effect can increase 8.82 in the genetic background of Nipponbare, and the yield per plant is significantly increased by 18.69% (18.842g vs. 15.875g) compared with Nipponbare. Thus, on the one hand, the multi-grain single fragment substitution line Z499 of qSP1 gene can be combined with two-line sterile lines (Xida 4S, Xida 3S and Xida 4S) to breed new multi-grain rice varieties; on the other hand, the coding sequence of qSP1 gene of the few-grain variety can be edited (knockout target sites are designed on the coding sequence of qSP1 and connected to the vector of CRISPR-Cas9, and then transformed into the variety) to make it vary to produce new multi-grain rice varieties. The qSP1 gene of the present application provides an important gene resource for the molecular breeding of the number of grains of rice. The technology has the advantages of precision, simplicity, rapidness and easy operation.

[0069] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions, which should be covered in the scope of the claims of the present application.

Claims

1. A rice multiple grain gene qSP1 characterized in that: The gene promoter sequence is shown as SEQ ID NO: 2, the wild type gene CDS nucleotide sequence of the rice multi-grain gene qSP1 The wild type gene CDS nucleotide sequence of the rice multi-grain gene is shown as SEQ ID NO: 3, and the sequence G base at the 604th position of the wild type gene CDS nucleotide is mutated to A base to obtain the rice multi-grain gene qSP1 .

2. Contains the rice multigrain gene as described in claim 1 qSP1 Recombinant vectors, expression cassettes, or recombinant bacteria.

3. The method of claim 1, qSP1 The application of the gene in breeding the multi-grain rice variety is characterized in that, The steps are as follows: S1: Knocking out the gene of claim 1 by CRISPR / Cas9 technology qSP1 gene; S2: The knockout plasmid is transformed into Agrobacterium; qSP1 The plasmid is transformed into Agrobacterium; S3: Positive Agrobacterium transformation of different rice few-grain varieties; S4: Screening of homozygous knockout transgenic lines as multiple-grain rice plants.

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