Rice multi-grain gene qSP1 and application thereof

Abstract

The invention relates to a rice multi-grain gene qSP1 and application thereof, and belongs to the technical field of biology. A promoter nucleotide sequence of the rice multi-grain gene qSP1 is shown as SEQ ID NO: 2, a CDS nucleotide sequence of the rice multi-grain gene qSP1 is shown as SEQ ID NO: 3, and a protein coded by the rice multi-grain gene qSP1 is a rice transcription co-inhibitor LEUNIG and has an amino acid sequence shown as SEQ ID NO: 4. The qSP1 can be used for regulating and controlling the grain number of the rice and increasing the yield of the rice. According to the invention, the grain number and yield of the rice are increased through a molecular marker-assisted selection technology or a gene editing technology, a new gene resource and technical approach are provided for genetic improvement and high-yield breeding of the rice, and the molecular marker has important application value and social benefit.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a rice multi-grain gene qSP1 and its application Background Art

[0002] Rice is one of the most important food crops in the world. About more than half of the world's population uses it as a staple food. Improving rice yield is of great significance for solving the food shortage problem caused by global climate change, continuous population growth and the year-on-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 my country and the reduction of arable land year by year. In order to make up for the rice yield lost due to land reduction, improving rice yield per unit area is still the primary goal of rice breeding. However, rice yield traits are complex quantitative traits controlled by multiple genes, which are mainly determined by three factors: effective tiller number per plant, number of grains per panicle and grain weight. These three internal elements complement each other and restrict each other. How to break through the mutual constraints between various factors and find the nodes where the factors interact and maintain balance has become a challenge faced by current rice scientific research and molecular design breeding (Guo et al, GRAINSIZEAND NUMBER 1 negatively regulates the OsMKKK10-OsMKK4-OsMPK6 cascade to coordinate the trade-off between grain number perpanicle and grain size in rice. The Plant Cell. 30, (2018) 871–888). The number of grains per panicle is one of the three elements of rice yield, which largely determines the final yield. Identifying the gene that controls the number of grains and studying its molecular regulatory mechanism are of great significance to rice high-yield design breeding. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a rice multi-grain gene qSP1 and a recombinant plasmid prepared using the gene, and use thereof in breeding multi-grain rice varieties.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a rice multi-grain gene qSP1, wherein the gene promoter sequence is shown in SEQ ID NO: 2, and the gene CDS nucleotide sequence is shown in SEQ ID NO: 3;

[0006] The amino acid sequence of the gene expression is shown in SEQ ID NO:4;

[0007] Further, a recombinant vector, expression cassette or recombinant bacterium containing the rice multi-grain gene qSP1

[0008] Furthermore, the present invention provides a recombinant vector pCAMBIA1301-qSP1, wherein the recombinant nucleotide sequence of the recombinant vector is the qSP1 full genome DNA sequence of SEQ ID NO: 1;

[0009] The application of qSP1 gene in breeding multi-grain rice varieties is characterized by the following steps:

[0010] S1: Knockout of qSP1 gene using CRISPR / Cas9 technology;

[0011] S2: Transform the knockout qSP1 plasmid into Agrobacterium;

[0012] S3: positive Agrobacterium transformed different rice varieties with few grains;

[0013] S4: The homozygous knockout transgenic line is a multi-grain rice plant.

[0014] The beneficial effects of the present invention are:

[0015] It provides a powerful tool for rice transgenic research and molecular marker-assisted selection breeding, promotes research on high-yield hybrid rice breeding, effectively increases the number of rice grains and yield through genetic engineering technology, and provides new genetic resources and technical approaches for rice genetic improvement and high-yield breeding, which has important application value and social benefits.

[0016] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0018] Figure 1Analysis of traits related to grain number per ear and QTL identification of the single segment substitution line Z499 containing the qSP1 gene. a: ear type of Nipponbare and Z499. Scale bar, 5 cm; b: ear length (b), number of primary stalks (c), number of stalks (d), and total number of grains per ear (e) of Nipponbare and Z499. Statistical analysis. Data are shown as mean ± SD (n = 10). μ represents the mean and ai represents the additive effect of QTL. P value < 0.05 indicates that there is a significant difference between Nipponbare and Z499 in the t test.

[0019] Figure 2 This is a comparison diagram of the qSP1 mutant gene and the wild-type gene sequence (Nipponbare).

[0020] Figure 3 Phenotypic analysis of qSP1 complementation, a: panicle type of Nipponbare (Nip), Z499 and LOC_Os01g08190 complemented plants at maturity. qSP1-COM-1 and qSP1-COM-2 represent an independent LOC_Os01g08190 complemented transgenic line, respectively. Scale bar, 5 cm; b: sequencing results of variant sites of LOC_Os01g08190 complemented plants; c: qRT-PCR analysis of Nipponbare (Nip), Z499 and LOC_Os01g08190 complemented plants, ACTIN was used as an internal reference for qRT-PCR, mean ± SD (n = 3); dh: statistical analysis of panicle length (d), number of primary branches (e), number of secondary branches (f), total number of grains per panicle (g) and yield per plant (h) of Nipponbare, 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 (dh), different lowercase letters indicate significant difference (P < 0.05) determined by one-way ANOVA and Duncan's multiple comparison.

[0021] Figure 4a , 4b , 4c are protein sequence alignments of qSP1 and its predicted family proteins, with relatively conserved sequences shown in red and black boxes.

[0022] Figure 5 Phylogenetic tree analysis of the qSP1 protein structure and its predicted family proteins containing conserved motifs and domains, a: Schematic diagram of the protein structure of qSP1; b: Phylogenetic tree analysis of conserved motifs and conserved domains. At: Arabidopsis; Os: Rice; Zm: Zea mays; FCD: Fig.

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

[0024] Figure 7 The interaction analysis between qSP1 and OsSEUs in yeast. SD-LT represents SD / -Leu-Trp deficiency medium; SD-LTHA represents SD / -Leu-Trp-His-Ade deficiency medium; Xa-gal represents SD / -Leu-Trp-His-Ade deficiency medium containing Xa-gal.

[0025] Figure 8 These are the electrophoretic diagrams during the construction of complementary vector plasmids: a: target fragment amplification electrophoretic diagram (divided into 3 segments); b: pCAMBIA1301 vector restriction electrophoretic diagram; c: bacterial liquid detection electrophoretic diagram.

[0026] Fig. 9 Construct maps for complementation vectors. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0028] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0029] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0030] Embodiment 1

[0031] The experimental methods for which specific conditions are not specified in the examples are generally performed according to conventional conditions, such as the conditions described in Molecular Cloning Laboratory Manual (3rd edition, written by J. Sambrook et al., translated by 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 multi-grain single segment substitution line Z499, all of which were cultivated by the inventor's laboratory; 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 IIIDNAMarker and DL5,000DNAMarker were purchased from TaKaRa; DNAMarker III was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Ampicillin (Ampicillin, Amp) and kanamycin (Kanamycin, Kan) are products of Sigma; primer synthesis and DNA sequencing were completed by Shanghai Yingjun Biotechnology Co., Ltd.; other chemical reagents were purchased from Beijing Dingguo Biotechnology Co., Ltd.; Escherichia coli DH5α and Agrobacterium LBA4404 were stored in the inventor's laboratory.

[0033] Through natural variation, 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 with japonica rice Nipponbare as the recipient parent and indica rice restorer line Xihui 18 as the donor. On this basis, the present invention uses the F2 population QTL positioning produced by hybridization of Nipponbare and Z1364 and the combined MAS method to select and breed a multi-grain single segment substitution line Z499. The present invention further detected the grain number QTL-qSP1 using Z499, which is located in the interval RM10198--RM1329-RM10224--RM6777 of chromosome 1, with a minimum substitution length of 50Kb, an estimated length of 0.20Mb, and a maximum substitution length of 0.35Mb. The ear length, number of primary branches, number of secondary branches and total number of grains per ear of Z499 were significantly increased compared with those of Nipponbare ( Figure 1 ae). Then, qSP1 (LOC_Os01g08190) located on chromosome 1 was cloned using Z499 as the material. The nucleotide sequence is shown in SEQ ID NO: 1. This gene encodes the transcriptional co-repressor LEUNIG protein. Compared with Nipponbare, LOC_Os01g08190 in Z499 has a SNP difference in the fourth exon (from G to A), which causes the encoded amino acid to be replaced from Ala in Nipponbare to Thr in Z499 ( Figure 2 The LEUNIG protein it encodes and OsSEU1, OsSEU2 or OsSEU3 form a qSP1-OsSEUs complex to regulate the size of rice grains ( Figure 7 ). Therefore, determining the nucleotide sequence of the qSP1 gene, including the promoter (nucleotide sequence as shown in SEQ ID NO: 2) and CDS sequence (nucleotide sequence as shown in SEQ ID NO: 3) and the encoded protein sequence (amino acid sequence as shown in SEQ ID NO: 4), will be beneficial to studying 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, the present invention firstly determined that the rice multi-grain gene qSP1 is LOC_Os01g08190 through gene prediction, homology search and gene sequence difference comparison. Subsequently, the present invention cloned the full-length CDS of the mutant gene qSP1 using the rice multi-grain single segment substitution line Z499 as the material. The sequencing results showed that the full-length CDS sequence of the mutant gene qSP1 was still 2634bp, consisting of 18 exons, encoding 877 amino acids, but compared with the receptor Nipponbare, it had a base substitution in the 4th exon, resulting in a change in the amino acid it encoded ( Figure 2 ). Through genetic complementation, the whole genome DNA of LOC_Os01g08190 from the recipient parent Nipponbare was introduced into the Z499 background, and sequencing and phenotypic analysis were performed. The results showed that the T0 generation complementary transgenic plants had double peaks 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. The number of secondary branches and total grains per ear of its complementary transgenic line was significantly lower than that of Z499, and had no significant difference with Nipponbare ( Figure 3 ah). This indicates that the LOC_Os01g08190 gene is the target gene for controlling multiple grains. Bioinformatics analysis of qSP1 showed that the N-terminus of the protein contained a conserved LisH sequence and the C-terminus contained 7 repeated WD40 domains, which were evolutionarily closely related to ZmLUG10 in maize (Figures 4-5). qRT-PCR showed that the qSP1 gene was expressed at different developmental stages of the ear, and was most highly expressed in the 1.5 cm ear, but was very low in roots, stems, leaves, and sheaths ( Figure 6 ). Compared with Nipponbare, the expression level of qSP1 in Z499 was significantly downregulated ( Figure 3 c).

[0035] In addition, library screening and yeast two-hybrid experiments revealed that qSP1 interacted with OsSEUs family proteins ( Figure 7). OSHI1 / SB1 directly inhibits the expression of DEP1 and IPA1 by interacting with OsSEU, affecting the development of grain number per panicle (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 atranscription repressor. Rice Science. 28, (2021) 243-256). In the present invention, qSP1 regulates the number of rice panicles by interacting with OsSEUs family proteins.

[0036] Based on the cultivated single segment 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), the present invention firstly uses online gene prediction (http: / / mendel.cs.rhul.ac.uk), BLAST online alignment ( http: / / blast.ncbi.nlm.nih.gov / ) and gene function complementation analysis ( Figure 2 , Figure 3), identified the rice multi-grain variant gene qSP1 as the transcriptional co-repressor LEUNIG protein (LOC_Os01g08190). LEUNIG protein is widely present in plants and belongs to a subclass of the 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 indirectly interacts with transcription factors through SEUSS to regulate the expression of AGAMOUS. Ectopic expression of AGAMOUS 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 LEUNIG-YABBY complex to promote the initiation of adaxial cells and embryo apical meristem (SAM) in Arabidopsis leaves and the maintenance of post-embryonic SAM (Stahle et al, YABBYs and the transcriptional core pressors 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 small. The present invention uses the multi-grain rice single segment substitution line Z499 to map clone the multi-grain gene qSP1 located on chromosome 1.qSP1 encodes the transcriptional co-repressor LEUNIG protein. The mutation of the coding sequence of this gene (G to A) in Z499 caused the amino acid it encoded to change, resulting in an increase in rice grain number and negatively regulating the development of grain number per panicle.

[0037] Subsequently, the present invention used rice multi-grain single segment substitution line Z499 as material, cloned the promoter and CDS sequence of the mutant gene qSP1, used bioinformatics methods to study the nucleotide sequence and encoded protein structure of the receptor wild-type gene qSP1, and constructed a wild-type gene qSP1 recombinant plant complementation vector for rice transgenic research.

[0038] 1. Cloning, sequencing and functional complementation analysis of the mutant gene qSP1

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

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

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

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

[0043] 5'-GACAGACCACCACTCCAAGAGGAAACC-3'

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

[0045] 5'-GGTTTCCTCTGGAGGTGGGTGGTCTGTC-3'

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

[0047] 5'-ACCATAGCCTGAACCGCACCAACTG-3'

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

[0049] 5'-CAGTTGGTGCGGTTCAGGCTATGGT-3'

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

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

[0052] Upstream primer for bacterial liquid detection (qSP1-detection-F):

[0053] 5'-AGCGTAGTGAAAGCAGTCATTTGC-3'

[0054] Bacterial liquid detection downstream primers (qSP1-detect-R):

[0055] 5'-TTAGTCAAACAGCTACTACATGCTGGA-3'

[0056] The whole seedlings of the rice recipient parent Nipponbare and the multi-grain single segment substitution line Z499, which had been cultured for two weeks, were taken and quickly ground into powder in liquid nitrogen, and the total DNA was extracted according to the CTAB method. The electrophoresis results of the obtained rice recipient Nipponbare and multi-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 to amplify the whole genome DNA sequence.

[0057] The obtained DNA of Nipponbare was used as a template, and PCR amplification was performed using specific primers qSP1-COM-F / qSP1-COM-R and high-fidelity DNA polymerase PFU. The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, renaturation at 55°C for 30 seconds, and extension at 72°C for 1 minute, for a total of 35 cycles; and finally extension at 72°C for 10 minutes. The RT-PCR product was detected by 1.0% (g / mL) agarose gel electrophoresis, and then gel-cutting and purification were performed according to the instructions of the DNA gel recovery kit; the purified DNA fragment was connected with the pCAMBIA1301 vector cut with enzymes, and the enzyme cutting sites were Sac I and Sal I, and connected at 50°C for 30 minutes under the action of the recombinase. The connection product was transformed into Escherichia coli DH5α competent cells, and positive clones were screened on LB plates containing kanamycin. The plasmid was extracted, and sequencing was performed after PCR identification to obtain the recombinant vector pCAMBIA1301-qSP1. The electrophoresis diagram is shown as follows Figure 7 As shown, the recombinant vector Fig. 9 shown.

[0058] The recombinant vector pCAMBIA1301-qSP1 plasmid obtained above was transferred into EHA105 Agrobacterium to obtain positive Agrobacterium colonies containing the target fragment, and then transformed into the single-fragment substitution line Z499. It was identified that the number of grains per ear of the positive transgenic plants was reduced and restored to a similar number of grains per ear as Nipponbare ( Figure 3 ah). The LOC_Os01g08190 gene was determined to be a multi-grain variant gene.

[0059] The CRISPR / Cas9 technology was used to knock out the qSP1 gene to obtain multi-grain rice plants. The specific steps are as follows: design qSP1 knockout target primers on CDS → first-round PCR amplification of pE-U3t and gRNA → second-round PCR to construct sgRNA expression cassette - recombining sgRNA expression cassette into CRISPR / Cas9 vector → transforming Escherichia coli → obtaining correct positive clone transformants → extracting plasmids → transforming Agrobacterium → positive Agrobacterium transforming different rice varieties with few grains → homozygous knockout transgenic lines are multi-grain rice plants.

[0060] 2. Bioinformatics analysis of the mutant gene qSP1

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

[0062] ORF Finder software analysis showed that the variant gene qSP1 consisted 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 , and the N-terminal and C-terminal sequences of qSP1 are highly homologous to those of other genes encoding proteins of the LEUNIG family in rice, Arabidopsis, and maize.

[0064] The phylogenetic tree and CDD analysis results of qSP1 are shown in Figure 5 As shown, qSP1 contains a LisH conserved domain and seven WD40 conserved domains. qSP1 and ZmLUG10 are in the same branch and have a close relationship.

[0065] 3. Rice qSP1 regulates the development of grain number by interacting with OsSEUs family proteins

[0066] The results of the interaction analysis between qSP1 and OsSEUs are as follows Figure 7 As shown, qSP1 interacts with OsSEUs family members OsSEU1, OsSEU2 and OsSEU3.

[0067] IV. Application of Rice Multi-grain Mutation Gene qSP1

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

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. Rice multi-grain gene qSP1, characterized in that: The gene promoter sequence is shown in SEQ ID NO:2, and the gene CDS nucleotide sequence is shown in SEQ ID NO:

3.

2. The rice multi-grain gene qSP1 according to claim 1, characterized in that: The amino acid sequence expressed by the gene is shown in SEQ ID NO:

4.

3. A recombinant vector, expression cassette or recombinant bacterium containing the rice multi-grain gene qSP1 according to claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is pCAMBIA1301-qSP1, and the recombinant nucleotide sequence of the recombinant vector is the qSP1 full genome DNA sequence shown in SEQ ID NO:

1.

5. Use of the qSP1 gene according to claim 1 or 2 in breeding multi-grain rice varieties, characterized in that: The steps are as follows: S1: Knockout of qSP1 gene using CRISPR / Cas9 technology; S2: Transform the knockout qSP1 plasmid into Agrobacterium; S3: positive Agrobacterium transformed different rice varieties with few grains; S4: The homozygous knockout transgenic line is a multi-grain rice plant.

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