Rice basic vegetative growth period regulatory factor OsMADS64 gene as well as encoding protein and application thereof

Abstract

The invention discloses a rice basic vegetative growth period regulatory factor OsMADS64 gene as well as an encoding protein and application thereof, and belongs to the technical field of gene engineering. The amino acid sequence of the protein coded by the gene OsMADS64 is as shown in SEQ ID NO. 2, and the nucleotide sequence is as shown in SEQ ID NO. 1. According to the invention, the OsMADS64 is located in a cell nucleus and cytoplasm; a spatio-temporal expression analysis result shows that the OsMADS64 gene is expressed in roots, stems, leaf sheaths and leaves in different periods, but the expression quantity in the roots is the highest; the osmads64 mutant obtained by a gene editing technology shows a heading delay phenotype under long-day and short-day conditions, which indicates that the regulation of the OsMADS64 on the heading period of the rice does not completely depend on a photoperiod. The invention enriches and perfects the rice heading stage regulation pathway to a certain extent, provides an important theoretical basis for molecular breeding for modifying the ripe stage of rice, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a rice basic vegetative growth period regulating factor OsMADS64 gene, its encoded protein and application. Background Art

[0002] The flowering period of rice, known as the heading period in agriculture, is an important agronomic trait that determines the variety's cultivation area and planting season. The length of the rice heading period is determined by the variety's photosensitivity, temperature sensitivity, and basic vegetative growth. Currently, research on the heading period of rice mainly focuses on the photoperiod pathway, and there are few reports on the genes and their regulatory mechanisms that affect the basic vegetative growth period of rice. Extending the basic vegetative growth period can enable rice plants to achieve sufficient vegetative growth, and sufficient dry matter accumulation is a basic condition for achieving high yields. Therefore, exploring and utilizing the relevant genes that affect the basic vegetative growth period of rice will help create rice germplasm resources with a long basic vegetative growth period and solve the problem of shortened growth period when introducing high-quality varieties from the north to the south. Summary of the Invention

[0003] One of the objectives of the present invention is to provide an application of the rice basic vegetative growth period regulatory factor OsMADS64 in regulating rice heading. The amino acid sequence of the protein encoded by the gene OsMADS64 is shown in SEQ ID NO.2.

[0004] In one embodiment of the present invention, the nucleotide sequence of the rice gene is shown as SEQ ID NO.1.

[0005] A second object of the present invention is to provide a method for delaying the heading period of rice, the method comprising: silencing or knocking out the gene OsMADS64 in rice by genetic engineering means; wherein the gene OsMADS64 is the same as above.

[0006] In one embodiment of the present invention, the method for silencing or knocking out the gene OsMADS64 in rice is any one of the following:

[0007] 1) Knockout of the OsMADS64 gene by homologous recombination;

[0008] 2) T-DNA insertion mutagenesis to knock out the OsMADS64 gene;

[0009] 3) RNA interference silencing of the OsMADS64 gene;

[0010] 4) CRISPR / Cas9 knockout of the OsMADS64 gene.

[0011] In one embodiment of the present invention, the 4) includes: designing a target site using the CDS sequence of the gene OsMADS64 as a template, constructing a CRISPR / Cas9 vector containing the OsMADS64 U3 target site, and transforming rice using Agrobacterium-mediated method to obtain transgenic rice with loss of gene function.

[0012] A third object of the present invention is to provide the application of the transgenic rice obtained by the above method in rice breeding.

[0013] In one embodiment of the present invention, the breeding method includes transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention used RNA-seq and ChIP-seq to explore downstream target genes that regulate the heading date of the rice SDG724-IP71-ROCL1 protein complex, identifying eight candidate genes, one of which was the MADS-box transcription factor OsMADS64. The rice transcription factor OsMADS64 gene was cloned from rice using PCR. The full-length coding region sequence of the OsMADS64 gene obtained by the present invention corresponds to LOC_Os04g31804 published in the Rice Genome Annotation Project. It was also discovered for the first time that OsMADS64 can positively regulate the heading date of rice.

[0016] (2) The present invention uses CRISPR / Cas9 knockout technology and genetic transformation to obtain the OsMADS64 gene knockout mutant, and found that the osmads64 mutant delayed heading under both long-day and short-day conditions, with a delay of 2.6-7 days under long-day conditions and 8.2-12.8 days under short-day conditions.

[0017] (3) The discovery of rice OsMADS64 as a positive regulatory factor for heading period has enriched and improved the regulatory pathway of rice heading period to a certain extent, provided an important theoretical basis for molecular breeding to improve rice maturity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1To explore candidate target genes co-regulated by SDG724-ROC1L-IP71 using RNA-seq and ChIP-seq; A, Number of co-downregulated genes in sdg724, roc1l and ip71 mutants by RNA-seq; B, Statistics of genes differentially enriched with H3K36me3 modification in sdg724 mutant; (C) Venn diagram showing the comparison between the number of co-downregulated genes in sdg724, roc1l and ip71 mutants and genes with decreased H3K36me3 modification in sdg724 mutant; (D) OsMADS64 transcription level and H3K36me3 modification level in NP and sdg724 mutants.

[0019] Figure 2A This is the subcellular localization result of OsMADS64 in Example 3.

[0020] Figure 2B This is the tissue-specific expression profile in Example 4.

[0021] Figure 3 These are the knockout types and sequencing results of the osmads64 mutant in Example 5, where m64-1 (SEQ ID NO.15): AGGAAGCCAGCAGAGCGTCCG (+T); Ref (SEQ ID NO.16): AGGAAGCCAGCAGAGCGCCG; m64-2 (SEQ ID NO.17): AGGAAGCCAG CAGAGC--CG (-GC); Ref (SEQ ID NO.16) NO.16):AGGAAGCCAGCAGAGCGCCG; m64-3(SEQ ID NO.17):AGGAAG--------------AGAT(-29bp); Ref(SEQ ID NO.18):AGGAAGCCAGCAGAGCGCCGAGGAGGAGGAGGAGGAGAT; m64-4(SEQ ID NO.19):AGGAAGCCAGCAGAGC-CCG(-G); Ref(SEQ ID NO.18):AGGAAGCCAGCAGAGCGCCG NO.16): AGGAAGCC AGCAGAGCGCCG.

[0022] Figure 4The osmads64-1000-1000-1000-100000000000100010010101010101000011000110100511110505010111111011011001100011111110150505050000010150100010101201011012011011010000101101101010110101101505050000002012211111101000051515101010101010.m64-1(SEQ IDNO.20):MGRRGRVVLRRIEDRVRGFRGRLKLEKLEKLKVLEKH VGFVVLSCSSGDDANPHFAAPATIENIVERYEHSQAAQKGVHGRCILQKRKSK DFQVLKETIDKGPINDDMRPIDEKDISTLNMDQISQIEILLEDELRWTRARKVV;m64-2(SEQ ID NO.21):MGRRGRVVLRRAGLEKLEKLEK AVLCDAHVGFVVLSCSSGDDANPHHFAAPATIENIVERYEHSQAAQKGVHGRCI LQKRKSKDFQVLKETIDKGPINDDMRPIDEKDISTLNMDQISQIEILLEDELRW TRARKVV:m64-3(SEQ ID NO.22):MGRRGRVVLRRIEDRVRRGICFRKRLAGLEKVEELAVLCDAHVGFVVLSCSSGDDANPHFAAPATIENIVERYEHSQA AQKGVHGRCILQKRKSKDFQVLKETIDKGPINDDMRPIDEKDISTLNMDQISQ IETRKVLRQ44m ID NO.23):MGRRGRVVLRRIED RVRRGICFRKRLAGLEKVEELAVLCDAHVGFVVLSCSSGDDANPHFAAPATI ENIVERYEHSQAAQKGVHGRCILQKRKSKDFQVLKETIDKGPINDDMRPIDEKDISTLNMDQISQIEILLEDELRWTRARKVV:OsMADS64(SEQ ID NO.24):MGRRGRVVLRRIEDRVRRGICFRKRLAGLEKKVELKVELVLCHVLCH GDDANPHHFAAPATIENIVERYEHSQAAQKGVHGRCILQKRKSKDFQVLKETI DKGPINDDMRPIDEKDISTLNMDQISQIEILLEDELRWTRARKVV:m64-1(SEQ ID NO.25): ADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGGSQ QSV----------------RGGGGGDGGGAQAPPEPGHRRP-----------------; m64-2 (SEQ ID NO.26): ADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGGSQQS-----------------RGGGGGDGGGAQAPPEPGHRRP----------------; m64-3 (SEQ ID NO.27): ADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGG--------------------------------RDGGGAQAPPEPGHRRP-----------------; m64-4 (SEQ ID NO.28): ADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGGSQQ S--------------------------PRRRRRRRWRWCSGTA-------------; OsMADS64 (SEQ ID NO.29): ADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGGSQQSA EEEEEEMEVVLRHRLSLGTGDRDDGGGGAAEQRHRTTPPPAVDLNVPCRDAGQLQ. .

[0023] Figure 5 The phenotypes of Hejiang 19 and osmads64 mutants under short-day and long-day conditions in Example 6 (bar: 10 cm).

[0024] Figure 6 These are the statistical data of heading period of Hejiang 19 and osmads64 mutants under short-day and long-day conditions in Example 6. DETAILED DESCRIPTION

[0025] Example 1 Mining of the OsMADS64 gene, a regulator of the basic vegetative growth phase of rice

[0026] The long growth period mutants sdg724, ip71 and roc1l, which are not completely dependent on the photoperiod, were used to explore potential target genes affecting the basic vegetative growth period of rice. Figure 1 As shown, mutants of each component of the SDG724-IP71-ROC1L protein complex downregulated 129 genes ( Figure 1A); 1801 genes with low H3K36me3 enrichment in sdg724 mutants ( Figure 1 B); Overlap analysis was performed on down-regulated genes and H3K36me3 low enriched genes to obtain 8 candidate genes ( Figure 1 C), one of which is the MADS-box transcription factor OsMADS64; Figure 1 D shows that the transcription level of OsMADS64 in the long growth period mutant sdg724 was significantly decreased, and the enrichment level of H3K36me3 level of the OsMADS64 gene was reduced.

[0027] Example 2 Cloning of the OsMADS64 gene, a positive regulator of rice heading period

[0028] 1. Using rice Nipponbare as the experimental material, total RNA was extracted from leaves according to the Invitrogen TRIzol kit operating instructions;

[0029] 2. Take 1 μg of total RNA from step 1 for cDNA synthesis. The cDNA synthesis operation was carried out according to the ReverTra The qPCR RT Master Mix with gDNA Remover (FSQ-301) kit was used according to its instruction manual to obtain cDNA;

[0030] Third, using the cDNA obtained above as a template, the OsMADS64 gene was amplified using forward primer F1 and reverse primer R1, according to the TOYOBO KOD Fx operating instructions. The PCR reaction conditions were as follows: initial denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 68°C for 60 seconds, for a total of 38 cycles; and a final extension at 68°C for 7 minutes. Finally, the PCR product was sequenced on an ABI3130 sequencer (ABI). The sequencing results showed that the OsMADS64 gene, a positive regulator of rice heading period, consists of 750 base pairs. Its nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO. 2.

[0031] OsMADS64 CDS sequence (SEQ ID NO. 1):

[0032] ATGGGGAGGCGGGGGCGGGTGGTGCTGCGGCGGATCGAGGACCGGGTGCGGCGCGGGATCTGCTTCCGGAAGAGGCTCGCCGGGCTGGAGAAGAAGGTGGAGGAGCTCGCCGTGCTCTGCGACGCCCACGTCGGCTTCGTCGTCCTCTCCTGCTCCGGCGACGACGCCAACCCCCACCACTTCGCCGCGCCCGCCACTATTGAAAACATTGTGGAACGTTATGAGCACTCTCAAGCAGCACAAAAAGGAGTACATGGCAGGTGCATTCTGCAAAAGAGGAAGAGCAAGGATTTTCAAGTTCTCAAGGAAACAATAGATAAAGGACCAATCAATGATGACATGCGACCTATTGATGAAAAAGACATATCCACATTAAACATGGACCAGATTAGCCAAATTGAAATATTATTGGAAGATGAACTGAGGTGGACAAGGGCAAGAAAGGTGGTGGCAGATAGAATTGCCAGGTTGCAGAAAAAGGTACAGAAGAAACCGGCGACTGCTGAGACAGAGAGCAACTCCACTGAGATGCCCTCTGATCATGAGAAGAAACAGGTAGCAGGAGGAAGCCAGCAGAGCGCCGAGGAGGAGGAGGAGGAGATGGAGGTGGTGCTCAGGCACCGCCTGAGCCTGGGCACCGGCGACCGTGACGATGGTGGCGGTGGCGCGGCGGAGCAACGGCACCGGACGACGCCGCCGCCGGCGGTCGACCTCAACGTGCCGTGCCGGGACGCGGGTCAGCTGCAGTAG

[0033] OsMADS64 Protein sequence (SEQ ID NO.2):

[0034] MGRRGRVVLRRIEDRVRRGICFRKRLAGLEKKVEELAVLCDAHVGFVVLSCSGDDANPHHFAAPATIENIVERYEHSQAAQKGVHGRCILQKRKSKDFQVLKETIDKGPINDDMRPIDEKDIST LNMDQISQIEILLEDELRWTRARKVVADRIARLQKKVQKKPATAETESNSTEMPSDHEKKQVAGGSQQSAEEEEEEMEVVLRHRLSLGTGDRDDGGGGAAEQRHRTTPPPAVDLNVPCRDAGQLQ

[0035] Forward primer F1 (SEQ ID NO. 3): 5′-ATGGGGAGGCGGGGGCGGGT-3′;

[0036] Reverse primer R1 (SEQ ID NO. 4): 5'-CTACTGCAGCTGACCCGCGTC-3'.

[0037] Example 3 OsMADS64 subcellular localization

[0038] 1. Construction of entry vector: Based on the CDS sequence of the rice OsMADS64 gene, primers (F2, R2) were designed using Primer 5 primer design software. Using Nipponbare cDNA as a template, TOYOBO's KOD Fx high-fidelity DNA polymerase was used, and the above primers were used for amplification according to its instructions. The PCR product was electrophoresed on a 1% agarose gel, and the target band was cut out. The target band was recovered according to the instructions of the agarose gel DNA recovery kit of Kangwei Century. The entry vector pQB-V3 (chloramphenicol resistance) was digested with EcoRV restriction endonuclease to recover a 3.3kb fragment. The recovered target product was ligated to the entry vector pQB-V3 according to the instructions of the ClonExpress II One Step Cloning Kit of Nanjing Novozymes Biotechnology Co., Ltd. The ligation product was transformed into Escherichia coli competent cell Top10. Positive clones were identified by colony PCR using primers F2 / R2. The identified positive clones were cultured overnight in LB containing chloramphenicol resistance, and the plasmids were sequenced. The sequencing primers were universal vector primers (T7, M13R). The plasmids that were sequenced correctly were placed in a -20°C refrigerator for later use.

[0039] Forward primer F2 (SEQ ID NO.5):

[0040] 5'-AAAGCAGGCTCAGGGGGATATCATGGGGAGGCGGGGGCGGGT-3';

[0041] Reverse primer R2 (SEQ ID NO.6):

[0042] 5'-AGCTGGGTGCAGGGCGATATCCTACTGCAGCTGACCCGCGT-3'.

[0043] 2. Construction of Gateway system expression vector: Use LR reaction to connect the correctly sequenced plasmid pQBV3-MADS64 to the subcellular localization vector PH7WGF2.0 (spectinomycin resistance). The method is based on Invitrogen's LR Clonase TM II Enzyme Mix instructions: Reaction system: 1 μl pQBV3-MADS64 (50 ng / μl) + 1 μl PH7WGF2.0 vector (50 ng / μl) + 0.5 μl LR Clonase II enzyme mix. Incubate at room temperature for 1–2 hours before transformation, identification of positive clones, plasmid extraction, sequencing, and sequence alignment analysis. The identified plasmid, PH7WGF2.0-MADS64, should be stored at -20°C until ready for use.

[0044] III. Subcellular Localization: PH7WGF2.0 and PH7WGF2.0-MADS64 vectors were transformed into Agrobacterium tumefaciens GV3101, respectively. Positive colonies were identified by colony PCR and selected in liquid LB medium supplemented with spectinomycin and rifampicin. Simultaneously, P19 bacteria were removed from the -80°C freezer and added at a 1:100 ratio to liquid LB medium supplemented with kanamycin (50 μg / ml) and rifampicin (50 μg / ml). The cultures were incubated at 180 rpm and 28°C for approximately 14 h. The above bacterial suspensions were then added at a 1:100 ratio to liquid LB medium supplemented with the corresponding antibiotics, rifampicin, 10 mM MES (pH 5.6), and 40 μM acetosyringone. Generally, 200 μL of bacterial suspension was added to the above 20 ml of liquid medium and incubated at 180 rpm and 28°C for approximately 16 h. Centrifuge at 5000 rpm at 28°C for 3 minutes, discard the supernatant, and resuspend the pH7WGF2.0 and pH7WGF2.0-MADS64 pellets in 10 mM MgCl2 to an OD of approximately 1.5 and that of P19 to approximately 1.0. Add acetosyringone to a final concentration of 200 μM and incubate at room temperature for at least 3 hours. Mix the target bacterial suspension with the P19 suspension in a 1:1 volume ratio. Inject the suspension into tobacco (N. benthamiana) leaves, injecting at least three leaves per combination. Observe the suspension under a laser confocal microscope three days later.

[0045] like Figure 2AThe four fields of view shown in the figure from left to right are green fluorescence (GFP fluorescence), autofluorescence (auto-fluorescence), bright-field field, and merged field of view. Empty GFP vector was used as a positive control. The results showed that the OsMADS64 gene was localized in the cell nucleus and cytoplasm.

[0046] Example 4 OsMADS64 tissue-specific expression

[0047] Different tissues at the tillering and booting stages were selected, mainly including roots, stems, leaves, leaf sheaths, and young panicles. Total RNA extraction and cDNA synthesis were performed as described in Example 1. Primer 5 primer design software was used to design OsMADS64 qRT-PCR primers (F3, R3). The qRT-PCR method was used to analyze the relative expression of the OsMADS64 gene at different developmental stages of the wild type to determine its spatiotemporal expression specificity. The reaction system was as follows: 0.2 μl F3 + 0.2 μl R3 + 5 μl SYBR Premix Ex Taq (2×) + 4.6 μl cDNA template (diluted 10-20 times with deionized water). The reaction procedure was as follows: pre-denaturation at 95°C for 30 s; PCR reaction: 95°C for 5 s, 60°C for 20 s; 40 cycles.

[0048] Forward primer F3 (SEQ ID NO. 7): 5′-CAAGAAAGGTGGTGGCAGAT-3′;

[0049] Reverse primer R3 (SEQ ID NO. 8): 5'-ATCAGAGGGCATCTCAGTGG-3'.

[0050] like Figure 2B As shown, OsMADS64 was expressed in roots, stems, sheaths and leaves at different stages, but the expression level was highest in roots.

[0051] Example 5 Obtaining osmads64 mutants

[0052] 1. Vector construction: The mads64 mutant was constructed using the CRISPR / Cas9 vector multi-target system. The construction method was carried out with reference to the CRISPR / Cas9 vector system and its operation method in the prior art (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, et al. (2015). A Robust CRISPR / Cas9 System for Convenient, High Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant, 8: 1274-1284.). Based on the CDS sequence of the OsMADS64 gene, the gRNA target site was designed using the CRISPR-GE website (http: / / skl.scau.edu.cn / targetdesign / ). Following the protocol described in the aforementioned literature, the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pubi-H containing the OsMADS64 gene U3 target was constructed. The nucleotide sequences of the targeting primers (F4 and R4) linked to the U3 promoter are as follows:

[0053] Forward primer F4 (SEQ ID NO. 9): 5′-GGCAGGAAGCCAGCAGAGCGCCG-3′;

[0054] Reverse primer R4 (SEQ ID NO. 10): 5'-AAACCGGCGCTCTGCTGGCTTCC-3'.

[0055] 2. The target vector was transformed into Agrobacterium EHA105 and then into Hejiang 19 by Agrobacterium-mediated method.

[0056] III. Identification of OsMADS64 Knockout Mutants: After transgenic seedlings have been differentiated, they must be identified to eliminate false positives. First, crude rice DNA is extracted. Using this crude DNA as a template, amplification is performed using hygromycin primers (F5 and R5) according to the instructions for Es Taq DNA Polymerase from Cosmed. Subsequently, target site sequencing primers (F6 and R6) are designed to detect the knockout variant.

[0057] Forward primer F5 (SEQ ID NO. 11): 5′-TGCGCCCAAGCTGCATCAT-3′;

[0058] Reverse primer R5 (SEQ ID NO. 12): 5′-TGAACTCACCGCGACGTCTGT-3′;

[0059] Forward primer F6 (SEQ ID NO. 13): 5′-TACAGAAGAAACCGGCGACT-3′;

[0060] Reverse primer R6 (SEQ ID NO. 14): 5'-CTGTGTTATGGGCTCGGATT-3'.

[0061] like Figure 3 As shown in FIG, the sequencing results of the OsMADS64 gene knockout mutants m64-1, m64-2, m64-3, and m64-4 are shown, which respectively contain inserted base T, deleted base GC, deleted 29 bp base, and deleted base G; Figure 4 As shown, the insertion and deletion bases in the m64 mutant caused subsequent frameshift mutations, which changed the expressed amino acids and caused the OsMADS64 protein to lose its function.

[0062] Example 6 Statistics of heading period of osmads64 mutant under different daylight lengths

[0063] Hejiang 19 and osmads64 mutants (m64-1, m64-2, m64-3 and m64-4) were planted under natural long-day (14 h light + 10 h dark) and short-day (10 h light + 14 h dark) conditions, and the heading dates of Hejiang 19 and osmads64 mutants were recorded and counted.

[0064] like Figure 5 As shown in the figure, the heading period of m64-1 and m64-3 was delayed compared with the control Hejiang 19. Figure 6 Data statistical results showed that under long-day conditions, the heading of the m64-1, m64-2, m64-3 and m64-4 mutants was delayed by 7, 2.8, 3.6 and 2.6 days respectively compared with the control; under short-day conditions, the heading of the four mutants was delayed by 10.4, 10, 8.2 and 12.8 days respectively compared with the control.

[0065] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of the rice basic vegetative growth period regulatory gene OsMADS64 in regulating rice heading, characterized in that: The amino acid sequence of the protein encoded by the gene OsMADS64 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The nucleotide sequence of the rice gene is shown in SEQ ID NO.

1.

3. A method for delaying the heading period of rice, characterized in that: The method comprises: utilizing genetic engineering means to silence or knock out the gene OsMADS64 in rice; wherein the gene OsMADS64 is the same as that described in claim 1 or 2.

4. The method according to claim 3, characterized in that The method for silencing or knocking out the gene OsMADS64 in rice is any one of the following: 1) Knockout of the OsMADS64 gene by homologous recombination; 2) T-DNA insertion mutagenesis to knock out the OsMADS64 gene; 3) RNA interference silencing of the OsMADS64 gene; 4) CRISPR / Cas9 knockout of the OsMADS64 gene.

5. The method according to claim 4, characterized in that The above 4) includes: designing a target site using the OsMADS64 CDS sequence as a template, constructing a CRISPR / Cas9 vector containing the U3 target site, and transforming rice using Agrobacterium-mediated method to obtain transgenic rice with the gene function deleted.

6. Use of the transgenic rice obtained by the method according to any one of claims 3 to 5 in rice breeding.

7. The use according to claim 6, characterized in that Breeding methods include transgenics, hybridization, backcrossing, selfing or asexual reproduction.

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