Application of rice driving protein gene KIN14M

Abstract

The invention belongs to the technical field of genetic engineering and plant genetic breeding, and particularly relates to application of a rice driving protein gene KIN14M. According to the invention, a CRISPR / Cas9 mediated gene knockout technology is utilized to construct a knockout vector of the OSKIN14M gene, and novel tetraploid rice Huaduo No.1 and diploid rice Nipponbare are respectively transformed. Phenotype identification shows that after the OSKIN14M gene is knocked out, the Huaduo No.1 shows that the fertility is obviously reduced, the pollen activity is reduced, the female gametophyte is abnormal in development, and the fertility of Nipponbare is not influenced. The biological function of the OSKIN14M gene is defined for the first time, it is proved that the OSKIN14M gene is a new gene for specifically regulating and controlling the fertility of tetraploid rice, only the fertility of the tetraploid rice is affected by function deficiency of the OSKIN14M gene, no obvious adverse effect is caused to other agronomic traits, and a brand-new gene target and resource support are provided for breeding of the tetraploid rice.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and plant genetic breeding technology, specifically involving the application of the rice kinesin gene KIN14M. Background Technology

[0002] Rice (Oryza sativa L.) is a vital food crop globally, and continuously increasing its yield has always been a core objective in rice breeding. After two major technological breakthroughs in dwarfing breeding and hybrid rice breeding, rice yields have achieved a leapfrog increase. However, achieving further substantial yield increases on this basis faces severe challenges. Therefore, some breeders have proposed a new breeding strategy to enhance rice yield by utilizing heterosis among subspecies of autotetraploid rice (Cai Detian et al., 2001, "Research Progress on Autotetraploid Rice"). Autotetraploid rice is a new polyploid germplasm created by doubling the chromosomes of diploid rice. Compared to diploid rice, it possesses superior characteristics such as strong stress resistance, high biological yield, and significant heterosis.

[0003] However, previous studies have found that autotetraploid rice suffers from problems such as fewer effective panicles, reduced grains per panicle, and decreased seed setting rate. These defects severely limit its direct application in breeding work (Song Wenchang and Zhang Yuhua, 1992, "An Overview of Rice Polyploid Breeding Research"; Dai Ximei, 2006, "Research Strategies for Homopolyploid Rice"). To address this technical bottleneck, our laboratory has successfully obtained a batch of new tetraploid rice germplasm with normal seed setting rate through extensive hybridization and screening of autotetraploid rice. The seed setting rate of its F1 hybrids can reach over 80%, and it has been named "New Tetraploid Rice" (Guo et al., 2017, Transcriptome analysis of neo-tetraploid ricereveals specific differential gene expressions associated with fertility and heterosis). This novel tetraploid rice possesses the outstanding advantages of high fertility, strong and stable heterosis, and shows great promise for production and application in the field of polyploid rice breeding (Guo et al., 2017, Transcriptome analysis of neo-tetraploid rice reveals specific differential gene expressions associated with fertility and heterosis; Liu Xiangdong et al., 2022, Innovation and Application of Polyploid Rice Breeding Technology).

[0004] Novel tetraploid rice represents a class of polyploid rice germplasm resources with excellent fertility; however, the genetic regulatory mechanisms underlying its high fertility remain poorly understood. In-depth analysis of the biological functions of fertility-related genes in novel tetraploid rice will not only help elucidate the molecular regulatory mechanisms of its reproductive and developmental processes but also provide theoretical support for the genetic improvement of polyploid rice. Against this backdrop, identifying fertility-regulating genes in novel tetraploid rice through resequencing combined with bioinformatics analysis has significant theoretical and practical value. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, this invention discovered and cloned a novel driver protein gene, OSKIN14M, that regulates the fertility of a novel tetraploid rice. Loss of function of this gene leads to a decrease in the seed setting rate of the novel tetraploid rice by approximately 30.30%. However, loss of function of OSKIN14M has little impact on the fertility of diploid rice, only causing an average decrease in the seed setting rate of diploid rice by 6.93%. Furthermore, loss of function of OSKIN14M has little impact on yield-related agronomic traits, demonstrating strong application value and potential for use in subsequent tetraploid rice breeding systems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the application of the rice gene OsKIN14M in regulating the fertility of tetraploid rice.

[0007] It should be understood that, considering the degeneracy of codons, modifications to the nucleotide sequence of the aforementioned encoding gene without altering the amino acid sequence also fall within the scope of protection of this invention. Furthermore, this invention also covers the protein encoded by the gene OsKIN14M, and derived proteins whose sequences have been substituted, deleted, or have one or more amino acids added, and which possess equivalent functions.

[0008] Preferably, regulating the fertility of tetraploid rice includes regulating the seed setting rate of tetraploid rice, regulating the yield per plant of tetraploid rice, regulating the pollen fertility of tetraploid rice, and regulating the embryo sac fertility of tetraploid rice.

[0009] More preferably, after the OsKIN14M mutation in rice, pollen fertility decreases and the frequency of abnormal embryo sac fertility increases, which in turn leads to a decrease in seed setting rate and a decrease in yield per plant.

[0010] Preferably, the tetraploid rice includes Huaduo No. 1.

[0011] The second aspect of the present invention also provides a method for reducing the fertility of tetraploid rice, specifically by using genetic engineering techniques to knock out the OSKIN14M gene in rice or reduce the expression level of the OSKIN14M gene in rice.

[0012] Preferably, the method for reducing the fertility of tetraploid rice specifically includes: designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment containing the encoding the sgRNA sequence into a vector carrying CRISPR / Cas9, transforming tetraploid rice, and screening for positive lines to obtain transgenic rice materials with reduced fertility.

[0013] Preferably, the nucleotide sequences of the sgRNA action site are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0014] More preferably, the designed CRISPR / Cas9-based sgRNA sequence is shown in SEQ ID No. 3.

[0015] Preferably, the vector carrying CRISPR / Cas9 includes the pYLCRISPR / Cas9 Pubi-H binary vector system.

[0016] The third aspect of the present invention also provides the application of transgenic rice materials obtained by the method described in the second aspect in rice breeding.

[0017] Preferably, the breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes CRISPR / Cas9-mediated gene knockout technology to systematically analyze the biological function of the rice OSKIN14M gene. The constructed gene knockout vector was transformed into a novel tetraploid rice variety, Huaduo 1, and a diploid rice variety, Nipponbare. Phenotypic identification showed that after OSKIN14M gene knockout, Huaduo 1 exhibited significantly reduced fertility, decreased pollen viability, and abnormal female gametophyte development, while Nipponbare's fertility was unaffected. This invention clarifies the biological function of the OSKIN14M gene for the first time, confirming it as a novel gene specifically regulating the fertility of tetraploid rice. Its functional loss only affects the fertility of tetraploid rice and has no significant adverse effects on other agronomic traits, providing a novel gene target and resource support for tetraploid rice breeding. Attached Figure Description

[0019] Figure 1Screening and gene expression pattern analysis of the OSKIN14M gene; A. Structure of the OSKIN14M gene; B. RT-qPCR analysis results of OSKIN14M in the anther tissue of Huaduo No. 1 rice; where PM represents interphase of meiosis; M represents meiosis; ESCP represents early stage of mononuclear microspores; MSCP represents metaphase of mononuclear microspores; LSCP represents late stage of mononuclear microspores; BCP represents bicellular pollen stage; A represents anther; O represents ovary; C. GUS staining results of OSKIN14M in rice florets of different lengths.

[0020] Figure 2 This study compares the phenotypic differences of OSKIN14M loss-of-function mutants and wild-type in a novel tetraploid rice variety. A. Comparison of plant phenotypic differences between the three OSKIN14M loss-of-function mutant lines and the wild-type; B. Differences in late-season seed setting rate between the three OSKIN14M loss-of-function mutant lines and the wild-type in 2020; C. Differences in early-season seed setting rate between the three OSKIN14M loss-of-function mutant lines and the wild-type in 2021; D. Differences in single-plant yield per plant in late-season 2020 between the three OSKIN14M loss-of-function mutant lines and the wild-type; E. Differences in single-plant yield per plant in early-season 2021 between the three OSKIN14M loss-of-function mutant lines and the wild-type. WT is the wild-type material, Huaduo 1; kin14m-t1, kin14m-t2, and kin14m-t3 are loss-of-function mutant lines of OSKIN14M in a novel tetraploid rice variety.

[0021] Figure 3 This study compares the phenotypic differences between OSKIN14M mutants and wild-types in diploid rice. A. Comparison of plant phenotypic differences between OSKIN14M loss-of-function mutant lines and wild-types in diploid rice; B. Comparison of panicle length differences between OSKIN14M loss-of-function mutant lines and wild-types in diploid rice; C. Comparison of seed setting rate differences between OSKIN14M loss-of-function mutant lines and wild-types in diploid rice; D. Comparison of yield per plant differences between OSKIN14M loss-of-function mutant lines and wild-types in diploid rice. WT is the wild-type material, Huaduo 1; kin14m-d1 and kin14m-d2 are OSKIN14M loss-of-function mutant lines in diploid rice.

[0022] Figure 4Comparison of pollen fertility and viability differences among three loss-of-function mutants of OSKIN14M and wild-type rice in a novel tetraploid rice variety; A. Mature pollen fertility of wild-type material Huaduo 1; B. Mature pollen fertility of the kin14m-t1 mutant; C. Mature pollen fertility of the kin14m-t2 mutant; D. Mature pollen fertility of the kin14m-t3 mutant; E. Pollen viability of wild-type material Huaduo 1; F. Pollen viability of the kin14m-t1 mutant; G. Pollen viability of the kin14m-t2 mutant; H. Pollen viability of the kin14m-t3 mutant; I. Pollen germination status of wild-type material Huaduo 1; J. Pollen germination status of the kin14m-t1 mutant; K. Pollen germination status of the kin14m-t2 mutant; L. Pollen germination of the kin14m-t3 mutant; among them, WT is the wild-type material, Huaduo No. 1; kin14m-t1, kin14m-t2 and kin14m-t3 are loss-of-function mutant lines of OSKIN14M in a novel tetraploid rice.

[0023] Figure 5 The differences between the OSKIN14M loss-of-function mutant kin14m-t1 and the wild type in rice embryo sac development are as follows: A. Functional megaspore formation stage in Huaduo 1; B. Uninucleate stage of embryo sac mitosis in Huaduo 1; C. Binucleate stage of embryo sac mitosis in Huaduo 1; D. Mature embryo sac stage in Huaduo 1; E. Functional megaspore formation stage in mutant kin14m-t1, with multiple functional megaspores present; F. Binucleate stage of embryo mitosis in mutant kin14m-t1, with the arrow indicating abnormal nucleus position; G. Tetranucleate stage of embryo mitosis in mutant kin14m-t1, with the arrow indicating abnormal nucleus position; H. Mature embryo sac stage in mutant kin14m-t1, with degeneration of the mature embryo sac. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0026] Example 1: Screening and spatiotemporal expression pattern analysis of the OSKIN14M gene OSKIN14M (MSU-RGAP: LOC_Os06g36080; RAP-DB: Os06g0554700) is a gene on rice chromosome 6 that encodes kinesin, but how it participates in the fertility regulation of novel tetraploid rice is still unclear.

[0027] Therefore, OSKIN14M was selected as the target gene in this embodiment. The prediction results from the National Rice Data Center database indicate that this gene contains 17 exons ( Figure 1 A). First, its gene expression regulation pattern in tetraploid rice was verified using qRT-PCR technology. Figure 1 B): Using anther and ovary cDNA from wild-type material Huaduo 1 at various stages of reproductive development as templates, specific primers were used to detect the expression level of this gene in different tissues and developmental stages.

[0028] The specific primers used for the OSKIN14M gene are as follows: OSKIN14M-F: 5'-CTCCAAGGGCGACAGCTAAA-3' (SEQ ID No. 4); OSKIN14M-R: 5'-ACATCCGACCCTGCTCTACT-3' (SEQ ID No. 5); The qRT-PCR amplification employed a two-step method with the following reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; and 58℃ extension for 20 s, for a total of 45 cycles; followed by melting curve analysis at 65℃ to 95℃. A negative control was included in the experiment, and the detection of each gene consisted of three technical replicates and three biological replicates.

[0029] The results showed that OSKIN14M was expressed in the anthers and corresponding ovaries of the novel tetraploid rice during interphase, meiosis, microspore stage, and bicellular pollen stage, with relatively higher expression levels in the anthers during the bicellular pollen stage and in the corresponding ovaries during the late microspore stage. Figure 1 B).

[0030] The expression site of the OSKIN14M gene was further clarified using GUS staining experiments. Figure 1C). Using wild-type Huaduo No. 1 as a template, the 2000bp sequence of the upstream promoter of the OSKIN14M gene was amplified using Takara's Primer STAR high-fidelity enzyme (SEQ ID No. 6). After obtaining the promoter sequence of the OSKIN14M gene by PCR amplification (amplification primers are shown in SEQ ID No. 7 and 8), it was ligated into the pCAMBIA1305.1 vector carrying the GUS gene. After sequencing verification, a genetic transformation vector fused with the OSKIN14M promoter sequence and the GUS gene was obtained. After genetic transformation of wild-type plants, positive plants carrying the OSKIN14M gene promoter sequence were obtained. Then, the spikelets of the positive plants at various stages of reproductive development were selected for GUS staining, and the material was completely immersed in the GUS staining solution. After staining at 37℃ in the dark for 2-8 hours, the plants were stored in fixative for observation and photography. The specific method for GUS staining experiments is the same as that of Lu et al. in our laboratory (Lu Z, Guo X, Huang Z, et al. Transcriptome and gene editing analyses reveal MOF1a defect alters the expression of genes associated with tapetum development and chromosome behavior at meiosis stage resulting in low pollen fertility of tetraploidrice[J]. International Journal of Molecular Sciences, 2020, 21(20): 7489.).

[0031] The 2000bp sequence of the upstream promoter of the OSKIN14M gene (SEQ ID No. 6):

[0032] The specific primers for amplifying the OSKIN14M promoter are as follows: OSKIN14M-promter-F: 5'-CTCCAAGGGCGACAGCTAAA-3' (SEQ ID No. 7); OSKIN14M-promter-R: 5'-ACATCCGACCCTGCTCTACT-3' (SEQ ID No. 8).

[0033] GUS staining results from the analysis of reproductive tissues such as florets, anthers, and ovaries during the reproductive development of rice showed that OSKIN14M was expressed in all stages and parts of the rice floret. Figure 1 C), this result is consistent with the results of the real-time quantitative PCR experiment. Figure 1 (B) This indicates that OSKIN14M is expressed in both the anthers and ovaries of rice, especially in the anthers at the bispore stage and the ovaries corresponding to the microspore stage. A blue GUS signal was detected in florets ranging in length from 3.0 to 7.0 mm. These results demonstrate that the OSKIN14M gene is expressed at a high level in reproductive organs, providing further experimental evidence for the functional verification of the target gene.

[0034] Example 2: Verification of the function of OSKIN14M using gene knockout To clarify the function of OSKIN14M in a novel tetraploid rice, the novel tetraploid rice variety Huaduo 1-4x was used as the research material, and the OSKIN14M gene was edited using CRISPR / Cas9 technology. Using the OSKIN14M gene LOC_Os06g36080 as the target, CRISPR / Cas9-based sgRNA sequences were designed [Target 1: 5'-GTCGGAGGACGACCTCAGGGCGG-3' (SEQ ID No. 1); Target 2: 5'-CGAGGGCACGGGCTCCGCGGCGG-3' (SEQ ID No. 2)]. DNA fragments (SEQ ID No. 1 and SEQ ID No. 2) containing the encoding the sgRNA sequences (SEQ ID No. 3) were ligated into a vector carrying CRISPR / Cas9 (the pYLCRISPR / Cas9 Pubi-H binary vector system provided by the laboratory of Academician Liu Yaoguang of South China Agricultural University, available from Addgene).Then, the novel tetraploid rice variety Huaduo 1 was transformed using Agrobacterium-mediated transformation, and the positive transgenic plants were identified using Sanger sequencing technology. Three independent transgenic knockout positive plants were obtained, all of which were homozygous mutations with large fragment deletions, and were named kin14m-t1, kin14m-t2 and kin14m-t3, respectively. The mutant kin14m-t1 has a 78 bp deletion between target sites 1 and 2, located 131-208 bp after the start codon ATG in the CDS region (specifically, the deleted sequence is: 5'-GGGCGGCGCTCGCGGACGGGGCGCTGCTCTGCGCCGCGCTCCGCAGGCTCGGCTGCGACCCCGCCGCCGCCTCCGACG-3'; SEQ ID No. 9); the mutant kin14m-t2 has a 79 bp deletion between target sites 1 and 2, located 133-211 bp after the start codon ATG in the CDS region (specifically, the deleted sequence is: 5'-GCGGCGCTCGCGGACGGGGCGCTGCTCTGCGCCGCGCTCCGCAGGCTCGGCTGCGACCCCGCCGCCGCCTCCGACGAGG-3'; SEQ ID No. 9). No. 10); The mutant kin14m-t3 has an 84bp deletion between target sites 1 and 2, located 126-209bp after the start codon ATG in the CDS region (the specific deleted sequence is: 5'-TCTCAGGGCGGCGCTCGCGGACGGGGCGCTGCTCTGCGCCGCGCTCCGCAGGCTCGGCTGCGACCCCGCCGCCGCCTCCGACGA-3'; SEQ ID No. 11). The above mutation type leads to premature termination of protein translation of OSKIN14M.

[0035] Phenotypic analysis of the OSKIN14M gene knockout homozygous mutant and the wild-type Huaduo 1 revealed that, compared with the wild type, the number of spikes and spike length of the OSKIN14M gene knockout mutant kin14m-t did not show significant changes. Figure 2 A), among which agronomic traits such as seed setting rate and yield per plant showed varying degrees of decline ( Figure 2(B-2E). Among them, the average decrease in plant height for kin14m-t1 over two seasons was 2.67cm, the average decrease in fruit set rate was 30.3%, and the average decrease in yield per plant was 4.73g. The average decrease in plant height for kin14m-t2 was 10.17cm, the average decrease in fruit set rate was 24.81%, and the average decrease in yield per plant was 5.82g. The average decrease in plant height for kin14m-t3 was 1.57cm, the average decrease in fruit set rate was 17.95%, and the average decrease in yield per plant was 3.17g.

[0036] In addition, the same gene-editing vector used in the transformation of a novel tetraploid rice, containing the target sequence shown in SEQ ID No. 1 and SEQ ID No. 2, was used to transform diploid rice Nipponbare using Agrobacterium-mediated transformation. The positive transgenic plants were identified using Sanger sequencing technology, and two independent transgenic knockout positive plants were obtained. Both plants were homozygous mutants with large fragment deletions, and were named kin14m-d1 and kin14m-d2, respectively. The mutant kin14m-d1 has a 78bp deletion between target sites 1 and 2, located 131-208bp after the start codon ATG in the CDS region (the specific deletion sequence is: 5'-GGGCGGCGCTCGCGGACGGGGCGCTGCTCTGCGCCGCGCTCCGCAGGCTCGGCTGCGACCCCGCCGCCGCCTCCGACG-3'; SEQ ID No. 9); the mutant kin14m-d2 has a 79bp deletion between target sites 1 and 2, located 133-211bp after the start codon ATG in the CDS region (the specific deletion sequence is: 5'-GCGGCGCTCGCGGACGGGGCGCTGCTCTGCGCCGCGCTCCGCAGGCTCGGCTGCGACCCCGCCGCCGCCTCCGACGAGG-3'; SEQ ID No. 10).

[0037] A comparative study of the phenotypes of homozygous gene knockout mutants of the OSKIN14M gene in diploid rice and their wild-type mutants revealed that the plant architecture and spikelet fertility of kin14m-d did not show significant changes compared to the wild-type. Figure 3 A). In the late 2020 crop, kin14m-d1 showed varying degrees of decline in panicle length, seed setting rate, and yield per plant. The average decrease in panicle length was 1.83 cm, the average decrease in seed setting rate was 5.46%, and the average decrease in yield per plant was 1.18 g. For kin14m-d2, the average decrease in panicle length was 1.20 cm, the average decrease in seed setting rate was 1.69%, and the average decrease in yield per plant was 1.41 g. Figure 3 (B-3D). The above results indicate that the loss of function of OSKIN14M does not significantly reduce the seed setting rate of diploid rice. It mainly participates in the regulation of fertility in novel tetraploid rice, which leads to a decrease in the seed setting rate of novel tetraploid rice.

[0038] Example 3: Observation and analysis of the fertility of rice pollen and embryo sac in the OSKIN14M tetraploid mutant To verify the fertility differences between the mutant kin14m-t and the wild-type Huaduo 1-4x, the pollen and embryo sac development processes of the mutant kin14m-t and the wild-type were observed using WE-CLSM (global eosin B staining transparent laser scanning confocal microscopy) technology (specific observation methods refer to "Li Xiang. Cellular and molecular genetic study on low fertility in autotetraploid rice [D]. South China Agricultural University, 2018."), and their pollen and embryo sac development processes were summarized.

[0039] The results of observing the pollen fertility of the mutant kin14m-t and the wild type using WE-CLSM technology showed that in the late season of 2020, the mature pollen fertility of kin14m-t1 was significantly reduced compared with the wild type Huaduo 1. Specifically, the decrease in mature pollen fertility was 28.15% for the mutant kin14m-t1, 19.91% for kin14m-t2, and 15.21% for kin14m-t3. Meanwhile, there were no significant abnormalities in pollen viability and pollen germination between the mutant and the wild type. Figure 4 A-4L).

[0040] Further observation of embryo sac fertility using WE-CLSM technology showed that the abnormal frequency of mature embryo sacs (female gametophytes) in the mutant kin14m-t1 was as high as 40%, while the abnormal frequency of mature embryo sacs in Huaduo No. 1 was only 8.75%. Compared with the wild-type Huaduo No. 1, kin14m-t1 showed abnormalities in the functional megaspore stage, binucleate embryo sac stage, tetranucleate embryo sac stage, and mature embryo sac stage during embryo sac development. Figure 5 A-5H).

[0041] The above research results indicate that OSKIN14M does indeed participate in the regulation of fertility in novel tetraploid rice. It mainly affects the fertility of novel tetraploid rice by participating in the regulation of pollen and embryo sac development, but this gene does not affect the regulation of fertility in diploid rice.

[0042] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of rice gene OsKIN14M in regulating the fertility of tetraploid rice.

2. The application according to claim 1, characterized in that, Regulating the fertility of tetraploid rice includes regulating the seed setting rate, the yield per plant, the pollen fertility, and the embryo sac fertility.

3. The application according to claim 2, characterized in that, After the OsKIN14M gene mutation in rice, pollen fertility decreased and the frequency of abnormal embryo sac fertility increased, which in turn led to a decrease in seed setting rate and yield per plant.

4. The application according to claim 1, characterized in that, The tetraploid rice includes Huaduo No.

1.

5. A method for reducing the fertility of tetraploid rice, characterized in that, Using genetic engineering techniques, the OSKIN14M gene in rice was knocked out or its expression level was reduced.

6. The method for reducing fertility in tetraploid rice according to claim 5, characterized in that, Design a CRISPR / Cas9-based sgRNA sequence, ligate a DNA fragment containing the sgRNA sequence into a vector carrying CRISPR / Cas9, transform tetraploid rice, and screen for positive lines to obtain transgenic rice materials with reduced fertility.

7. A method for reducing the fertility of tetraploid rice according to claim 6, characterized in that, The nucleotide sequences of the sgRNA action sites are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

8. A method for reducing fertility in tetraploid rice according to claim 6, characterized in that, Vectors carrying CRISPR / Cas9 include the pYLCRISPR / Cas9 Pubi-H binary vector system.

9. The application of transgenic rice materials obtained by the method according to any one of claims 5-8 in rice breeding.

10. The application according to claim 9, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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