Rice OsSYP71b gene and application in breeding

Abstract

The invention discloses a pollen specific expression gene OsSYP71b and a function of the pollen specific expression gene OsSYP71b in a rice development process, and belongs to the field of botany and genetic engineering. The rice OsSYP71b is knocked out on the basis of expression pattern analysis and a CRISPR / Cas9 system, specific expression of the rice OsSYP71b in pollen is found, the activity of the pollen in an Ossyp71b mutant is remarkably reduced, and the seed setting rate is reduced. The invention has reference significance for researching rice gene functions and crossbreeding, and provides a new target gene for creating a rice male sterile line.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering breeding, and specifically relates to a gene OsSYP71b that specifically expresses in pollen cells and regulates pollen fertility, and the application of this gene in the cross-breeding of monocotyledonous rice. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and more than half of the world's population takes rice as the staple food. Hybrid breeding using heterosis has greatly increased rice yield. As a self-pollinating and hermaphroditic plant, the creation of male sterile lines is required for rice hybrid breeding. Currently, the commonly used hybrid breeding methods in rice are the "three-line method" breeding based on the CMS (Cytoplasmic male sterility) line and the "two-line method" breeding based on the PTGMS (Photo-thermosensitive genic male sterile line) (Wu et al., 2016. Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnology Journal, 14: 1046–1054.).

[0003] The creation of male sterile materials based on pollen fertility regulation is the key to rice hybrid breeding. Studying the related regulatory genes during pollen development can better reveal the molecular mechanism of male fertility maintenance. The continuous excavation of male sterile genes is beneficial to the utilization of heterosis and the improvement of rice yield.

[0004] Although in Arabidopsis thaliana, after the SYP72 gene mutates, the pollen viability of the mutant significantly decreases, and the pollen cannot maintain cell integrity during the hydration process, resulting in pollen rupture and inability to germinate. In rice, the function of the homologous gene of SYP72 is not clear. (Zhou X, et al. 2022. SYP72 interacts with the mechanosensitive channel MSL8 to protect pollen from hypoosmotic shock during hydration. Nature Communications, 13:73.). Currently, the research on rice male sterility genes mainly relies on EMS mutagenesis, and relevant genes are explored based on the phenotype of pollen sterility. The function of the homologous gene of SYP72 in rice is unknown.

[0005] Rice male sterility is the core link in rice hybrid breeding. The cloning and functional analysis of its sterility genes can not only analyze the genetic mechanism of the formation of dominant male nuclear sterility in rice and further improve the genetic mechanism of rice male sterility, but also provide gene resources and theoretical basis for the application of rice male sterility. Whether it is to enrich rice fertility resources or improve breeding efficiency, the research on rice male sterility genes is important and urgent. Summary of the Invention

[0006] The first object of the present invention is to provide a new gene that regulates pollen viability in rice and is used to create a rice male sterile line.

[0007] The second object of the present invention is to provide a new protein that regulates pollen viability in rice and is used to create a rice male sterile line.

[0008] The third object of the present invention is to provide a method for generating rice male sterility.

[0009] The fourth object of the present invention is the application of pollen-specific expression genes and proteins in breeding.

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] The present invention discloses a rice pollen-specific expression gene OsSYP71b, whose nucleotide sequence is shown in SEQ ID NO.1. This gene is specifically expressed in pollen, and knocking out or silencing this gene can lead to rice male sterility.

[0012] For the rice pollen-specific expression gene OsSYP71b disclosed in the present invention, knocking out or silencing the OsSYP71b gene by the CRISPR / Cas9 technology results in rice male sterility.

[0013] The present invention also discloses a rice pollen-specific expression protein, the amino acid sequence of which is shown in SEQ ID NO. 2. By knocking out or silencing the OsSYP71b gene through the CRISPR / Cas9 technology, a knockout or silencing mutant is constructed, resulting in male sterility of rice.

[0014] The present invention also discloses a method for preparing an Ossyp71b mutant rice plant, the steps of which include: constructing an OsSYP71b knockout plasmid, genetically transforming it with Nipponbare rice as the background, and obtaining an Ossyp71b mutant plant.

[0015] Further, the method for preparing an Ossyp71b mutant rice plant includes the steps of:

[0016] ① According to the gene sequence of OsSYP71b, use the online website CRISPR-P2.0 to analyze the knockout sites, and select two target sites with higher scores and appropriate positions;

[0017] Target sequence:

[0018] T1: 5’-CCGTCGAGGTCGATCGATCA-3’,

[0019] T2: 5’-CCAAGAAGGTGACCGGAACT-3’;

[0020] ② According to the target sequence and the vector sequence of the sgRNA expression cassette, design amplification primers. Use U6a-F and DT1-R as paired primers to amplify the U6a promoter fragment, use DT1-F and DT2-R as paired primers to amplify the U6b promoter fragment, and use DT2-F and SG-R as paired primers to amplify the sgRNA fragment.

[0021] Primer sequence:

[0022] U6a-F: ATATATGGTCTCGctcgtggaatcggcagcaaagg,

[0023] SG-R: ATATATGGTCTCGaccgtccatccactccaagctc,

[0024] DT1-F: ATATATGGTCTCGGTCGATCGATCAgttttagagctagaaata,

[0025] DT1-R: ATATATGGTCTCGCGACCTCGACGGcggcagccaagccagca,

[0026] DT2-F: ATATATGGTCTCGGTGACCGGAACTgttttagagctagaaata,

[0027] DT2-R: ATATATGGTCTCGTCACCTTCTTGGcaacacaagcggcagcg;

[0028] ③ The fragment was ligated to the MH plasmid by restriction enzyme digestion and ligation, and the constructed MH knockout vector was used for genetic transformation with Nipponbare rice as the background to obtain Ossyp71b mutant plants.

[0029] Furthermore, the present invention discloses a method for constructing a pOsSYP71b::GUS expression pattern analysis vector, performing genetic transformation, screening positive lines, and confirming that OsSYP71b is a pollen-specific expression gene through GUS staining experiments. The method includes the following steps:

[0030] ① The promoter region of 2989 bp before the ATG of pOsSYP71b was amplified from the rice genome by PCR, and the pOsSYP71b promoter was ligated into the pC1300-GUS vector to obtain the pOsSYP71b::GUS expression pattern analysis vector;

[0031] ② The pOsSYP71b::GUS expression pattern analysis vector was sent to Boyuan Biotechnology Company for genetic transformation to obtain transgenic plants;

[0032] ③ Plants expressing GUS signal in pollen were screened by GUS staining, that is, positive plants;

[0033] ④ Primers were designed on HygR, and the genomic DNA of positive plants was amplified by PCR to screen positive homozygous plants with vector segregation;

[0034] ⑤ Different tissues and pollen of the screened positive plants were subjected to GUS staining to identify the expression pattern of OsSYP71b.

[0035] Further, the present invention discloses a method for constructing rice OsSYP71b knockout mutants by CRISPR / Cas9 technology, screening and creating three homozygous transgenic lines, and analyzing the phenotypes of the mutant lines. The results confirmed that the mutation of the OsSYP71b gene would lead to a significant decrease in rice pollen viability, rupture during the hydration process, and a decrease in seed setting rate; this indicates that OsSYP71b is related to pollen viability and seed setting rate, providing a reference for the creation of male sterile lines and the preparation of hybrid seeds. The method includes the following experimental steps:

[0036] ① The target site was ligated into the MH plasmid by the method of cutting and ligating simultaneously, and heat shock transformed into Escherichia coli to obtain the MH-OsSYP71b knockout vector;

[0037] ② Send the MH-OsSYP71b knockout vector to Boyuan Biotech for genetic transformation to obtain transgenic plants;

[0038] ③ Extract genomic DNA from the transgenic plants, perform PCR amplification and sequencing on the fragment ranges where target 1 and target 2 are located, and compare the sequencing results with the OsSYP71b gene in Nipponbare. If the sequence has a specific single-peak feature at the target position and there is a base insertion or deletion in this sequence, then this strain is of homozygous genotype; if the mutant sequence has a double-peak feature at the target position, then this strain is of heterozygous genotype;

[0039] ④ Design primers at the Cas9 position to identify the separation of the MH-OsSYP71b knockout vector in the T1 generation transgenic plants, and screen for homozygous genotype mutant plants with the separation of the MH-OsSYP71b knockout vector;

[0040] ⑤ Conduct phenotypic identification on the screened homozygous genotype mutant plants, observe the phenotypes such as pollen and seed setting rate of the mutant plants, and compare them with the wild type;

[0041] ⑥ Obtain OsSYP71b mutant plants. The pollen viability of this mutant is reduced, the seed setting rate is low, and the pollen will rupture during the hydration process and cannot germinate.

[0042] Furthermore, the present invention also discloses the function of the homologous gene of the pollen-specific expression gene SYP72 in the monocotyledonous plant rice, and it is confirmed by yeast two-hybrid experiments that it may function through a mechanosensitive ion channel protein.

[0043] The present invention discloses the application of the rice pollen-specific expression gene OsSYP71b in the preparation of rice male sterile lines.

[0044] The present invention discloses the application of the rice pollen-specific expression protein in the preparation of rice male sterile lines.

[0045] In the present invention, "SNARE" refers to Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptors, which are highly conserved membrane transport proteins that play important roles in vesicle transport in species such as plants, animals, and microorganisms. (Gu et al. 2020. Vesicle Transport in Plants: A Revised Phylogeny of SNARE Proteins. Evolutionary Bioinformatics Online, 16: 1612702879.)

[0046] The term "SYP72" used in this article refers to the SYP72 gene in Arabidopsis thaliana, which is specifically expressed in pollen. After mutation, the pollen viability of Arabidopsis thaliana plants is significantly abnormal. During pollen hydration, the cell integrity cannot be maintained, resulting in rupture, and the siliques are significantly smaller, and the seed setting rate is significantly reduced. (Zhou, et al. 2022. SYP72 interacts with the mechanosensitive channel MSL8 to protect pollen from hypoosmotic shock during hydration. Nature Communications, 13: 73.)

[0047] The term "gene editing" used in this article is a relatively precise emerging genetic engineering technology that can modify target genes in the genome of organisms. This technology can perform site-directed "editing" on target genes to achieve modification of specific DNA fragments. Gene editing relies on genetically engineered nucleases, also known as "molecular scissors", to generate site-specific double-strand breaks (DSBs) at specific positions in the genome, inducing organisms to repair DSBs through non-homologous end joining (NHEJ) or homologous recombination (HR). Since this repair process is error-prone, it leads to targeted site-directed mutations.

[0048] The term "CRISPR / Cas9" used in this article refers to an endonuclease that uses the sgRNA guide strand to target the endonuclease cleavage site, a site-directed mutagenesis technique widely used in recent years.

[0049] In this article, the homologous genes of SYP72 in angiosperms are defined as genes with the following characteristics:

[0050] (1) Having a conserved domain of the SNARE protein family;

[0051] (2) The encoded protein is secretory.

[0052] (3) Specifically expressed in pollen.

[0053] The biomaterials used in the present invention can all be purchased from companies, or contact the State Key Laboratory of Hybrid Rice, Wuhan University, or can be obtained from published articles.

[0054] Advantages of the present invention:

[0055] Using the CRISPR / Cas9 technology to construct knockout mutants of Ossyp71b in rice, revealing the function of Ossyp71b in the late process of pollen development, providing reference for the creation of rice male sterile lines and hybrid breeding. The advantages of the present invention are mainly reflected in the following aspects:

[0056] (1) Revealed the function of the SNARE protein OsSYP71b in the process of rice pollen development;

[0057] (2) Confirmed that OsSYP71b is a pollen-specifically expressed gene, and the loss of gene function will lead to a significant decrease in pollen viability and seed setting rate, which has important reference significance for the creation of rice male sterile lines and hybrid breeding.

[0058] (3) Provided new ideas for revealing the biological function of SNARE family proteins related to vesicle transport in the process of pollen development of monocotyledonous rice. Description of the Drawings

[0059] Figure 1 It is a figure showing the specific expression of OsSYP71b in pollen. The expression of OsSYP71b in tissues such as rice seedlings (A, E), roots (B, F), stems (C, G), leaves (D, H), spikelets (I, M), ovaries (J, N), anthers (K, O), and pollen (L, P) of wild-type and transgenic plants is specifically expressed only in pollen.

[0060] Figure 2 It is the MH plasmid map and the positions of each element after the vector construction is completed.

[0061] Figure 3 It is the homozygous mutants of three different editing methods identified.

[0062] Figure 4 It is the I2-KI staining map of wild-type and mutant pollen. 10× and 20× represent images under different backgrounds.

[0063] Figure 5FDA staining images of wild-type and mutant pollen. FDA indicates fluorescein diacetate staining; BF indicates bright-field view; Merge indicates overlapping channels.

[0064] Figure 6 Seed setting rates of wild-type and mutant. * indicates aborted seeds.

[0065] Figure 7 Phenotypic analysis of the hydration process of wild-type and mutant. Changes in the pollen morphology of wild-type and Ossyp71b mutant pollen over time in liquid medium. 0, 10 min, 20 min, 30 min, 60 min indicate hydration time, and * indicates ruptured pollen.

[0066] Figure 8 Interaction relationship diagram between OsSYP71b and OsMSL5. Detailed implementation mode

[0067] To better understand the present invention, the main content of the present invention is further explained below in conjunction with specific embodiments. These examples are only used to illustrate the present invention but do not limit the present invention. Unless otherwise specified, the experimental methods in the following examples are all conventional methods, and the reagent materials used are all purchased from conventional biochemical reagent companies.

[0068] Example 1: Creation of pOssyp71b::GUS expression pattern plants

[0069] (1) Identification of homologous genes of SYP72 in rice

[0070] Screen for SNARE protein domains through the Pfam database, screen SANRE family proteins on the RGAP website and TBtools, and construct a phylogenetic tree of SNARE proteins in Arabidopsis and rice using the neighbor-joining method (NJ) in MEGA7.

[0071] In rice, the SYP7 subfamily has only two members: OsSYP71a and OsSYP71b. Extract the gene expression profiles from the rice tissue expression profile data generated in the laboratory and analyze them. It is found that OsSYP71b is specifically expressed in pollen, and OsSYP71a is expressed in different tissues. OsSYP71b is selected as the research object for subsequent experiments.

[0072] (2) Analysis of OsSYP71b expression pattern

[0073] To study the expression patterns of OsSYP71b in different tissues of rice and at different stages of pollen development, a pOsSYP71b::GUS expression pattern analysis vector was constructed and genetically transformed using Nipponbare as the background to obtain transgenic materials. Genomic DNA was extracted from the transgenic materials and identified using specific primers designed on the vector to screen for transgenic positive plants. After obtaining the T0 generation seeds, stable transgenic positive plants were obtained after screening for two generations. The expression of the OsSYP71b gene in different tissues of rice was observed by GUS staining, and the results showed that OsSYP71b was specifically expressed in pollen (attached Figure 1 ).

[0074] Example 2: Construction of Ossyp71b knockout plasmid and obtaining mutant plants

[0075] According to the genomic sequence information of OsSYP71b, target sites were predicted on the CRISPR-P2.0 website. Two target sites with higher scores and appropriate positions were selected, and the two target sites were located on the first exon and the second exon respectively. Amplification primers were designed according to the target site sequences and the vector sequences of the sgRNA expression cassette. The vector construction method was as follows: Using the MH plasmid (attached Figure 2 ) as a template, the U6a promoter fragment was amplified using U6a-F and DT1-R as paired primers, the U6b promoter was amplified using DT1-F and DT2-R as paired primers, and the sgRNA fragment was amplified using DT2-F and SG-R as paired primers. The above three fragments were ligated to the MH plasmid by a method of simultaneous digestion and ligation, and the plasmid with correct sequencing alignment was the constructed knockout plasmid. The plasmid was sent to Boyuan Biotechnology Co., Ltd. in Wuhan for genetic transformation using the wild-type rice Nipponbare as the background to obtain CRISPR / Cas9 (abbreviated as Ossyp71b) transgenic plants of OsSYP71b.

[0076] Target site sequences:

[0077] T1: 5’-CCGTCGAGGTCGATCGATCA-3’ (SEQ ID NO.3)

[0078] T2: 5’-CCAAGAAGGTGACCGGAACT-3’ (SEQ ID NO.4)

[0079] Primer sequences:

[0080] U6a-F: ATATATGGTCTCGctcgtggaatcggcagcaaagg

[0081] (SEQ ID NO.5)

[0082] SG-R: ATATATGGTCTCGaccgtccatccactccaagctc

[0083] (SEQ ID NO.6)

[0084] DT1-F: ATATATGGTCTCGGTCGATCGATCAgttttagagctagaaata

[0085] (SEQ ID NO.7)

[0086] DT1-R: ATATATGGTCTCGCGACCTCGACGGcggcagccaagccagca

[0087] (SEQ ID NO.8)

[0088] DT2-F: ATATATGGTCTCGGTGACCGGAACTgttttagagctagaaata

[0089] (SEQ ID NO.9)

[0090] DT2-R: ATATATGGTCTCGTCACCTTCTTGGcaacacaagcggcagcg

[0091] (SEQ ID NO.10)

[0092] Table 1 Restriction Enzyme-Ligation Reaction System

[0093]

[0094] Table 2 PCR Reaction Program

[0095]

[0096] Example 3: Identification of Knockout Sites in Transgenic Rice Plants

[0097] (1) Extract genomic DNA of mutants

[0098] Grind approximately 2 cm leaves of the transgenic plants of the T0 generation in liquid nitrogen; add 500 μL of CTAB extraction solution, incubate at 65 °C for 30 min, and invert and mix evenly every 10 min; add 300 μL of chloroform and mix well, then centrifuge at 12000 rpm for 10 min at room temperature; transfer 500 μL of the supernatant to a 1.5 ml centrifuge tube, add 500 μL of isopropanol and mix well, let stand at -20 °C for 20 min, and centrifuge at 12000 rpm for 10 min at room temperature; discard the supernatant, add 500 μL of 75% ethanol, centrifuge at 12000 rpm for 5 min, and repeat the washing once after discarding the supernatant; after sucking dry the ethanol in the tube, air dry it in a laminar flow hood, and add 50 μL of ddH2O for dissolution.

[0099] (2) Identification of the editing method of mutant plants

[0100] Using genomic DNA as a template for target amplification and sequencing, for the sequence with a specific single-peak feature starting from the target sequence, if the sequence has a mutation, the genotype of this strain is a homozygous genotype (both OsSYP71b on two chromosomes have mutations); if it is a sequence with a double-peak feature, the genotype of this strain is a heterozygous genotype (only the OsSYP71b gene on one chromosome has a mutation). All the plants of the T0 generation are heterozygous plants.

[0101] (3) Screening of homozygous plants

[0102] For the T1 generation plants, first design primers on Cas9 for the identification of CRISPR / Cas9 vector separation, perform target amplification and sequencing on the plants with Cas9 vector separation, and analyze the sequencing results. Select three different homozygous genotype editing methods (attached Figure 3 ), and take Ossyp71b#3 as the research object, select homozygous genotype plants and harvest seeds, and use the homozygous mutant plants to observe the phenotypes in the T2 generation to obtain mutant plants. The OsSYP71b mutant plants have the defects shown in Example 4.

[0103] Example 4: Phenotypic identification of plants with OsSYP71b gene function defects

[0104] (1) Observation of mutant pollen viability by I2-KI staining method

[0105] Rice pollen accumulates a large amount of starch after maturity. I2-KI staining solution can stain starch blue, which is one of the common methods for detecting rice pollen viability. During the rice pollen maturity period, observe and count the pollen viability of wild-type plants and Ossyp71b mutant lines by I2-KI staining.

[0106] It was found that the wild-type pollen grains were plump and spherical, and all could be stained blue by I2-KI. Some of the pollen grains in the mutant were shriveled, and some of the plump pollen grains could not be stained blue. After statistics, it was found that the pollen viability of the mutant was only about 50% (attached Figure 4 ). At the same time, it indicates that there may be abnormalities in the pollen starch deposition process of Ossyp71b.

[0107] (2) Observation of mutant pollen grain viability by FDA staining method

[0108] FDA staining can detect the integrity of cells. Viable cells can decompose FDA to produce fluorescein and present green, while non-viable cells cannot produce green fluorescence. Therefore, during the rice pollen maturation period, FDA staining was used to supplement and verify the results of I2-KI staining.

[0109] The results showed that all wild-type pollen grains were green and spherical, and about half of the pollen grains had no green fluorescence. The statistical results showed that the pollen viability of the mutant was about 50%, which was consistent with the results of I2-KI staining (attached Figure 5 ).

[0110] (3) Statistics of the seed setting rate of Ossyp71b mutants

[0111] To further verify whether the abnormal pollen viability of the Ossyp71b mutant would affect the seed setting rate of the offspring, we harvested seeds from individual plants of the mutant line and the wild-type plants and counted the seed setting rate.

[0112] The results showed that almost all the wild-type plants had plump seeds, and the seed setting rate was above 90%; most of the mutants had shriveled seeds, and the seed setting rate was only about 15%. It indicates that in addition to abnormal pollen viability, there may be other defects that prevent viable pollen from completing the fertilization process, thereby leading to a significant decrease in the seed setting rate (attached Figure 6 ).

[0113] (4) Mutant pollen ruptures during the hydration process

[0114] We observed the pollen hydration process. After 1 h of hydration in distilled water, both wild-type pollen and mutant pollen showed obvious rupture phenomena. Since hydration in distilled water is an extreme condition, we observed the pollen hydration status in a pollen liquid medium. With the passage of time, fewer wild-type pollen ruptured; while mutant pollen began to rupture at 10 min of hydration, and with the passage of time, the proportion of ruptured pollen gradually increased. At 60 min, the proportion of unruptured pollen was about 25% (attached Figure 7 ).

[0115] (5) OsSYP71b interacts with OsMSL5

[0116] The results of the yeast two-hybrid experiment showed that there was an interaction between OsSYP71b and OsMSL5. There might be a relationship similar to that in Arabidopsis between OsSYP71b and OsMSL5, that is, OsSYP71b might function through the mechanosensitive channel protein OsMSL5 (attached Figure 8 ).

Claims

1. A rice pollen-specifically expressed gene OsSYP71b, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The gene OsSYP71b according to claim 1, characterized in that: Knocking out or silencing the OsSYP71b gene using CRISPR / Cas9 technology resulted in rice male sterility.

3. A rice pollen-specific expression protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. The pollen-specific expression protein according to claim 3, characterized in that: The OsSYP71b gene was knocked out or silenced using CRISPR / Cas9 technology to construct knockout or silence mutants, resulting in rice male sterility.

5. The gene OsSYP71b according to claim 2, characterized in that: Knocking out the OsSYP71b gene using CRISPR / Cas9 technology resulted in male sterility in rice.

6. The gene OsSYP71b according to claim 2, characterized in that: Silencing the OsSYP71b gene through CRISPR / Cas9 technology resulted in rice male sterility.

7. A method for preparing an Ossyp71b mutant rice plant, comprising the steps of: The OsSYP71b knockout plasmid was constructed and genetically transformed into the rice Nipponbare background to obtain Ossyp71b mutant plants.

8. The method for preparing an Ossyp71b mutant rice plant according to claim 7, comprising the steps of: ① According to the gene sequence of OsSYP71b, analyze the knockout site and select two appropriate target sites; Target sequence: T1: 5'-CCGTCGAGGTCGATCGATCA-3', T2: 5'-CCAAGAAGGTGACCGGAACT-3'; ② Design amplification primers according to the target sequence and the vector sequence of the sgRNA expression cassette, use U6a-F and DT1-R as paired primers to amplify the U6a promoter fragment, use DT1-F and DT2-R as paired primers to amplify the U6b promoter fragment, and use DT2-F and SG-R as paired primers to amplify the sgRNA fragment. Primer sequences: U6a-F:ATATATGGTCTCGctcgtggaatcggcagcaaagg, SG-R:ATATATGGGTCTCGaccgtccatccactccaagctc; DT1-F:ATATATGGTCTCGGTCGATCGATCAgttttagagctagaaata, DT1-R:ATATATGGTCTCGCGACCTCGACGGcggcagccaagccagca, DT2-F:ATATATGGTCTCGGTGACCGGAACTgttttagagctagaaata, DT2-R:ATATATGGTCTCGTCACCTTCTTGGcaacacaagcggcagcg; ③ The fragment was connected to the MH plasmid using enzyme ligation, and the constructed MH knockout vector was used for genetic transformation in the rice Nipponbare background to obtain Ossyp71b mutant plants.

9. Use of the gene according to claim 1 in preparing rice male sterile lines.

10. Use of the protein according to claim 3 in preparing rice male sterile lines.

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