Application of rice pollen fertility related gene OsMS3 in breeding of male sterile line plants

Abstract

The application discloses application of a rice pollen fertility related gene OsMS3 in plant breeding of a male sterile line. The application provides a rice pollen fertility related gene OsMS3, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. Research shows that the OsMS3 gene can affect rice fertility and has the function of regulating rice pollen fertility. After knocking out the OsMS3 gene in a rice plant, the rice pollen viability is significantly reduced, the rice fertility is significantly reduced, and the OsMS3 gene can be used for creating a rice sterile line and preparing hybrid seeds. The application provides more gene resources for constructing a new hybrid breeding system, and has important significance for rice male sterile line breeding.

Description

Technical Field

[0001] The present invention belongs to the field of molecular breeding technology, and more specifically relates to the application of rice pollen fertility-related gene OsMS3 in the breeding of male sterile plants. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's major grain crops and my country's leading crop, accounting for approximately 40% of the country's total grain output. Rice plays an irreplaceable role in ensuring national food security and agricultural industry safety. In the 1960s, rice breeders achieved a "Green Revolution" in dwarfing, increasing my country's rice yield by approximately 20%. The "Second Green Revolution" of three-line hybrid breeding, which began in the 1970s, further increased rice yields, reaching 430 kilograms per mu. Hybrid rice exhibits significant advantages over conventional varieties in stress and disease resistance. The "three-line" hybrid breeding method requires the development of a comprehensive set of sterile, maintainer, and restorer lines. Hybrid rice breeding currently faces challenges such as high seed production costs, long production cycles, and complex technical requirements. In particular, there is a persistent shortage of germplasm resources for male sterile lines.

[0003] The key to hybrid rice seed production lies in the selection and breeding of male sterile lines. Male sterile lines are a special type of rice whose male organs are underdeveloped and unable to produce normal pollen, while their female organs develop normally. Consequently, these lines cannot reproduce on their own and require external pollen to produce seeds. The "three-line" rice method primarily involves the breeding of sterile lines, maintainer lines, and restorer lines to achieve hybrid rice production. The selection of pollen-sterile germplasm is the foundation of the "three-line" rice hybrid breeding process. The development of rice anthers is complex, encompassing eight stages: microspore mother cell formation, microspore mother cell meiosis, early microspore stage, mid-microspore stage, late microspore stage, early dispore stage, late dispore stage, and mature pollen stage. Pollen development involves numerous genes, and elucidating the specific functions of these genes in pollen development will have significant theoretical and practical implications.

[0004] Pollen fertility decline or pollen abortion refers to developmental disorders or stagnation during pollen development, which results in the inability of pollen to develop normally or the formation of abnormal pollen. Pollen abortion is an important factor affecting the success of rice pollen dispersal and pollination, and is also one of the difficulties affecting rice germplasm innovation. Pollen abortion can improve the yield and quality of rice hybrids. In rice breeding, pollen fertility disorder lines are often hybridized with ordinary varieties to obtain high-yield and high-quality variant materials, thereby achieving the goal of breeding high-yield and high-quality hybrids. Due to the current lack of genes related to rice pollen fertility, the development of more gene resources related to rice pollen fertility is of great significance for creating more germplasm resources of male sterile lines and cultivating more male sterile plants. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the current shortage of genes related to rice pollen fertility and provide the application of genes related to rice pollen fertility in the breeding of male sterile plants.

[0006] The first object of the present invention is to provide an application of the OsMS3 gene in regulating rice pollen fertility.

[0007] The second object of the present invention is to provide a use of a preparation for knocking out or silencing the expression of the OsMS3 gene.

[0008] The third object of the present invention is to provide a method for reducing rice pollen fertility.

[0009] The fourth object of the present invention is to provide a method for breeding rice male sterile plants.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides the use of the rice pollen fertility-related gene OsMS3 in the breeding of male sterile plants. The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the encoded amino acid sequence is shown in SEQ ID NO. 2. Studies have shown that the OsMS3 gene can affect rice fertility and regulate rice pollen fertility. Knocking out the OsMS3 gene in rice plants significantly reduces rice pollen viability and fertility. This gene can be used to create sterile lines and prepare hybrid seeds, providing more genetic resources for the construction of new hybrid breeding systems and having important implications for the breeding of male sterile rice lines.

[0012] Therefore, the present invention provides the following applications of the OsMS3 gene:

[0013] Application in regulating rice pollen fertility.

[0014] Application of the invention in reducing the vigor of rice pollen or in preparing products for reducing the vigor of rice pollen.

[0015] Application in breeding of rice male sterile lines.

[0016] Since knocking out the OsMS3 gene in wild-type rice can significantly reduce rice pollen fertility, the resulting mutant can be used in theoretical research on rice fertility regulation, creation of sterile lines and preparation of hybrid seeds.

[0017] Therefore, the present invention provides the following uses of a preparation for knocking out or silencing OsMS3 gene expression:

[0018] Application in reducing rice anther fertility.

[0019] Application in the preparation of products for reducing rice anther fertility.

[0020] Application in creating rice male sterile plants.

[0021] Preferably, the preparation for knocking out or silencing the expression of the OsMS3 gene is a plasmid, vector, or recombinant bacterium containing the knockout or inhibition of the expression of the OsMS3 gene.

[0022] The present invention provides a method for reducing rice pollen fertility, which comprises knocking out or silencing the OsMS3 gene in rice, or treating rice plants with a preparation for knocking out or silencing the expression of the rice OsMS3 gene.

[0023] The present invention also provides a method for breeding rice male sterile line plants, which knocks out or silences the rice OsMS3 gene to obtain rice male sterile line plants.

[0024] Preferably, gene editing technology is used to knock out the rice OsMS3 gene. The "mutation" can be a point mutation or a DNA fragment deletion or insertion mutation.

[0025] More preferably, a recombinant gene CRISPR / Cas9 knockout vector containing the OsMS3 gene is constructed and transformed into wild-type rice to obtain rice male sterile plants.

[0026] Preferably, the CRISPR / Cas9 knockout vector is constructed by inserting the target fragment of the OsMS3 gene into the BsaI recombination site of the pYLCRISPR / Cas9Pubi-H vector to obtain a recombinant plasmid.

[0027] Furthermore, the sequences of the primer set used to construct the CRISPR / Cas9 knockout vector are shown in SEQ ID NOs. 3 to 4.

[0028] The present invention has the following beneficial effects:

[0029] The application provides application of a rice pollen fertility related gene OsMS3 in breeding of male sterile line plants, and the nucleotide sequence of the OsMS3 gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. Research shows that the OsMS3 gene can affect rice fertility and has the function of regulating rice pollen fertility. After knocking out the OsMS3 gene in the rice plant, the pollen vitality of the rice is significantly reduced, the fertility of the rice is significantly reduced, the creation of sterile lines and hybrid seed preparation can be assisted, more gene resources are provided for construction of a new hybrid breeding system, and the application has important significance for rice male sterile line breeding. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Male fertility related genes are screened based on expression analysis of a public database.

[0031] Figure 2 A candidate gene expression pattern diagram.

[0032] Figure 3 OsMS3 gene quantitative analysis results in different anther development periods.

[0033] Figure 4 A CRISPR / Cas9 knockout target site is designed on the exon of the OsMS3 gene, and target site analysis of the material.

[0034] Figure 5 A wild type rice (WT) and ms3-ko mutant heading stage plant type diagram.

[0035] Figure 6 A wild type rice (WT) and ms3-ko mutant anther morphology observation diagram.

[0036] Figure 7 A wild type rice (WT) and ms3-ko mutant pollen staining analysis result diagram. DETAILED DESCRIPTION

[0037] The application will be further described below in combination with the drawings and specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1: Screening and quantitative analysis of rice fertility related genes

[0040] In the rice database, genes specifically expressed in rice anthers were searched and screened according to the tissue specificity of gene expression. The collected genes were then clustered and a heat map was drawn. The expression profiles of the candidate genes were analyzed using the RiceXPro rice database (https: / / ricexpro.dna.affrc.go.jp / ).

[0041] The results screened in the database were analyzed by cluster analysis, such as Figure 1 As shown in Figure 2, most candidate genes were expressed at higher levels in rice anthers. Figure 2 As shown, the expression levels of the OsMS3 gene in rice stems, anthers, and pistils were all high. Therefore, OsMS3 was selected as a key gene in the anther development period of rice. The nucleotide sequence of the OsMS3 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2 for subsequent studies.

[0042] Based on the nucleic acid sequence of the OsMS3 gene, quantitative primers (SEQ ID NOs. 5-6) were designed and synthesized. The specific primer sequences are shown in Table 1. Quantitative analysis of the selected target genes was performed at different stages of anther development using cDNA from the rice variety Tianfeng B (stored in our laboratory) at different stages (SC: sporulation stage; MMC: sporocyst stage; Meiosis: meiosis stage; EM: early uninucleate stage; LM: late uninucleate stage; BCP: dinucleate stage) as templates. The RT-quantitative PCR amplification system was as follows: 10 μL 2× ChamQ Universal SYBR qPCR Master Mix, 0.5 μL 10 μM Forward Primer, 0.5 μL 10 μM Reverse Primer, 1 μL template cDNA, and ddH2O to make up the system to 20 μL. Reaction procedure: 94°C for 10 min, 1 cycle (pre-denaturation); 94°C for 10 s (denaturation), 60°C for 15 s (annealing), 72°C for 15 s (extension), 40 cycles; the temperature range of the melting curve was set to gradually increase from 65°C to 94°C, and during this process, the fluorescence signal value was recorded once every 0.5°C increase.

[0043] Table 1 Quantitative primers

[0044]

[0045] Quantitative test results such as Figure 3 As shown, the results showed that the OsMS3 gene was highly expressed during the MMC period.

[0046] Example 2 Construction of knockout vector

[0047] A single target knockout was performed on the first exon of the OsMS3 gene nucleic acid sequence. A knockout primer pair (SEQ ID NO. 3-4) was designed and synthesized. The specific primer sequences are shown in Table 2. A CRISPR / Cas9 knockout target was designed on the OsMS3 gene exon. Figure 4 As shown. Subsequently, referring to the existing method for constructing the pYLCRISPR / Cas9Pubi-H vector: Ma et al., 2015; Molecular Plant 8, 1274-1284Ma X, Zhang Q, Zhu Q, et al. A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants [J]. Molecular Plant, 2015, 8(8): 1274-1284, a single-target CRISPR / Cas9 vector pYLCRISPR / Cas9Pubi-OsMS3 was constructed.

[0048] Table 2 Knockout primers

[0049]

[0050] The recombinant plasmid pYLCRISPR / Cas9Pubi-OsMS3 was then transformed into Agrobacterium tumefaciens EHA105 by electroporation to obtain a recombinant strain. The plasmid was then extracted for PCR and enzyme digestion identification. The recombinant strain that was correctly identified by PCR and enzyme digestion was named pYLCRISPR / Cas9Pubi-OsMS3. The genetic transformation method of Agrobacterium infection of mature rice embryos was then used. The specific method is referenced to: Nishimura A, Aichi I, Matsuoka MA protocol for Agrobacterium-mediated transformation in rice. Nature Protocols. 2006, 1(6): 2796-2802. pYLCRISPR / Cas9Pubi-OsMS3 was transformed into the indica rice variety Tianfeng B (preserved by this laboratory) to obtain transgenic T0 plants with OsMS3 gene knockout.

[0051] Example 3 Identification of editing sites in transgenic plants

[0052] An upstream detection primer (SEQ ID NO. 7) was designed and synthesized for the CDS region of the Cas9 protein on the recombinant plasmid pYLCRISPR / Cas9Pubi-OsMS3, and a downstream detection primer (SEQ ID NO. 8) was designed and synthesized for the NOS terminator. The specific primers are shown in Table 3. PCR amplification was performed using these detection primers to detect transgene expression in T0 plants. Genomic DNA from T0 plants was extracted using SDS microgenomic DNA extraction. The genomic DNA was used as a template. The PCR reaction system consisted of 1 μL DNA (20 ng / μL), 0.4 μL upstream detection primer (10 pmol / μL), 0.4 μL downstream detection primer (10 pmol / μL), 10 μL 2× Buffer, 0.4 μL dNTPs (10 mM), 0.4 μL rTaq (5 U / μL), and ddH2O added to 20 μL. PCR amplification program: 94°C for 3 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0053] Table 3 Detection primers

[0054]

[0055] The results showed that all T0 plants carrying the transgenic marker were successfully edited. Subsequently, primers (SEQ ID NOs. 9-10) were designed upstream and downstream of the target site in the genomic sequence corresponding to the OsMS3 gene. The specific primers are shown in Table 3. Amplification was performed using genomic DNA from the T0 plants as a template. The reaction system consisted of: 1 μL DNA (20 ng / μL), 0.4 μL upstream primer (10 pmol / μL), 0.4 μL downstream primer (10 pmol / μL), 10 μL 2× Buffer, 0.4 μL dNTP (10 mM), 0.4 μL rTaq (5 U / μL), and ddH2O to 20 μL. The amplification program was as follows: 94°C for 3 min; 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec; and 72°C for 5 min. After completion of amplification, the products were subjected to Sanger sequencing to detect mutations at the different target sites. The sequencing peaks were then decoded using the CRISPR-GE online tool. For details, please refer to: Liu, Weizhi, Xianrong Xie, Xingliang Ma, Jun Li, Jie-hu Chen and Yaoguang Liu. “DSDecode: A Web-Based Toolfor Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations.” Molecular plant, 2015, 8(9): 1431-1433. Xie X, Ma X, Zhu Q, et al. CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing. Molecular Plant, 2017, 10(9): 1246-1249.

[0056] The results are as follows Figure 4 As shown, the knockout target site underwent a frameshift mutation resulting in deletion.

[0057] Example 4 Plant type, anthers and pollen of transgenic T0 generation plants

[0058] The transgenic T0 plants with OsMS3 gene knockout created in Example 2 and wild-type rice Tianfeng B plants were planted at the Rice Experiment Station of South China Agricultural University. The environmental conditions were kept consistent during the planting period. The pollen of the different rice plants was then stained with I2-KI after flowering.

[0059] Different rice plant types at heading stage Figure 5As shown in the results, the plant morphology of the pYLCRISPR / Cas9Pubi-OsMS3 transgenic plants was not significantly different from that of the wild type. Figure 6 As shown in Figure 2, the anther appearance of the transgenic plants was not significantly different from that of the wild type. Figure 7 As shown, transgenic T0 plants exhibited reduced pollen viability and a phenotype of decreased pollen fertility, with pollen fertility significantly reduced. This suggests that the OsMS3 gene can influence rice fertility and regulate pollen fertility. Knocking out the OsMS3 gene in rice plants significantly reduced rice fertility, facilitating the development of sterile lines and hybrid seed preparation. This provides additional genetic resources for the construction of novel hybrid breeding systems and is of great significance for the breeding of male sterile rice lines.

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Knockout of the gene shown in SEQ ID NO.1 OsMS3 Application of genes in reducing rice pollen fertility.

2. Knockout of the gene shown in SEQ ID NO.1 OsMS3 Application of the gene in reducing rice pollen vigor or in preparing products for reducing rice pollen vigor.

3. Knockout of the gene shown in SEQ ID NO.1 OsMS3 Application of genes in breeding of male sterile rice plants.

4. Knockout of the gene shown in SEQ ID NO.1 OsMS3 Application of gene preparations in reducing anther fertility in rice.

5. Knockout of the gene shown in SEQ ID NO.1 OsMS3 Application of gene preparation in preparing product for reducing rice anther fertility.

6. Knockout of the gene shown in SEQ ID NO.1 OsMS3 The application of gene preparations in creating rice male sterile plants.

7. A method for reducing rice pollen fertility, characterized in that: Knockout rice OsMS3 knockout rice OsMS3 The rice plants are treated with a gene preparation; OsMS3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. A method for breeding rice male sterile plants, characterized in that: Knockout rice OsMS3 Gene, obtain rice male sterile plants; OsMS3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that Using gene editing technology to OsMS3 Knockout mutation of genes.

10. The method according to claim 9, characterized in that: Build OsMS3 The recombinant gene CRISPR / Cas9 knockout vector of the gene was used to transform wild-type rice to obtain rice male sterile plants.

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