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

Abstract

The invention discloses application of a rice pollen fertility related gene OsMS3 in male sterile line plant breeding. The invention provides a gene OsMS3 related to rice pollen fertility, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the coded amino acid sequence is as shown in SEQ ID NO.2. Researches show that the OsMS3 gene can influence rice fertility and has the effect of regulating and controlling rice pollen fertility. After the OsMS3 gene is knocked out from a rice plant, the activity of rice pollen can be remarkably reduced, so that the rice fertility is remarkably reduced, and the gene can be used for creating a rice sterile line and preparing hybrid seeds. The invention provides more gene resources for constructing a novel cross breeding system, and has important significance on breeding of a rice male sterile line.

Description

Technical Field

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

[0002] Rice (Oryza sativa L.) is one of the world's major food crops and the largest food crop in my country, accounting for about 40% of the country's total grain output. Rice plays an irreplaceable role in ensuring national food security and agricultural industry security. In the 1960s, breeders increased my country's rice yield by about 20% through the "Green Revolution" of dwarfing breeding. The "Second Green Revolution" brought about by three-line hybrid breeding starting in the 1970s further increased rice yields, reaching 430 kilograms per mu. Hybrid rice shows obvious advantages over ordinary varieties in terms of stress resistance and disease resistance, and the "three-line method" hybrid breeding is to breed matching sterile lines, maintenance lines and recovery lines. At present, hybrid rice breeding faces challenges such as high seed production costs, long cycles, and complex technical requirements, especially the lack of germplasm resources for male sterile lines.

[0003] The key to rice hybrid seed production is the selection of male sterile lines. Male sterile rice lines are a special type of rice. The male organs in their own floral organs are not fully developed and cannot form normal pollen, but the female organs develop normally. Therefore, male sterile lines cannot reproduce themselves and need to rely on foreign rice pollen to produce seeds. The "three-line" rice mainly achieves hybrid rice production by breeding sterile lines, maintainer lines and restorer lines. Among them, the selection of pollen sterile germplasm materials is the basis of the "three-line" rice hybrid breeding. The development process of rice anthers is relatively complex, and the development process includes 8 stages, namely, microspore mother cell formation stage, microspore mother cell meiosis stage, early microspore stage, mid-microspore stage, late microspore stage, early dicellular pollen stage, late dicellular pollen stage and mature pollen stage. The pollen development process involves many genes. Elucidating the specific functions of these genes in pollen development will have great value in theory and practice.

[0004] Pollen fertility decline or pollen abortion refers to the developmental disorder or stagnation phenomenon that occurs 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 dissemination 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 purpose 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 shortage of existing genes related to rice pollen fertility and provide application of genes related to rice pollen fertility in 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 the 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 an application of a rice pollen fertility-related gene OsMS3 in the breeding of male sterile plants, wherein 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 has the function of regulating rice pollen fertility. Knocking out the OsMS3 gene in rice plants will significantly reduce the vigor of rice pollen, and can significantly reduce the fertility of rice. It is used for the creation of sterile lines and the preparation of hybrid seeds, providing more gene resources for the construction of a new hybrid breeding system, and is of great significance to the breeding of rice male sterile lines.

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

[0013] Application in regulating rice pollen fertility.

[0014] Application 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, the creation of sterile lines and the 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 knocking out or inhibiting expression of the OsMS3 gene.

[0022] The 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 rice male sterile line plant breeding method, which knocks out or silences the rice OsMS3 gene to obtain the rice male sterile line plant.

[0024] Preferably, the rice OsMS3 gene is knocked out and mutated using gene editing technology. 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 line 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 sets 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 present invention provides an application of a rice pollen fertility-related gene OsMS3 in the breeding of male sterile plants. The nucleotide sequence of the OsMS3 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 has the function of regulating rice pollen fertility. Knocking out the OsMS3 gene in rice plants will significantly reduce the vigor of rice pollen and significantly reduce rice fertility, which can help the creation of sterile lines and the preparation of hybrid seeds, provide more gene resources for the construction of a new hybrid breeding system, and is of great significance to the breeding of rice male sterile lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is based on expression analysis of public databases to screen male fertility-related genes.

[0031] Figure 2 It is a graph of candidate gene expression patterns.

[0032] Figure 3 This is a diagram showing the results of quantitative analysis of the OsMS3 gene at different stages of anther development.

[0033] Figure 4 The goal is to design a CRISPR / Cas9 knockout target on the exon of the OsMS3 gene and perform target analysis on the material.

[0034] Figure 5 The plant morphology of wild-type rice (WT) and ms3-ko mutant at heading stage.

[0035] Figure 6 These are the anther morphology observation pictures of wild-type rice (WT) and ms3-ko mutant.

[0036] Figure 7 This is a graph showing the results of pollen staining analysis of wild-type rice (WT) and ms3-ko mutant. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[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 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, most of the candidate genes were expressed at higher levels in rice anthers. Figure 2 As shown, it was shown that the expression levels of OsMS3 gene in rice stem, anther and pistil were relatively high. Therefore, OsMS3 was selected as the key gene in the anther development period of rice. The nucleotide sequence of 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 research.

[0042] Further, based on the nucleic acid sequence of the OsMS3 gene, quantitative primers (SEQ ID NO. 5-6) were designed and synthesized. The specific primer sequences are shown in Table 1. At different stages of anther development, cDNA of rice variety Tianfeng B (preserved by this laboratory) at different stages (SC: sporulation period; MMC: sporocyst period; Meiosis: meiosis period; EM: early mononucleate period; LM: late mononucleate period; BCP: binucleate development period) was used as a template to quantitatively analyze the selected target gene. The amplification system of RT-quantitative PCR 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, ddH 2 The O supplementation system was 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 value of the fluorescence signal was recorded 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] The first exon in the nucleic acid sequence of the OsMS3 gene was single-targeted and knocked out. 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 exon of the OsMS3 gene. Figure 4 As shown. Subsequently, referring to the existing method for constructing the pYLCRISPR / Cas9Pubi-H vector: Ma et al., 2015; Molecular Plant 8, 1274-1284 Ma 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] Then, the recombinant plasmid pYLCRISPR / Cas9Pubi-OsMS3 was transformed into the Agrobacterium EHA105 strain by electroporation to obtain a recombinant strain, and then the plasmid was extracted for PCR and enzyme digestion identification. The recombinant strain with correct PCR and enzyme digestion identification was named pYLCRISPR / Cas9Pubi-OsMS3. The genetic transformation method of Agrobacterium infection of mature rice embryos was used again. 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 a transgenic T with OsMS3 gene knockout. 0 Generation of plants.

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

[0052] An upstream detection primer (SEQ ID NO.7) was designed and synthesized in 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 on its NOS terminator. The specific primers are shown in Table 3. PCR amplification was performed using the detection primers to detect T 0 The SDS microgenomic DNA extraction method was used to extract T 0 The genomic DNA of the first generation plants was used as the template. The PCR reaction system was as follows: DNA (20 ng / μL) 1 μL, upstream detection primer (10 pmol / μL) 0.4 μL, downstream detection primer (10 pmol / μL) 0.4 μL, 2× Buffer 10 μL, dNTP (10 mM) 0.4 μL, rTaq (5 U / μL) 0.4 μL, ddH 2 0 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 test results showed that the T 0 Subsequently, primers (SEQ ID NO.9-10) were designed at the upstream and downstream of the target position of the genomic sequence corresponding to the OsMS3 gene. The specific primers are shown in Table 3. 0 The genomic DNA of the first generation plants was used as a template for amplification. The reaction system was: DNA (20 ng / μL) 1 μL, upstream primer (10 pmol / μL) 0.4 μL, downstream primer (10 pmol / μL) 0.4 μL, 2×Buffer 10 μL, dNTP (10 mM) 0.4 μL, rTaq (5 U / μL) 0.4 μL, ddH 2O to 20 μL. The amplification program was: 94°C for 3 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, 30 cycles; 72°C for 5 min. After the amplification, the product was subjected to Sanger sequencing to detect mutations at different target sites. The sequencing peak graph was decoded using the CRISPR-GE online tool. For specific reference: 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 TargetedMutations.” 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 that resulted in deletion.

[0057] Example 4 Transgenic T 0 Plant type, anthers and pollen of the next generation plants

[0058] The OsMS3 gene knockout transgenic T cells created in Example 2 were 0 The generation plants and the wild-type rice Tianfeng B plants were planted at the Rice Experimental Station of South China Agricultural University. The environmental conditions were kept consistent during the planting period. Then the pollen of different rice plants after flowering was observed by I2-KI staining.

[0059] Different rice plant types at heading stage Figure 5 As shown in the figure, the results showed that the plant type of the transgenic plants of pYLCRISPR / Cas9Pubi-OsMS3 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 edited T 0The pollen vitality of the first generation plants decreased, and the phenotype of decreased pollen fertility appeared, and the pollen fertility decreased significantly. This shows that the OsMS3 gene can affect rice fertility and has the function of regulating rice pollen fertility. Knocking out the OsMS3 gene in rice plants can significantly reduce rice fertility, which can help the creation of sterile lines and the preparation of hybrid seeds, provide more genetic resources for the construction of a new hybrid breeding system, and is of great significance to the breeding of rice male sterile 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of the OsMS3 gene as shown in SEQ ID NO.1 in regulating rice pollen fertility.

2. Use of the OsMS3 gene as shown in SEQ ID NO.1 in reducing rice pollen vigor, or in preparing a product for reducing rice pollen vigor.

3. Application of the OsMS3 gene as shown in SEQ ID NO.1 in breeding of rice male sterile lines.

4. Use of a preparation for knocking out or silencing the expression of the OsMS3 gene as shown in SEQ ID NO. 1 in reducing the fertility of rice anthers.

5. Use of a preparation for knocking out or silencing the expression of the OsMS3 gene as shown in SEQ ID NO. 1 in the preparation of a product for reducing rice anther fertility.

6. Use of a preparation for knocking out or silencing the expression of the OsMS3 gene as shown in SEQ ID NO. 1 in creating rice male sterile plants.

7. A method for reducing rice pollen fertility, characterized in that: The OsMS3 gene in rice is knocked out or silenced, or the rice plant is treated with a preparation for knocking out or silencing the expression of the rice OsMS3 gene; the nucleotide sequence of the OsMS3 gene is shown in SEQ ID NO.

1.

8. A method for breeding rice male sterile plants, characterized in that: The rice OsMS3 gene is knocked out or silenced to obtain rice male sterile line plants; the nucleotide sequence of the OsMS3 gene is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that: Using gene editing technology, the rice OsMS3 gene was knocked out and mutated.

10. The method according to claim 9, characterized in that: A recombinant gene CRISPR / Cas9 knockout vector containing the OsMS3 gene was constructed and transformed into wild-type rice to obtain rice male sterile line plants.

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