Application of rice fertility gene MS2

Abstract

The invention discloses application of a rice fertility gene MS2. A gene MS2 for regulating and controlling rice fertility is found in a rice genome database, high expression is achieved in the rice anther development period, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO.2. Researches show that the MS2 gene is related to rice fertility and affects rice pollen fertility, the MS2 gene is knocked out from wild rice, so that the rice pollen fertility is remarkably reduced, and the MS2 gene can be applied to creation of rice sterile lines and preparation of hybrid seeds. Meanwhile, the male sterility line generated by MS2 gene mutation is not influenced by environmental conditions such as illumination and temperature, the fertility is stable, a new gene resource is provided for constructing a rice crossbreeding system, and the germplasm resource of the male sterility line is expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding and genetic engineering, and more specifically, relates to an application of rice fertility gene MS2. Background Art

[0002] Rice (Oryza sativa L.) is one of the three major food crops in the world, providing food for nearly 50% of the world's population. Rice is the largest staple food crop in my country, accounting for one-third of the annual grain planting area, so rice production is directly related to our country's food security. The "Green Revolution" of rice dwarfing breeding in the 1960s and the "Second Green Revolution" of hybrid breeding have greatly increased rice production. Hybrid rice has genetic differences and complementarity. It not only has advantages in yield, but also shows obvious advantages over ordinary varieties in terms of stress resistance and disease resistance. However, hybrid rice breeding faces challenges such as high seed production costs and complex technical requirements, especially the lack of germplasm resources of male sterile lines.

[0003] The selection and breeding of male sterile lines is a key link in hybrid seed production. Male sterility diversity is an effective way to ensure the diversity of hybrid rice germplasm and prevent production risks caused by germplasm homogeneity. At present, male sterility can be divided into cytoplasmic male sterility (CMS) and genic male sterility (GMS) according to different gene sources and inheritance modes. CMS is widely present in higher plants. It is generally caused by loss of function or abnormality caused by mitochondrial gene mutations. It is manifested as maternal inheritance, normal pistil function, but pollen abortion. In most cases, male sterility can be restored by the fertility restoration gene (Rf) encoded in the nucleus. Unlike CMS caused by cytoplasmic sources (generally mitochondrial gene abnormalities), male sterility caused by gene abnormalities from the nucleus is called GMS. The sterile lines constructed using recessive genic male sterility genes can remove the constraints of environmental factors on hybrid breeding and eliminate potential risks in production. They are also suitable for most varieties, which can greatly improve the utilization of hybrid vigor resources and solve the problem of resource utilization of hybrid vigor.

[0004] Since there is still a lack of genetic resources that can be used to construct male sterile lines, it is necessary to develop more genes related to rice fertility, which is of great significance to the creation of rice sterile lines. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the insufficiency of the existing gene resources for constructing male sterile lines and to provide an application of a rice fertility gene MS2.

[0006] The purpose of the present invention is to provide application of rice MS2 gene.

[0007] Another object of the present invention is to provide the use of an agent for knocking out or inhibiting the expression of rice MS2 gene.

[0008] Another object of the present invention is to provide a method for changing rice breeding properties or cultivating / creating rice male sterile lines.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention found a gene that regulates rice fertility in the rice genome database, and obtained the MS2 (MALE STERILITY 2) gene that is highly expressed during the rice anther development period through bioinformatics analysis and screening. This gene is not a light-temperature sensitive gene, and its nucleotide sequence 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 MS2 gene is related to rice fertility and will affect rice pollen fertility. Knocking out this gene in wild-type rice can significantly reduce rice pollen fertility, and can be better applied to the creation of rice sterile lines and the preparation of hybrid seeds. The male sterile line produced by the MS2 gene mutation provided by the present invention has stable fertility and is not affected by environmental conditions. It can help produce rice sterile lines that are not affected by light and temperature, and provides new gene resources for the construction of more rice hybrid breeding systems, while also expanding the germplasm resources of male sterile lines.

[0011] Therefore, the present invention provides the use of rice MS2 gene in negatively regulating rice fertility.

[0012] The invention provides application of rice MS2 gene in reducing pollen fertility.

[0013] The present invention provides an application of rice MS2 gene as a target in cultivating / creating rice male sterile line plants, specifically knocking out the MS2 gene in rice.

[0014] The present invention provides application of an agent for knocking out or inhibiting rice MS2 gene expression in reducing rice anther fertility.

[0015] The present invention provides application of an agent for knocking out or inhibiting rice MS2 gene expression in preparing a product for reducing rice anther fertility.

[0016] The present invention provides application of a reagent for knocking out or inhibiting rice MS2 gene expression in cultivating / creating rice male sterile line plants.

[0017] Preferably, the reagent is a plasmid, a vector, or a recombinant bacterium containing a knockout or suppressed rice MS2 gene expression.

[0018] More preferably, the reagent is a rice MS2 gene CRISPR / Cas9 knockout vector, or a recombinant bacterium containing the knockout vector.

[0019] Furthermore, the primer set used to construct the rice MS2 gene CRISPR / Cas9 knockout vector includes Primer1 (SEQ ID NO.3) / Primer2 (SEQ ID NO.4), Primer3 (SEQ ID NO.5) / Primer4 (SEQ ID NO.6).

[0020] The present invention provides a method for changing rice breeding or cultivating / creating rice male sterile lines, knocking out or inhibiting the expression of rice fertility gene MS2 to obtain plants with reduced fertility.

[0021] Preferably, gene editing technology is used to knock out and mutate the rice fertility gene MS2 to obtain plants with reduced fertility.

[0022] More preferably, a recombinant gene CRISPR / Cas9 knockout vector containing the MS2 gene target sequence is constructed and transformed into wild-type rice to obtain plants with reduced fertility.

[0023] Further preferably, the method for constructing the CRISPR / Cas9 knockout vector is: inserting the target fragment of gene MS2 into the BsaI recombination site of the pYLCRISPR / Cas9Pubi-H vector to obtain a recombinant plasmid.

[0024] The present invention has the following beneficial effects:

[0025] The present invention found a gene that regulates rice fertility in the rice genome database, and obtained the MS2 (MALE STERILITY 2) gene that is highly expressed during the rice anther development period through bioinformatics analysis and screening. The gene is not a light-temperature sensitive gene, and its nucleotide sequence 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 MS2 gene is related to rice fertility, and the gene can affect rice pollen fertility. Knocking out the gene in wild-type rice can significantly reduce rice pollen fertility, which can be better applied to the creation of rice sterile lines and the preparation of hybrid seeds. At the same time, the decrease in pollen fertility caused by the MS2 gene mutation is not affected by environmental conditions such as light and temperature, which provides new gene resources for the construction of more rice hybrid breeding systems, and also expands the germplasm resources of male sterile lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The expression pattern of the MS2 candidate gene (a) and the quantitative analysis results at different stages of anther development (b).

[0027] Figure 2 Heat map of male fertility-related genes screened for expression analysis based on public databases.

[0028] Figure 3 Design of two CRISPR / Cas9 knockout targets in the MS2 gene exon and sequencing results of target editing.

[0029] Figure 4 The plant morphology of the wild-type rice variety Tianfeng B (WT) and the ms2-ko mutant at the heading stage.

[0030] Figure 5 These are the anther morphology observation pictures of wild-type Tianfeng B (WT) and ms2-ko mutant.

[0031] Figure 6 This is the result of iodine staining analysis of pollen of wild-type Tianfeng B (WT) and ms2-ko mutant.

[0032] Specific implementation party

[0033] 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.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] Example 1 Obtaining Rice Fertility-Related Genes and Constructing Knockout Vectors

[0036] 1. Acquisition of rice genes

[0037] First, the RiceXPro rice database (https: / / ricexpro.dna.affrc.go.jp / ) was used to search and screen genes specifically expressed in rice anthers, such as Figure 1 Then the collected genes were clustered and analyzed based on gene expression and a heat map was drawn, as shown in Figure 2 As shown, a gene with a high expression level during the anther development period of rice was screened out, which is not a light-temperature sensitive gene and is named MS2 (MALE STERILITY 2). The nucleic acid sequence of the MS2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0038] 2. Quantitative analysis

[0039] According to the nucleic acid sequence of the MS2 gene, the primer pair Primer5 (SEQ ID NO.7): 5'-GAATCTGGCGCTCCACCCCT-3', Primer6 (SEQ ID NO.8): 5'-ACAGCGTCCCTGATGCTGC-3' was designed and synthesized, and the selected target gene was quantitatively analyzed. The cDNA of rice samples from different periods (SC: sporulation period; MMC: sporocyst period; Meiosis: meiosis period; EM: early mononucleate period; LM: late mononucleate period; BCP: binucleate development period) was diluted 10 times as a template to determine the expression status of the target gene. The rice gene OsUFC1 was selected as an internal reference. Three parallel control experiments were set up for each group of samples each time. The specific expression of the target gene was analyzed using the relative quantitative method; the relative value of the target gene to UFC1 was calculated using the expression of UFC1 as a reference, and the calculation method was 2 -△Ct The relative expression value was calculated as 2 -△Ct (2- [Cq目的-Cq内参] ).

[0040] The amplification system of RT-quantitative PCR is as follows: 10μL 2×ChamQ Universal SYBR qPCR MasterMix, 0.5μL 10μM Forward Primer, 0.5μL 10μM Reverse Primer, 1μL template cDNA, ddH2O supplement system to 20μL. Reaction program: 94℃ reaction for 10min, 1cycle (pre-denaturation); 94℃ reaction for 10s (denaturation), 60℃ reaction for 15s (annealing), 72℃ reaction for 15s (extension), 40cycles; the temperature range of the melting curve is set to gradually increase from 65℃ to 94℃, and in this process, the value of the fluorescence signal is recorded every 0.5℃ increase.

[0041] Quantitative test results such as Figure 1 As shown in b, the MS2 gene is expressed in multiple stages of anther development, with the highest expression level in the sporangium stage.

[0042] 3. Construction of knockout vector

[0043] Double target knockout was performed on two sites T1 (SEQ ID NO.9): 5'-GATGGACACGATCCTCCACTTGG-3', and T2 (SEQ ID NO.10): 5'-CCTCATCGACGGCGACCTCTTCC-3' on the 2nd and 6th exons of the MS2 gene, such as Figure 3 As shown, the two targets have a common feature, with NGG (N is any base of A, T, C, or G) at the 3' end.

[0044] Two pairs of primers were designed and synthesized for these two target sites: Primer1 (SEQ ID NO.3): 5'-ATGGACACGATCCTCCACTgtttcagagctagaaat-3', Primer2 (SEQ ID NO.4): 5'-AGTGGAGGATCGTGTCCATCggcagccaagccagca-3'; Primer3 (SEQ ID NO.5): 5'-GAAGAGGTCGCCGTCGATGgtttcagagctagaaat-3', Primer4 (SEQ ID NO.6): 5'-CATCGACGGCGACCTCTTCCggcagccaagccagca-3'.

[0045] Referring to the method for constructing the pYLCRISPR / Cas9Pubi-H vector (Ma X, Zhang Q, Zhu Q, et al. Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8 (8): 1274-1284), a CRISPR / Cas9 vector pYLCRISPR / Cas9Pubi-MS2 containing the above two targets was constructed. The recombinant plasmid pYLCRISPR / Cas9Pubi-MS2 was transformed into the Agrobacterium EHA105 strain by electroporation to obtain a recombinant strain, and the plasmid was extracted for PCR and enzyme digestion identification. The recombinant strain with correct PCR and enzyme digestion identification was named pYLCRISPR / Cas9Pubi-MS2.

[0046] Using the genetic transformation method of Agrobacterium infecting mature rice embryos (Nishimura A, Aichi I, Matsuoka M. A protocol for Agrobacterium-mediated transformation in rice. Nature Protocols. 2006, 1(6): 2796-2802), pYLCRISPR / Cas9Pubi-MS2 was transformed into the indica rice variety Tiangfeng B (TFB; derived from germplasm preserved in this laboratory) to obtain transgenic T0 generation plants with MS2 gene knockout.

[0047] Example 2 Identification and decoding analysis of editing sites in transgenic plants

[0048] The T0 generation plant genomic DNA obtained in Example 1 was extracted by the SDS microgenomic DNA extraction method. The genomic DNA was used as a template to design an upstream detection primer Primer7 (SEQ ID NO.11): 5'-CTGACGCTAACCTCGACAAG-3' in the CDS region of the Cas9 protein on pYLCRISPR / Cas9Pubi-MS2, and a downstream detection primer Primer8 (SEQ ID NO.12): 5'-CCGATCTAGTAACATAGATGACACC-3' was designed on the NOS terminator. PCR amplification was then performed using Primer7 and Primer8 primers to detect whether the T0 generation plants carried the transgene.

[0049] PCR reaction system: DNA (20 ng / μL) 1μL, Primer7 (10 pmol / μL) 0.4μL, Primer8 (10 pmol / μL) 0.4μL, 2×Buffer 10μL, dNTP (10 mM) 0.4μL, rTaq (5 U / μL) 0.4μL, add ddH2O to 20μL. PCR amplification program: 94℃3min; 94℃30sec, 56℃30sec, 72℃1min, 30 cycles; 72℃5min.

[0050] The test results showed that all T0 generation plants carrying transgenic markers were successfully edited.

[0051] Subsequently, primers were designed upstream and downstream of the two target positions of the genomic sequence corresponding to the MS2 gene, including the upstream primer Primer9 (SEQ ID NO.13) 5'-CCATGATAGCCAAACCCTAG-3' and the downstream primer Primer10 (SEQ ID NO.14) 5'-GGATGAGGAGGAAGTGAAGG-3' for the first target, and the upstream primer Primer11 (SEQ ID NO.15) 5'-GAGGATCGCATCAGCGCCCT-3' and the downstream primer Primer12 (SEQ ID NO.16) 5'-CGACAGCGTCCCTGATGCT-3' for the second target.

[0052] The T0 generation plant genomic DNA was used as a template for amplification. The PCR 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, add ddH2O to 20 μL. PCR amplification program: 94℃3min; 94℃30sec, 56℃30sec, 72℃30sec, 30 cycles; 72℃5min. After amplification, Sanger sequencing was performed to detect mutations at different target sites.

[0053] Then, the CRISPR-GE online tool was used to decode the sequencing peak graph (Liu, Weizhi, Xianrong Xie, Xingliang Ma, Jun Li, Jie-hu Chen and Yaoguang Liu. "DSDecode: A Web-Based Tool for 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.). The sequencing results are as follows: Figure 3 As shown, frameshift mutations caused by deletion / insertion were found in different target sites, and a transgenic strain with MS2 gene knockout was successfully constructed.

[0054] Example 3 Phenotypic analysis of transgenic plants

[0055] The transgenic T0 plants with MS2 gene knockout and the wild-type Tianfeng B were planted in the Rice Experimental Station of South China Agricultural University. The water and fertilizer conditions were consistent during the planting period, and the pollen of the plants after flowering was observed by I2-KI staining.

[0056] The results showed that there was no significant difference in plant type between the transgenic plants of pYLCRISPR / Cas9Pubi-MS2 and the wild-type plants at the heading stage. Figure 4 There is no obvious difference in anther morphology between different plants, such as Figure 5 However, the pollen vigor of the transgenic plants was reduced, and the phenotype of decreased pollen fertility appeared, such as Figure 6As shown, knocking out the MS2 gene significantly reduces the fertility of rice anthers, which can be used to create rice sterile lines. Since the MS2 gene is not a light-temperature sensitive gene, the sterile line constructed by the MS2 gene mutation has stable fertility and is not affected by the environment. Knocking out the MS2 gene can be used to prepare rice male sterile lines, helping to produce more rice sterile lines that are not affected by light and temperature, and providing more methods for constructing sterile lines with recessive nuclear sterile genes.

[0057] In summary, the present invention discovered a gene that regulates rice fertility. The MS2 gene, which is highly expressed during the rice anther development period, was screened through bioinformatics analysis. After knocking out the gene, the rice anther fertility was significantly reduced, which can be better applied to the creation of rice sterile lines and the preparation of hybrid seeds. At the same time, the sterile lines constructed by MS2 gene mutation have reduced pollen fertility, which can help to construct more male sterile line materials and provide new gene resources for rice hybrid breeding.

[0058] 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 rice MS2 gene in negatively regulating rice fertility, characterized in that: The nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

2. Application of rice MS2 gene in reducing pollen fertility, characterized in that: The nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

3. The use of rice MS2 gene as a target in the cultivation / creation of rice male sterile plants, characterized in that: The MS2 gene is knocked out in rice; the nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

4. Use of an agent for knocking out or inhibiting rice MS2 gene expression in reducing rice anther fertility, characterized in that: The nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

5. Use of an agent for knocking out or inhibiting the expression of rice MS2 gene in the preparation of a product for reducing rice anther fertility, characterized in that: The nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

6. Use of an agent for knocking out or inhibiting rice MS2 gene expression in cultivating / creating rice male sterile plants, characterized in that: The nucleotide sequence of the MS2 gene is shown in SEQ ID NO.

1.

7. The use according to any one of claims 4 to 6, characterized in that: The reagent is a plasmid, a vector or a recombinant bacterium containing a knockout or inhibited rice MS2 gene expression.

8. A method for changing rice breeding properties or cultivating / creating rice male sterile lines, characterized in that: Knocking out or inhibiting the expression of the rice fertility gene MS2 can obtain plants with reduced fertility.

9. The method according to claim 8, characterized in that: Using gene editing technology, the rice fertility gene MS2 was knocked out and mutated to obtain plants with reduced fertility.

10. The method according to claim 9, characterized in that: A recombinant gene CRISPR / Cas9 knockout vector containing the MS2 gene target sequence was constructed and transformed into wild-type rice to obtain plants with reduced fertility.

Images