Gene OsMATE12 for regulating and controlling rice grain shape as well as encoding protein and application of gene OsMATE12

Abstract

The invention discloses a gene OsMATE12 for regulating and controlling a rice grain shape as well as an encoding protein and application thereof, and belongs to the technical field of molecular biology. According to the invention, knockout mutants OsMATE12-KO1 and OsMATE12-KO2 of the OsMATE12 gene are constructed through a CRISPR / Cas9 system, then phenotypes of the knockout mutants OsMATE12-KO1 and OsMATE12-KO2 are identified, and it is found that compared with a wild type, the quantity of glume cells of the OsMATE12-KO1 and OsMATE12-KO2 is increased, but the cell length and width are reduced, and the grain length is also reduced. It is shown that the OsMATE12 gene affects the grain length by regulating and controlling expansion and proliferation of rice grain cells. A new research material is provided for rice MATE family gene research, and a new genetic resource is provided for rice molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a gene for regulating rice grain shape. OsMATE12 and its encoded protein and application. Background Art

[0002] Rice is the most important food crop in my country, and the stability and improvement of its yield directly affect national food security. The three elements of rice yield are the number of grains per panicle, 1000-grain weight and the number of effective panicles, among which 1000-grain weight is mainly affected by grain length, grain width and grain thickness. With the continuous development of genome sequencing and molecular biology technology, a large number of grain shape regulatory genes have been reported. The regulatory pathways of grain shape mainly include G protein signal transduction, ubiquitin proteasome degradation, mitogen-activated protein kinase, plant hormone signal transduction and transcriptional regulation pathways.

[0003] G protein-coupled receptors are a general term for a class of protein receptors, which contain 7 transmembrane α-helical domains, an extracellular N-terminal and an intracellular C-terminal tail. G proteins are composed of Gα, Gβ and Gγ subunits. GS3 encodes an atypical Gγ protein that has a negative regulatory effect on rice grain size and weight (Fan et al., 2006). The ubiquitin proteasome degradation pathway mainly includes ubiquitin, ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3 and 26S proteasome complex. GW2 encodes a RING-type E3 ubiquitin ligase, which can negatively regulate cell division by directing its substrate to the proteasome to regulate protein hydrolysis, thereby increasing the number of spikelet shell cells and promoting the increase of grain width and grain weight (Song et al., 2007). Mitogen-activated protein kinase is a member of the serine / threonine protein kinase family with 11 conserved domains, which transmits signals inside and outside the cell. SMG1 encodes the mitogen-activated protein kinase OsMAKK4. The phenotypic defects of smg1 mutants promote cell proliferation and present an erect panicle and shorter plant height, with smaller grains (Duan et al., 2014). Plant hormones are trace organic molecules that play an important role in plant growth, development, aging, dormancy and stress resistance. Common plant hormones that regulate grain shape in rice are cytokinins, auxins and brassinolide. Transcription factors play a key role in the process of plant growth and development. OsSPL16 / GW8 is a transcription factor containing an SBP domain that regulates rice grain width and can directly bind to the GW7 promoter and inhibit its expression (Wang et al., 2015). The transcription factor OsSPL13 positively regulates the size of lemma cells, leading to increased rice grain length and weight. Its high level of expression is associated with large grains in tropical japonica rice. Further analysis showed that the large-grain allele of OsSPL13 in tropical japonica rice was introgressed from indica rice varieties through artificial selection (Si et al., 2016).

[0004] In summary, although many grain shape regulatory genes or QTLs have been cloned and some key signal pathways regulating rice grain shape have been identified, the regulatory network of grain shape is still far from clear. Therefore, it is necessary to continuously explore new grain shape regulatory genes, clarify their regulatory molecular mechanisms, and clarify the upstream and downstream relationships of each gene, so as to gradually establish connections between different regulatory pathways and continuously improve the grain shape regulatory network, thereby providing a more detailed theoretical reference for molecular design breeding of high-yield and high-quality rice. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the present invention aims to provide a gene for regulating rice grain shape. OsMATE12 Its encoded protein and application, as a new gene that can regulate rice grain shape.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a rice OsMATE12 Gene, rice OsMATE12 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.1.

[0007] The present invention provides a rice OsMATE12 Gene encoding protein, rice OsMATE12 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0008] The present invention provides a rice OsMATE12 Application of genes in regulating rice grain shape.

[0009] Further, regulating rice grain shape is a knockout rice OsMATE12 The length of rice grains was reduced after the gene was modified.

[0010] Further, regulating rice grain shape is a knockout rice OsMATE12 The number of rice husk cells increased after the gene was added.

[0011] Further, regulating rice grain shape is a knockout rice OsMATE12 The length and width of rice husk cells were reduced after the gene was expressed.

[0012] The present invention provides a preparation for regulating rice grain shape, the preparation comprising the above-mentioned rice OsMATE12 Gene or the above rice OsMATE12 Protein encoded by a gene.

[0013] The present invention also provides a method for preparing a small-grain rice variety by knocking out the rice OsMATE12 Genes to produce small-grain rice varieties.

[0014] The present invention has the following beneficial effects: the present invention obtains a new MATE gene and knocks it out in the japonica rice material Nip using the CRISPR / Cas9 system to construct OsMATE12 Knockout mutants OsMATE12-KO1 and OsMATE12- KO2 , and then its phenotype was identified and found that compared with the wild type, OsMATE12-KO1 and OsMATE12-KO2 The number of husk cells increased, but the cell length and width decreased, and the kernel length also decreased. OsMATE12 The gene affects the grain length by regulating the expansion and proliferation of rice grain cells. The invention provides new research materials for the study of rice MATE family genes and new genetic resources for rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For rice OsMATE12 Schematic diagram of gene CRISPR / Cas9 vector; Figure 2 for OsMATE12 Gene knockout target site and grain length phenotype; Figure 3 For wild type and OsMATE12 Glume cytological analysis of knockout plants; Figure 4 For wild type and OsMATE12 Analysis of cell cycle-related gene expression in the glumes of knockout plants; Figure 5 for OsMATE12 Gene expression profile and subcellular localization. DETAILED DESCRIPTION

[0016] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] Example 1: Construction of OsMATE12 gene editing vector, transformation and plant phenotype observation (1) OsMATE12 Gene sequence acquisition The rice OsMATE12 gene coding region and the amino acid sequence of the encoded protein (as shown in SEQ ID NO.1 and SEQ ID NO.2) were obtained using the database Rice Resource Center (http: / / ricerc.sicau.edu.cn).

[0018] (2) OsMATE12Gene editing vector construction and transformation The guide RNA (gRNA) sequence of the gene editing target site was designed using the website (http: / / skl.scau.edu.cn / ), and the target site was selected after analysis. OsMATE12 On the second exon, the target site sequence was GCCGAGCGCCTACATCTCCG (SEQ ID NO. 3), and the promoter of OsU6a was selected to construct the gRNA into the pYLCRISPR / Cas9 Pubi-H vector with plant resistance to hygromycin (see Figure 1 ), the specific method refers to (Yao-Guang Liu, Current Protocols in Molecular Biology , 2016). After the vector was successfully constructed, it was transformed. The recipient material was japonica rice Nipponbare (Nip). The genetic transformation experiment was completed by Wuhan Boyuan Biotechnology Co., Ltd.

[0019] (3) OsMATE12 Phenotypic analysis of knockout mutant plants For the transgenic plants obtained, a single leaf was taken and DNA was extracted using the CTAB method as a template. Amplification primers across the target site were designed, and PCR reaction was performed using DNA polymerase from Bioengineering. After the correct target band was obtained, it was sent to a sequencing company for sequencing; the amplification primer sequences are as follows: F: TTCCACCGTGCTGCTCAC (SEQ ID NO.4); R: AGAACGCCTTGGACGAGA (SEQ ID NO. 5).

[0020] The obtained sequencing results were single-peaked single plant sequences, which were aligned by DNAMAN5 software (the control was the wild-type Nip sequence), and two transgenic lines were obtained. The two lines were named OsMATE12-KO1 and OsMATE12-KO2 , OsMATE12-KO1 and OsMATE12-KO2 The deletions were T and CATCGTGGCGCCGAGCGCCTACATCT (SEQ ID NO.6), respectively. The deletions caused the premature termination of translation of the amino acids encoded by the genes after positions 66 and 169. The two lines were planted to the T2 generation for phenotypic observation, and it was found that the grain length of the two transgenic lines was significantly reduced compared with the wild-type Nipponbare (see Figure 2 ).

[0021] (4) OsMATE12 Cytological observation of glume of knockout mutant Take Nip and OsMATE12-KO1The materials with lemma development and finalization were fixed with 3% glutaraldehyde. To improve the fixation effect, the fixed samples were immediately vacuumed for 15 minutes, and then fixed at 4°C for 3 hours before the fixative was aspirated. The fixative was added with 0.1 mol / L pH 6.8 phosphate buffer and rinsed for 1 hour, during which the rinsing solution was changed 3 times. After the liquid was aspirated, it was dehydrated and dried with ethanol gradient, i.e. 30%→50%→70%→80%→90%→100%→100%, and the sample stayed in each level of ethanol for 30 minutes. Isoamyl acetate was replaced 3 times, each time for 20 minutes. After the lemma was dried with anhydrous ethanol, the lemma and anther were cut open with a dissecting needle, and the pollen grains were collected on a smooth paper with a brush, and observed by scanning electron microscopy after gold spraying. In Nip and OsMATE12-KO1 The photos were taken at the same position of the husk, and the cell size and number were statistically analyzed using ImageJ software. Figure 3 Compared with the wild type OsMATE12-KO1 The number of cells increased and the length and width of cells decreased.

[0022] (4) OsMATE12 Analysis of cell cycle-related gene expression in glume Take field-grown Nip and OsMATE12-KO1 Total RNA was extracted from young panicles of 5-7 cm using the Plant RNA Extraction Kit of Fuji Company; the concentration of the extracted total RNA was measured using the instrument Nanodrop 2000, and then RNA (500 ng) was reverse transcribed using the reverse transcription kit (RT EasyTM II) of Fuji Company to obtain cDNA. Quantitative PCR used the PerfectStart® Green qPCR SuperMix Kit of Quanshijin Company, and real-time fluorescence quantitative PCR (RT-qPCR) analysis was performed using the BIO-RAD CFX96TM Real-TimeSystem quantitative PCR instrument. OsUBQ5 was used as an internal reference, and the primers are shown in Table 1.

[0023] Depend on Figure 4 Compared with the wild type, the cell expansion gene EXPA5 , EXPA19 , EXPA24 , EXPB3 , EXPB6 and EXPB7 exist OsMATE12-KO1 In addition, the expression of cell cycle protein genes CYCA1;4 , CYCA3;1 , CYCD3;1 , CYCD4;1 , CYCD5;2 and CYCH1;1 exist OsMATE12-KO1 These results indicate that OsMATE12It affects the expression of cell expansion and cell cycle related genes, thereby regulating rice grain shape.

[0024] Table 1 Primer sequences for real-time fluorescence quantitative PCR

[0025] Example 2: OsMATE12 gene expression profile and subcellular localization (1) OsMATE12 Gene expression profiling Different tissue samples of rice at different developmental stages were collected in the field, and RNA extraction, reverse transcription and real-time fluorescence quantitative release were performed according to the method in Example 1 of the present invention; wherein R is a young root; L1 is a leaf at the seedling stage; L2 is a leaf at the booting stage; YP3, YP6 and YP10 are young ears of 3 cm, 6 cm and 10 cm; S is a stem at the filling stage; E5, E10 and E15 are caryopsis developed 5, 10 and 15 days after fertilization. Figure 5 As we know, OsMATE12 The gene was mainly expressed in leaves at the seedling stage, leaves at the booting stage and young ears.

[0026] (2) OsMATE12 Protein subcellular localization The constructed vector (OsMATE12:GFP, DG1:RFP) plasmid was transferred into Agrobacterium GV3101. A single colony was picked and transferred to 10 mL LB liquid medium containing two types of antibiotics, rifampicin and kanamycin sulfate, and cultured for 16 hours. Acetosyringone (final concentration 50μmol / L) and MES (2-morpholineethanesulfonic acid, final concentration 10mmol / L) were added. The bacteria were collected by centrifugation at room temperature for 15 minutes, and the bacteria were gently resuspended in 10mL MgCl2 solution (containing 10mmol / L MES and 150μmol / L acetosyringone) and allowed to stand at room temperature for 2 hours. The two bacterial solutions were prepared into an infiltration solution in a 1:1 ratio, and then a needleless syringe was used to inoculate tobacco leaves. The injected tobacco was cultured at room temperature in the dark for 2 days. The cultured tobacco leaves were placed on a slide and photographed under a laser confocal microscope (Leica, STELLARIS STED / EM CPD300, Germany), and it was found that it was located on the cell membrane (see Figure 5 B).

[0027] In the present invention OsMATE12 The coding region and protein sequences of the genes are as follows: (1) Coding region sequence: (2) Amino acid sequence: MATTAASPEIAGAARLYVVGLIPQIFAYAANFPIQKFLQAQSIVAPSAYISAATLAAHVALSWFAVYKLGLGLLGASLILSLSWWVIVLAQFAYIVVSDRCRLTWAGFSSKAFSGLPEFLQLSAASAVMLCLETWYFQVTVLIAGLLKDPEIALDSLAVCMSISGWVFMVSVGFNAA ASVRVSNELGAGNPRAAAFSVKVVTSLSLIVAAIIAAIVMCLREYLSYVFTQGEEVARAVSSMTPLLAVTIVLNGIQPVLSGVAVGCGWQAFVAYVNIGCYYIIGVPFGCVLGFHFDLGAMGIYGGMIVGLFVQTLILVYVTFRTDWNREVGEAKKRLNKWGDIAKPLLANED (SEQ ID NO.2).

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kind of rice OsMATE12 A gene characterized by The rice OsMATE12 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.

1.

2. The rice according to claim 1 OsMATE12 The protein encoded by the gene is characterized in that The rice OsMATE12 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The rice according to claim 1 OsMATE12 Application of genes in regulating rice grain shape.

4. The use according to claim 3, characterized in that: The rice grain type is regulated by knockout rice OsMATE12 The length of rice grains was reduced after the gene was modified.

5. The use according to claim 3, characterized in that: The rice grain type is regulated by knockout rice OsMATE12 The number of rice husk cells increased after the gene was added.

6. The use according to claim 3, characterized in that: The rice grain type is regulated by knockout rice OsMATE12 The length and width of rice husk cells were reduced after the gene was expressed.

7. A preparation for regulating rice grain shape, characterized in that: The preparation comprises the rice according to claim 1 OsMATE12 The gene or the rice according to claim 2 OsMATE12 Protein encoded by a gene.

8. A method for preparing a small-grain rice variety, characterized in that: Knockout rice OsMATE12 Genes to produce small-grain rice varieties.

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