Gene osmate12 for regulating grain shape of rice, and encoding protein and application thereof

Abstract

The application discloses a kind of genes for regulating rice grain type OsMATE12 And its encoding protein and application, belong to molecular biology technical field.The application is constructed by CRISPR / Cas9 system OsMATE12 Gene knockout mutant OsMATE12-KO1 And OsMATE12-KO2 Then its phenotype is identified, and compared with wild type, OsMATE12-KO1 And OsMATE12-KO2 Husk cell quantity increases, but cell length and width reduce, and grain length also reduces.It is explained that OsMATE12 Gene affects grain length by regulating rice grain cell expansion and proliferation.The application provides new research material for rice MATE family gene research, and provides new genetic resources for rice molecular breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a gene for regulating grain type of rice OsMATE12 and an encoded protein and application thereof. BACKGROUND

[0002] Rice is the most important food crop in China, and its stable and improved yield directly affects the national food security. The three elements of rice yield are the number of grains per panicle, 1000-grain weight and effective panicle number, and the 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 type regulation genes have been reported. The regulation pathways of grain type mainly include G protein signaling, ubiquitin proteasome degradation, mitogen-activated protein kinase, plant hormone signaling and transcriptional regulation pathways.

[0003] G protein-coupled receptors are a general term for a class of protein receptors, which contain 7 transmembrane alpha-helix domains and an extracellular N-terminal and an intracellular C-terminal tail, and G proteins are composed of Gα, Gβ and Gγ subunits. GS3 encodes an atypical Gγ protein, which negatively regulates the size and weight of rice grains (Fan et al, 2006). The ubiquitin proteasome degradation pathway mainly includes ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase E3 and 26S proteasome complex. GW2 encodes a RING-type E3 ubiquitin ligase, which can negatively regulate cell division by conducting its substrate to the proteasome to regulate proteolysis, thereby increasing the number of lemma and palea cells, promoting grain width and weight increase (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 mitogen-activated protein kinase OsMAKK4, and the phenotype defects of smg1 mutants promote cell proliferation and exhibit erect panicles and short stature, and small grains (Duan et al, 2014). Plant hormones are a kind of trace organic molecules, which play an important role in plant growth, development, senescence, dormancy and stress resistance, and the common plant hormones regulating grain type in rice are cytokinin, auxin and brassinosteroid. Transcription factors play a key role in the process of plant growth and development. OsSPL16 / GW8 is a transcription factor containing SBP domain, which regulates rice grain width and can directly bind to the GW7 promoter and inhibit its expression (Wang et al, 2015). Transcription factor OsSPL13 positively regulates the size of lemma and palea cells, leading to increased length and weight of rice grains, and its high-level expression is related to large grains of tropical japonica rice, and further analysis shows that the large grain allele of OsSPL13 of tropical japonica rice is introduced from indica rice varieties by artificial selection (Si et al, 2016).

[0004] In summary, although many grain type regulatory genes or QTLs have been cloned, and some key signal pathways for regulating rice grain type have been determined, the regulatory network of grain type is still far from clear. Therefore, new grain type regulatory genes need to be continuously excavated, the molecular mechanisms thereof need to be clarified, the upstream and downstream relationships of each gene need to be determined, different regulatory pathways need to be gradually connected, and the grain type regulatory network needs to be continuously improved, so as to provide more detailed theoretical references for molecular design breeding of high-yield and high-quality rice. SUMMARY

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a kind of rice grain type regulatory gene OsMATE12 And its encoding protein and application, with a new kind of rice grain type regulatory gene.

[0006] The technical scheme for solving the above-mentioned technical problems of the present application is as follows: a kind of rice OsMATE12 Gene, rice OsMATE12 The nucleotide sequence of the coding region of the rice

[0007] The present application provides a kind of protein encoded by the above-mentioned rice OsMATE12 Gene, rice OsMATE12 The amino acid sequence of the protein encoded by the rice

[0008] The present application provides a kind of application of the above-mentioned rice OsMATE12 Gene in regulating rice grain type.

[0009] Further, the regulation of rice grain type is to knock out the rice OsMATE12 Gene, the length of rice grain is reduced.

[0010] Further, the regulation of rice grain type is to knock out the rice OsMATE12 Gene, the number of rice glume cells is increased.

[0011] Further, the regulation of rice grain type is to knock out the rice OsMATE12 Gene, the length and width of rice glume cells are reduced.

[0012] The present application provides a kind of preparation for regulating rice grain type, and the preparation contains the above-mentioned rice OsMATE12 Gene or the protein encoded by the above-mentioned rice OsMATE12 Gene.

[0013] The present application also provides a kind of preparation method of small-seeded rice variety, which is prepared by knocking out the rice OsMATE12 Gene to prepare small-seeded rice variety.

[0014] The present application has the following beneficial effects: a new MATE gene is obtained, and the gene is knocked out in japonica rice material Nip using a CRISPR / Cas9 system to construct OsMATE12 Gene knockout mutant OsMATE12-KO1 And OsMATE12- KO2 Then the phenotype is identified, and it is found that, compared with the wild type, OsMATE12-KO1 And OsMATE12-KO2 The number of hull cells is increased, but the cell length and width are reduced, and the grain length is also reduced. It is proved that OsMATE12 The gene affects grain length by regulating rice grain cell expansion and proliferation. The present application provides new research materials for rice MATE family gene research and new genetic resources for rice molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For rice OsMATE12 Gene CRISPR / Cas9 vector schematic diagram

[0016] Figure 2 For OsMATE12 Gene knockout target site and grain length phenotype

[0017] Figure 3 For wild type and OsMATE12 Knockout plant cell analysis

[0018] Figure 4 For wild type and OsMATE12 Analysis of the expression amount of cell cycle related genes in the hull of knockout plants

[0019] Figure 5 For OsMATE12 Gene expression profile and subcellular localization DETAILED DESCRIPTION

[0020] The following examples are only used to explain the present application and are not used to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0021] Example 1: OsMATE12 gene editing vector construction, transformation and plant phenotype observation

[0022] (1) OsMATE12 Gene sequence acquisition

[0023] The coding region of rice OsMATE12 gene and the amino acid sequence of the coded protein (as shown in SEQ ID NO. 1 and SEQ ID NO. 2) were obtained by using the database Rice Resource Center (http: / / ricerc.sicau.edu.cn).

[0024] (2) OsMATE12 Gene editing vector construction and transformation

[0025] The guide RNA (gRNA) sequence of the gene editing target site was designed by using the website (http: / / skl.scau.edu.cn / ), and after analysis, the target site was selected on the second exon, and the target site sequence was GCCGAGCGCCTACATCTCCG (SEQ ID NO. 3). The gRNA was constructed into the pYLCRISPR / Cas9 Pubi-H vector with the promoter of OsU6a (see OsMATE12 ), and the specific method was referred to (Yao-Guang Liu, Figure 1 , 2016). After the vector was successfully constructed, it was transformed, and the receptor material was japonica rice Nipponbare (Nip). The genetic transformation experiment was completed by Wuhan Boyuan Biotechnology Co., Ltd. Current Protocols in Molecular Biology (3)

[0026] (3) OsMATE12 Phenotype analysis of knockout mutant plants

[0027] The obtained transgenic plants were taken single leaf, and the DNA was extracted by CTAB method as a template. The amplification primers crossing the target site were designed, and the PCR reaction was carried out by using the DNA polymerase of GenScript. After the correct target band was obtained, the sequencing company was sent for sequencing. The amplification primer sequences are as follows:

[0028] F: TTCCACCGTGCTGCTCAC (SEQ ID NO. 4);

[0029] R: AGAACGCCTTGGACGAGA (SEQ ID NO. 5).

[0030] The obtained sequencing results were single peak single plant sequences which were compared by using the DNAMAN5 software (the control was the wild type Nip sequence), and two transgenic lines were obtained, which were named OsMATE12-KO1 and OsMATE12-KO2 , OsMATE12-KO1 and OsMATE12-KO2T and CATCGTGGCGCCGAGCGCCTACATCT (SEQ ID NO. 6), respectively, which caused the premature termination of translation at the 66th and 169th amino acids, respectively. Two transgenic lines were planted to T2 generation for phenotype observation. It was found that the grain length of both transgenic lines was significantly reduced compared with wild type Nipponbare (see Figure 2 ).

[0031] (4) OsMATE12 Cytological observation of the mutant glume

[0032] Nip and OsMATE12-KO1 The materials for glume development setting were fixed with 3% glutaraldehyde. In order to improve the fixation effect, the fixed samples were immediately vacuumed for 15 minutes, and then the fixed liquid was absorbed after the samples were fixed at 4°C for 3 hours. The samples were rinsed with 0.1 mol / L pH 6.8 phosphate buffer for 1 hour, and the rinsing liquid was replaced 3 times. After the liquid was absorbed, the samples were dehydrated and dried with ethanol gradient, i.e. 30%→50%→70%→80%→90%→100%→100%, and the samples were stopped in each ethanol for 30 min. The samples were replaced with isoamyl acetate for 3 times, each time for 20 min. The anthers of the glumes dried with anhydrous ethanol were dissected with a dissection needle, and the pollen grains were concentrated on a smooth paper sheet with a brush. After spraying gold, the samples were observed by scanning electron microscopy. In Nip and OsMATE12-KO1 The same position of the glume was photographed, and the cell size and number were statistically analyzed by ImageJ software. As can be seen from Figure 3 compared with the wild type, the cell number of OsMATE12-KO1 increased, and the cell length and width decreased.

[0033] (4) OsMATE12 Analysis of the expression amount of cell cycle related genes in the glume

[0034] Nip and OsMATE12-KO1 The 5-7 cm young panicles of the field grown materials were used to extract total RNA with the plant RNA extraction kit of Qiagen. The concentration of the extracted total RNA was determined by the instrument Nanodrop 2000, and then the RNA (500 ng) was reversely transcribed to obtain cDNA by using the reverse transcription kit (RT EasyTM II) of Qiagen. The PerfectStart® Green qPCR SuperMix kit of Quanta was used for quantitative PCR, and the real-time fluorescent quantitative PCR (RT-qPCR) analysis was performed by using the BIO-RAD CFX96TM Real-Time System quantitative PCR instrument. OsUBQ5 was used as an internal reference, and the primers are shown in Table 1.

[0035] As can be seen from Figure 4 compared with the wild type, the cell expansion genesEXPA5 , EXPA19 , EXPA24 , EXPB3 , EXPB6 and EXPB7 exist OsMATE12-KO1 Expression levels were significantly downregulated. In addition, cyclin genes... CYCA1;4 , CYCA3;1 , CYCD3;1 , CYCD4;1 , CYCD5;2 and CYCH1;1 exist OsMATE12-KO1 The levels were also significantly downregulated. These results indicate that... OsMATE12 It affects the expression of genes related to cell expansion and cell cycle, thereby regulating rice grain shape.

[0036] Table 1 Primer sequences for real-time quantitative PCR

[0037]

[0038] Example 2: OsMATE12 gene expression profile and subcellular localization

[0039] (1) OsMATE12 Gene expression profiling analysis

[0040] Different tissue samples were collected from rice at different developmental stages in the field. RNA extraction, reverse transcription, and real-time quantitative PCR were performed according to the method described in Example 1 of this invention. Wherein, R represents young roots; L1 represents seedling leaves; L2 represents leaves during the booting stage; YP3, YP6, and YP10 represent young panicles of 3cm, 6cm, and 10cm respectively; S represents stems during the grain-filling stage; and E5, E10, and E15 represent grains that have developed for 5, 10, and 15 days after fertilization. Figure 5 A indicates that... OsMATE12 The gene is mainly expressed in the leaves during the seedling stage, the leaves during the booting stage, and the young spikelets.

[0041] (2) OsMATE12 Protein subcellular localization

[0042] The constructed vector (OsMATE12:GFP, DG1:RFP) plasmid was transferred into Agrobacterium GV3101. Single colonies were picked and transferred to 10 mL of LB liquid medium containing two types of antibiotics, rifampicin and kanamycin sulfate, and cultured for 16 hours, and then acetyl eugenol (final concentration 50 μmol / L) and MES (2-morpholinoethanesulfonic acid, final concentration 10 mmol / L) were added. The bacterial cells were collected by centrifugation at room temperature for 15 min, and then 10 mL of MgCl2 solution (containing 10 mmol / L MES and 150 μmol / L acetyl eugenol) was used to gently resuspend the bacterial cells, and the bacterial cells were allowed to stand at room temperature for 2 h. Two types of bacterial liquid were mixed at a ratio of 1:1 to prepare a dipping liquid, and then a needle-free syringe was used to inject the liquid into tobacco leaves. The injected tobacco was cultured at room temperature in the dark for 2 days. The cultured tobacco leaves were placed on a glass slide, and then photographed under a laser confocal microscope (Leica, STELLARIS STED / EM CPD300, Germany). It was found that it was located on the cell membrane (see Figure 5 B}.

[0043] In the present application OsMATE12 The coding region and protein sequence of the gene are as follows:

[0044] (1) Coding region sequence:

[0045]

[0046] (2) Amino acid sequence:

[0047] MATTAASPEIAGAARLYVVGLIPQIFAYAANFPIQKFLQAQSIVAPSAYISAATLAAHVALSWFAVYKLGLGLLGASLILSLSWWVIVLAQFAYIVVSDRCRLTWAGFSSKAFSGLPEFLQLSAASAVMLCLETWYFQVTVLIAGLLKDPEIALDSLAVCMSISGWVFMVSVGFNAAASVRVSNELGAGNPRAAAFSVKVVTSLSLIVAAIIAAIVMCLREYLSYVFTQGEEVARAVSSMTPLLAVTIVLNGIQPVLSGVAVGCGWQAFVAYVNIGCYYIIGVPFGCVLGFHFDLGAMGIYGGMIVGLFVQTLILVYVTFRTDWNREVGEAKKRLNKWGDIAKPLLANED (SEQ ID NO. 2).

[0048] The above description is merely that of the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. Oryza sativa OsMATE12 application of the gene in regulating grain type in rice, characterized in that, The method comprises the following steps: introducing a gene into a rice plant to regulate the grain type of the rice plant, wherein the gene is a gene encoding a protein having the amino acid sequence shown as SEQ ID NO. 2; and the gene is a gene encoding a protein having the amino acid sequence shown as SEQ ID NO.

2. OsMATE12 After the gene is knocked out, the length of the rice grain is reduced, the number of pericarp cells of the rice is increased, and the length and width of the pericarp cells of the rice are reduced. OsMATE12 The nucleotide sequence of the coding region of the gene is shown as SEQ ID NO.

1. OsMATE12 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.

2.

2. A method of making a small-grain rice variety, the method comprising: A small grain rice variety is prepared by knocking out the rice OsMATE12 gene; the rice OsMATE12 OsMATE12 The nucleotide sequence of the coding region of the rice gene is shown as SEQ ID NO. 1.

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