Gene OsMATE16 for regulating and controlling cracking of rice anther as well as encoding protein and application of gene OsMATE16

Abstract

The invention discloses a gene OsMATE16 for regulating and controlling cracking of rice anther as well as an encoding protein and application thereof, and belongs to the technical field of molecular biology. According to the invention, knockout mutant OsMATE16-KO1 and OsMATE16-KO2 plants of the OsMATE16 gene are constructed through a CRISPR / Cas9 system, then phenotypes of the OsMATE16-KO1 and OsMATE16-KO2 plants are identified, it is found that compared with ZH11, the anther cracking rate and the maturing rate of the OsMATE16-KO1 and the OsMATE16-KO2 are both reduced, and it is indicated that the OsMATE16 gene influences the rice yield by regulating and controlling rice anther cracking. 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 anther dehiscence. OsMATE16 and its encoded protein and application. Background Art

[0002] Grain setting rate is one of the main factors affecting rice yield, and rice anther dehiscence has an important influence on grain setting. In the late stage of stamen development, anther dehiscence releases mature pollen grains for pollination, fertilization and seed production. In flowering plants, this process is strictly regulated so that anther dehiscence, pollen tube maturation and elongation proceed synchronously to promote pollen-stigma interaction and achieve fertilization at the optimal time. At present, the research progress of rice anther dehiscence is mainly focused on the plant hormone pathway, mainly auxin (IAA) and jasmonic acid (JA). In the late stage of anther development, OsFTIP7 mediates the nuclear and cytoplasmic distribution of OSH1, thereby directly inhibiting the auxin biosynthesis gene OsYUCCA4, thereby downregulating the level of auxin, promoting anther dehiscence, and ultimately controlling the fertility of rice (Song et al, 2018). DAO encodes a predicted 2-oxoglutarate and ferrous iron-dependent dioxygenase responsible for oxidizing active IAA to inactive OxIAA. Its mutants have high auxin content, resulting in anther indehiscence (Zhao et al., 2013). In addition, studies have found that the OsTIE1-OsTCP1 module plays a key role in anther development by finely regulating JA biosynthesis (Fang et al., 2024). OsJAR1 is a jasmonic acid-amino acid synthase that is involved in JA-regulated rice anther dehiscence (Xiao et al., 2014).

[0003] In summary, previous studies have reported several genes that can regulate rice anther dehiscence through IAA and JA pathways, but there have been no reports of MATE family genes regulating anther dehiscence. With the development of gene editing technology, researchers can use genetic engineering to create excellent new varieties. Therefore, finding new genes that affect rice fruiting rate can provide a theoretical basis and new gene resources for breeding new rice varieties. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the present invention aims to provide a gene for regulating rice anther dehiscence. OsMATE16 and its encoded protein and application, so as to provide a new gene for regulating rice anther dehiscence.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a rice OsMATE16 Gene, rice OsMATE16 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.1.

[0006] The present invention provides a rice OsMATE16 Gene encoding protein, rice OsMATE16 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0007] The present invention provides a rice OsMATE16 Application of genes in regulating anther dehiscence in rice.

[0008] Further, regulating rice anther dehiscence is a new method for knocking out rice OsMATE16 The anther dehiscence rate of rice was reduced after the gene was mutated.

[0009] Further, regulating rice anther dehiscence is a new method for knocking out rice OsMATE16 The rice seed setting rate is reduced after the gene is modified.

[0010] The present invention also provides a preparation for regulating rice anther dehiscence, the preparation comprising the above-mentioned rice OsMATE16 Gene or the above rice OsMATE16 Protein encoded by a gene.

[0011] The present invention has the following beneficial effects: The present invention constructs a CRISPR / Cas9 system OsMATE16 Knockout mutants OsMATE16-KO1 and OsMATE16-KO2 The plants were then phenotyped and found to be OsMATE16-KO1 and OsMATE16-KO2 The anther dehiscence rate and fruit setting rate of the rice varieties were both reduced. OsMATE16 The gene affects rice yield by regulating rice anther dehiscence. The invention provides new research materials for rice MATE family gene research and new genetic resources for rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 for OsMATE16 Schematic diagram of gene CRISPR / Cas9 vector; Figure 2 for OsMATE16 Gene knockout target site and plant phenotype diagram; Figure 3 For ZH11 and OsMATE16 Observation of anther dehiscence in knockout plants; Figure 4 for OsMATE16 Gene expression profiling. DETAILED DESCRIPTION

[0013] 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.

[0014] Embodiment 1: OsMATE16 Construction of gene knockout vector, transformation and plant phenotype observation (1) OsMATE16 Gene sequence acquisition Using the Rice Resource Center database ( http: / / ricerc.sicau.edu.cn ), obtain rice OsMATE16 The coding region of a gene and the amino acid sequence of the encoded protein.

[0015] (2) OsMATE16 Gene 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 gRNA was constructed into the pYLCRISPR / Cas9 Pubi-H vector with plant resistance to hygromycin (see Figure 1 ), for specific methods, please refer to (Yao-Guang Liu, Current Protocols in Molecular Biology , 2016), the gene editing vector transformation material recipient was japonica rice Zhonghua 11 (ZH11), and the genetic transformation experiment was completed by BioGene Company.

[0016] (3) OsMATE16 Phenotypic analysis of knockout mutant plants For the transgenic plants obtained, the leaves of individual plants were taken and DNA was extracted using the CTAB method as a template. Amplification primers across the target sites were designed. After amplification using the DNA of individual plants as a template, the amplification was sent to Sangon Biotech Co., Ltd. for sequencing. The sequences of the amplification primers are as follows: F: TCAACGTGCTCCTCGTGT (SEQ ID NO.3); R: TTGGGATGCAGTGACTGATA (SEQ ID NO. 4).

[0017] The obtained sequences were compared with DNAMAN5 software, and the results showed that two transgenic plants with premature termination of protein translation were obtained, which were named OsMATE16-KO1 and OsMATE16-KO2 . The two harvested T0 generations OsMATE16 The loss-of-function plants were sown in plots to form T1 transgenic lines and phenotypes were observed. It was found that the seed setting rate of knockout transgenic plants was significantly reduced compared with ZH11ZH11 (see Figure 2 ).

[0018] (4) OsMATE16 Observation of anther dehiscence phenotype of knockout mutants ZH11 and OsMATE16-KO1 Plants, mark the florets that bloomed on the day with a marker at around 11 noon. Mark 15 plants of each material, and mark 20 florets for each individual plant. When it grows to around 16:00 in the afternoon of the same day (5 hours of growth), take the florets and observe the anther dehiscence under a microscope. Each floret has 6 anthers, and the number of anther dehiscences from 1 to 6 is counted as dehiscence, and the number of dehiscences is 0, which is not dehiscence. Every 5 individual plants have 100 florets, which is a biological replicate, and there are 3 biological replicates in total. The results show that compared with ZH11, OsMATE16-KO1 The anther dehiscence rate of OsMATE16-KO1 The anther dehiscence rate of ZH11ZH11 was significantly lower than that of ZH11 (see Figure 4 ).

[0019] Embodiment 2: OsMATE16 Expression profiling analysis Different tissue samples of rice at different developmental stages were collected in the field, and the samples were fully ground in a liquid nitrogen pre-cooled grinder. About 50 mg of the sample was taken in a 2 mL RNAase-free EP tube, and the total RNA was extracted using the RNA extraction kit of QIAGEN; 500 ng of RNA was reverse transcribed using the reverse transcription kit of Takara, and the reverse transcription product was diluted 5 times. Gene-specific primers and OsUbiquitin were used as internal reference controls, and qPCR reactions were performed on a real-time quantitative system (qTOWER3G, Analytik, Jena, Germany). Three biological replicates were set for each sample, and statistical analysis was performed after the reaction was completed; among them, C2 is the booting stage; LB1 is the seedling leaf; LB2 is the booting leaf; YP2, YP3, YP5, YP9 and YP12 are 2, 3, 5, 9, and 12 cm long panicles; H9, H12 and H15 are 9, 12 and 15 cm long husks; E5, E10, E15, E20, E25 and E30 are caryopsis developed 5, 10., 15, 20, 25 and 30 days after fertilization; Anther is anther. The specific primers and OsUbiquitin primer sequences are as follows: OsMATE16 -qPCR-F: TGACAGGGGCTGAATTAGA (SEQ ID NO.5); OsMATE16 -qPCR-R:CCAGTGCTTGTAGCTATA (SEQ ID NO.6); OsUbiquitin-qPCR-F:TTCATGGCCAACCACTT (SEQ ID NO.7); OsUbiquitin-qPCR-R:AGCCTGCTGGTTGTAGA (SEQ ID NO. 8).

[0020] Depend on Figure 4 It can be seen that OsMATE16 It is mainly highly expressed in stems, leaves and young panicles at different developmental stages.

[0021] The coding region and protein sequence of the OsMATE16 gene in the present invention are as follows: (1) Coding region sequence: (2) Protein sequence: MAIPLQGKAQQQQGEGGKGDGAVDDDGDDQPSVASELRELWGMAAPITALNCVVYLRAMVSVLCLGRLGPLDLAGGALAIGLTNITGHSVLFGLASGLEPLCAQAFGSKNYDLLTLSLQRAVLLLTLAAL PIALLWLHVGPILVALGQDPTISASAAAYAAYALPDLAASAVLQPLRVYLRSQGITKPMAACSAIAVALHVPLNVLLVFGLGFGVRGVAAAQALTNTNMVLFLLAYIRWSRACDATWKGWARPAAVASGL AGLVRLAVPSCVGVCLEWWWYEVVTVLAGYLPDPAAAVGAAGVLIQTTSLMYTVPMALAACVSTRVGNELGGGKPRRARMAAMVALGCAVVIGVVHVAWTAAFSREWVELFTREAAVVRLAAAAMPILGL CELGNCPQTTGCGVLRGTARPAVGARINLLSFYLVGTPVAVTLAFGARVGFGGLWYGLLSAQAACVALVLLAVVWRTDWHLEALRAKKLTGLEMIAAAAEGDDDECKRLIAPLPPPGHDVAVDVV (SEQ IDNO.2).

[0022] 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 OsMATE16 A gene characterized by The rice OsMATE16 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.

1.

2. The rice according to claim 1 OsMATE16 The protein encoded by the gene is characterized in that The rice OsMATE16 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 OsMATE16 Application of genes in regulating anther dehiscence in rice.

4. The use according to claim 3, characterized in that: The method for regulating rice anther dehiscence is to knock out rice OsMATE16 The anther dehiscence rate of rice was reduced after the gene was mutated.

5. The use according to claim 3, characterized in that: The method for regulating rice anther dehiscence is to knock out rice OsMATE16 The rice seed setting rate is reduced after the gene is modified.

6. A preparation for regulating rice anther dehiscence, characterized in that: The preparation comprises the rice according to claim 1 OsMATE16 The gene or the rice according to claim 2 OsMATE16 Protein encoded by a gene.

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