METS2 gene for regulating and controlling rice grain shape, protein and application of METS2 gene and protein

Abstract

The invention relates to the technical field of gene engineering, and discloses an METS2 gene for regulating and controlling the grain shape of rice, a protein and application of the METS2 gene, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO.1. The invention further discloses application of the METS2 gene in regulation and control of the rice grain shape. According to the METS2 gene for regulating and controlling the rice grain shape, the protein and the application of the METS2 gene, the METS2 gene is overexpressed in a rice plant, short-grain-shaped rice can be obtained, the short-grain-shaped rice is not prone to breakage in the processing process, and the quality of a finished product is high; a basis is provided for further clarification of the molecular mechanism of the rice grain shape, and new gene resources and breeding resources are provided for rice breeding.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a method for regulating rice grain shape. METS2 Genes, proteins and their applications. Background Art

[0002] Rice grain development determines its appearance quality (grain shape, chalkiness, etc.), which is a core agronomic trait that determines yield and market competitiveness. The abundant rice resources contain many genes that affect grain shape. In the discovery of rice grain shape genes and analysis of their molecular mechanisms, researchers have cloned several genes related to grain shape regulation. Among them, GS3 It encodes a transmembrane protein consisting of 232 amino acids, which controls rice grain weight and length, rice grain width and grain fullness; GW5 / qSW5 Encodes a calmodulin-binding protein that degrades the transcriptional repressor OsBAK1 through the ubiquitin-proteasome pathway, relieving its inhibition on cell expansion and thereby increasing granule width; GNP12 Encodes a NB-ARC gene that affects the development of rice grain length and number of grains per panicle; Gnp4 is a regulatory factor for grain length. Gnp4 / LAX2 As a component of the auxin response pathway, it may play a role in the OsIAA3-OsARF25-OsERF142 pathway to regulate grain length. GS5 and GLW7 They affect grain shape by regulating the cell cycle and the activity of cell wall relaxases, respectively. Currently, my country's high-quality rice breeding faces two major challenges: a narrow genetic basis and insufficient resources for appearance quality genes, and the genetic network of related traits is still unclear. Therefore, exploring rice grain development genes and analyzing their regulatory mechanisms for grain shape formation can not only provide a theoretical basis for genetic improvement of quality, but also broaden genetic resources and assist in molecular design breeding. This has important theoretical and practical significance for the development of new high-quality rice varieties. In the future, it is necessary to further discover and identify more excellent grain shape regulatory genes to broaden the gene source and enrich the genetic background for rice breeding. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for regulating rice grain shape METS2 Genes, proteins and their applications, overexpressed in rice plants METS2 Genes can produce short-grain rice, which is not easy to break during processing and has high-quality finished products. It not only provides a basis for further elucidating the molecular mechanism of rice grain shape, but also provides new genetic resources and breeding resources for rice breeding.

[0004] To achieve the above object, the present invention provides a method for regulating rice grain shape. METS2 Gene, METS2The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0005] Furthermore, the present invention also provides METS2 Application of genes in regulating rice grain shape, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, when applied, overexpression in rice plants METS2 Gene, reducing rice grain length and width.

[0007] Furthermore, the present invention also provides a METS2 protein for regulating rice grain shape. The amino acid sequence of the METS2 protein is shown in SEQ ID NO. 2. The gene encoding the METS2 protein is METS2 Gene, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the present invention also provides the use of METS2 protein in regulating rice grain shape. The gene encoding METS2 protein is METS2 Gene, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] Furthermore, the present invention also provides a recombinant vector comprising the above-mentioned METS2 Gene, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] Furthermore, the recombinant vector is a plant overexpression vector.

[0011] A biological material contains the above-mentioned recombinant vector, wherein the biological material is a recombinant microorganism, a transgenic plant cell line or a transgenic plant tissue.

[0012] The recombinant microorganism is a bacterium, yeast, algae or fungus; the bacterium is Escherichia ( Escherichia ), Erwinia ( Erwinia ), Agrobacterium tumefaciens ( Agrobacterium ), Flavobacterium ( Flavobacterium ), Alcaligenes spp. ( Alcaligenes ), Pseudomonas spp. ( Pseudomonas ) or Bacillus spp. ( Bacillus ) is one of the following.

[0013] Furthermore, the present invention also provides the use of the above-mentioned recombinant vector in regulating rice grain shape.

[0014] Furthermore, the present invention also provides a method for cultivating short-grain transgenic rice, METS2 The gene was overexpressed in rice plants, and short-grain transgenic rice was obtained through screening and cultivation; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] The method for regulating rice grain shape of the present invention METS2 The advantages and positive effects of genes, proteins and their applications are: 1. The present invention discloses a method related to rice grain shape. METS2 Gene, through experiments, it was shown that rice grain shape-related coding genes METS2 Under the condition of enhanced function or increased expression, short-grain rice will be obtained, which proves that the rice-related coding genes METS2 The results not only provide a basis for further elucidating the molecular mechanism of rice grain shape, but also provide new genetic resources and breeding resources for rice breeding.

[0016] 2. The present invention discloses overexpression in rice METS2 The invention discloses a method for obtaining short-grain transgenic rice, wherein the obtained short-grain rice is not easy to break during processing and the finished product is of high quality.

[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In the embodiment of the present invention METS2 -OE1, -Schematic diagram of the relative expression levels of overexpressed genes in OE2 and its parent rice Guanghui 998 (abbreviated as GH998) plants; METS2 is the coding gene related to rice grain shape in the embodiment of the present invention Figure 2 Bar graph of grain length and width of overexpressing transgenic rice, where A is grain length and B is grain width; METS2 is the coding gene related to rice grain shape in the embodiment of the present invention Figure 3 Grain length and width phenotypes of overexpressing transgenic rice. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0021] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0022] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0023] Example 1 Construction of rice grain shape-related gene overexpression vector: 1. Construction of overexpression vector: 1.1 METS2 Acquisition of genes: The common wild rice Y11 ( METS2 Griff.) DNA was used as a template and the target gene was amplified by PCR using the following primers: Primer1 and Primer2: primer1: 5' - atggcgtctcacattgttgg-3' (SEQ ID NO.3); primer2: 5'-tcacttcgcgctagcgag-3' (SEQ ID NO. 4).

[0024] The PCR product was recovered and purified, then connected to the Zero sequencing vector (purchased from Beijing Quanshijin Company), transformed into DH5α competent cells, and positive clones were selected for sequencing.

[0025] The sequencing results showed that the sequence of the PCR product was as shown in SEQ ID NO.1, with a length of 2301 bp, and it was named Oryza rufipogon Gene.

[0026]

[0027] The amino acid sequence of METS2 protein is shown in SEQ ID NO.2: MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMADAGIKYIPSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGFSTYFSMARGNATVPAMEMTKWFDTNYHFIVPELGPNTKFSYSSHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKPAKGVEKSFALLSLLSSILPVYKEV IAELKAAGATWIQFDEPTLVLDLDSHQLAAFSAAYTELESALSGLNVLIETYFADIPAESYKTLTSLNSVTAYGFDLIRGFKTLDLVKSAGFPSGKYLFAGVVDGRNIWADDLAASLTTLESLEAIVGKDKLVVSTSCSLMHTAVDLVNETKLDSEIKSWLAFAAQKVVEVNALAKALAGQKDEAYFAANTA AQASRRSSPRVTNEEVQKAAAALRGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTVELRRVRREYKAKKISEDEYVSAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGSRCVKPPIIYGDVSRPNAMTVFWSKMAQSMTSRPMKGMLTGPVTILNWSFVRND QPRFETCYQIALAIKKEVEDLEAGGIQVIQIDEAALREGLPLRKAEHAFYLDWAVHSFRITNCGVQDTTQIHTHMCYSNFNDIIHSIINMDADVITIENSRSDEKLLSVFREGVKYGAGIGPGVYDIHSPRIPSTEEIADRINKMLAVLDTNILWVNPDCGLKTRKYTEVKPALTNMVLAAKLIRTQLASAK (SEQ ID NO.2).

[0028] 1.2 Genes related to grain shape in common wild rice METS2 Overexpression vector (i.e., recombinant expression vector PMDC32-OE- METS2 ) build: 1) Use primer 1 and primer 2 to amplify wild rice cDNA and obtainMETS2 The sequence of the gene was connected to the vector Zero to obtain the recombinant Zero- METS2 The positive clones were digested with restriction endonucleases KpnI and PacI to form the recombinant vector Zero- METS[2]] Obtain OE- METS2 fragment; 2) The expression vector PMDC32 was digested with restriction endonucleases Kpn I and Pac I to obtain a linear expression vector PMDC32, and the linear fragment was recovered; the fragment OE- METS2 The homologous recombination directional cloning method was used to integrate it into the linear expression vector PMDC32 (for specific methods, please refer to the PMDC32 instruction manual) to obtain the homologous recombination product 1 (ie: PMDC32-OE- METS2 , is the gene of the present invention METS2 The homologous recombination product 1 was then transformed into DH5α competent cells and cultured at 37°C overnight; 3) The recombinant vector PMDC32-OE- METS2 Sequencing results showed that the recombinant vector had the nucleotide sequence shown in SEQ ID NO.1 inserted in the forward direction of the Kpn I restriction enzyme cleavage site of the expression vector PMDC32, that is, the DNA sequence between the Kpn I and PacI recognition sites (recognition sequence) of PMDC32 was successfully replaced with the DNA sequence shown in SEQ ID No.1.

[0029] 1.3 Conversion: Heat shock method to transform E. coli: take 5 μL of recombinant vector PMDC32-OE- METS2 , heat shock method to transform Escherichia coli, the specific steps refer to the Tiangen transformation kit.

[0030] 1.4 Bacterial liquid PCR verification: In a clean bench, a single colony was picked as a template for PCR amplification using the Taq DNA polymerase PCR amplification system and procedure.

[0031] 1.5 Plasmid extraction: Refer to the instructions of Tiangen Plasmid Extraction Kit to extract the plasmid and detect the concentration to obtain the recombinant expression vector PMDC32-OE- METS2 .

[0032] Example 2 Cultivating genes encoding rice grain shape, growth and development METS2 Overexpression vector transgenic plants and identification of transgenic plants: 1. Cultivation METS2 Gene overexpression transgenic plants: The recombinant vector PMDC32-OE- METS2Transformation of GH998 indica rice was mediated by Agrobacterium tumefaciens EHA105. The specific method is as follows: 1. Plasmid transformation: The recombinant vector PMDC32-OE- METS2 The recombinant vector PMDC32-OE- METS2 recombinant Agrobacterium tumefaciens EHA105; containing the recombinant vector PMDC32-OE- METS2 The recombinant Agrobacterium tumefaciens EHA105 was cultured at 28°C for 16 h, and the cells were collected. The cells were diluted with N6 liquid medium (Sigma, catalog number C1416) containing 100 μM acetosyringone to obtain a diluted bacterial solution with an OD600 of ≈ 0.5. 2. Infection: One-month-old embryogenic callus of GH998 indica rice with mature embryos was mixed with the diluted bacterial solution obtained in step 1 and inoculated for 30 minutes. The bacterial solution was dried with filter paper and then transferred to N6 solid co-cultivation medium. The callus was co-cultivated at 24°C for 3 days to obtain the co-cultivated callus. 3. Screening: 3.1 The callus tissue after the co-cultivation treatment in step 2 was inoculated onto N6 solid screening medium containing 150 mg / L hygromycin (hygromycin was added to N6 solid screening medium to obtain N6 solid screening medium, and the mass concentration of hygromycin in N6 solid screening medium was 150 mg / L) for the first screening; 3.2 On the 16th day after the first screening, healthy calli were selected and transferred to N6 solid screening medium containing 200 mg / L hygromycin (hygromycin was added to N6 solid screening medium to obtain N6 solid screening medium, and the hygromycin concentration in N6 solid screening medium was 200 mg / L) for a second screening. Subculture was performed every 15 days for a total of one subculture to obtain resistant calli. 4. Differentiation culture to obtain overexpression positive plants: The resistant callus obtained in step 3 was selected and transferred to the differentiation medium containing 150 mg / L hygromycin (differentiation medium: 2 mg 6-BA, 0.2 mg NAA, 4 g N6, 1 g hydrolyzed casein, 0.1 g inositol, 25 g sucrose, 2.4 g sorbitol, 7 g agar powder, 1 L deionized water) for differentiation. The culture was carried out at 24 °C for 45 days (at this time, the height of the aboveground part of the plant was about 15 cm). The seedlings were opened and hardened for 3 days, and then transplanted to the greenhouse for cultivation. This is the PMDC32-OE- METS2Plants (denoted as T0 generation). Two plants were obtained by transforming callus tissue and were named as follows: METS2 -OE1, METS2 -OE2 plants.

[0033] two, METS2 PCR identification of transgenic plants: Extract the above METS2 -OE1, METS2 -OE2 plants T1 generation seedlings and recipient parent rice GH998 plant seedlings leaf genomic DNA, and primers hyg-F and hyg-R were used for PCR molecular detection to identify positive seedlings. After PCR verification, the single plant with hyg positive bands was the one mentioned above. METS2 -OE1, METS2 -OE2, identified as a transgenic individual.

[0034] hyg-F: 5'-AAAAGTTCGACAGCGTCTCCGACC-3' (SEQ ID NO.5), hyg-R: 5'-TCTACACAGCCATCGGTCCAGACG-3' (SEQ ID NO. 6).

[0035] The PCR system is shown in Table 1: Table 1 PCR system ;

[0036] The amplification program is shown in Table 2: Table 2 Amplification procedures ;

[0037] three, METS2 In transgenic plants METS2 Identification of gene expression levels: Extract the above METS2 -OE1, METS2 -OE2 plant T1 seedlings and recipient parent rice GH998 plant leaves, set as internal reference METS2 , using internal reference primers Actin -F and [[ID=]]Actin -R, and Actin Gene-specific quantitative primers METS2 -qRT-F and METS2 -qRT-R fluorescence quantitative PCR reaction to detect different transgenic plants METS2 Changes in gene expression levels.

[0038] The results showed that ( METS2), and then transferred into the recombinant vector PMDC32-OE- Figure 1 In the positive plants METS2 The expression levels of genes were higher than those of the control strain (GH998) METS2 The expression level of the gene increased significantly. The primers are as follows: METS2 -qRT-F: 5' -GCGCTGAGGATTTGGAGAAG-3' (SEQ ID NO.7) METS2 -qRT-R: 5' -ATAGCAGGGACAGTGGCATT-3' (SEQ ID NO.8) METS2 -F: 5' - ATTTGGCACCACACATTCTAC-3' (SEQ ID NO.9) Actin R: 5' - ATAACCTTCGTAGATTGGGACT-3' (SEQ ID NO.10) Four, Actin- Phenotypic identification of transgenic plants: Respectively METS2 -OE1, METS2 -OE2 and GH998 plants were planted in the transgenic experimental greenhouse of Guangxi Academy of Agricultural Sciences. After maturity, seeds were harvested and observed. METS2 -OE1, METS2 -Differences in grain shape phenotype between OE2 and GH998. The results of the measurements are shown in Table 3. METS2 and Figure 2 shown.

[0039] Table 3 Genes encoding rice grain shape, growth and development Figure 3 Grain shape expression in overexpression plants ;

[0040] Compared with GH998 plants, PMDC32-OE- METS2 Plants ( METS2 -OE1, METS2 -OE2) showed a phenotype of significantly reduced rice grain length and width compared with the control group (GH998) ( P <0.05), thus proving METS2 Genes involved in controlling rice grain shape, i.e. METS2 The gene is related to rice grain shape and is overexpressed METS2 The gene can significantly reduce rice grain length and width.

[0041] Therefore, the present invention adopts the above-mentioned method for regulating rice grain shape METS2Genes, proteins and their applications, overexpressed in rice plants METS2 METS2 Genes can produce short-grain rice, which is not easy to break during processing and has high-quality finished products. It not only provides a basis for further elucidating the molecular mechanism of rice grain shape, but also provides new genetic resources and breeding resources for rice breeding.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Regulation of rice grain shape METS2 A gene characterized by: METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. As claimed in claim 1 METS2 Application of genes in regulating rice grain shape.

3. The use according to claim 2, characterized in that: Overexpression in rice plants METS2 Gene, reducing rice grain length and width.

4. The METS2 protein that regulates rice grain shape is characterized by: The amino acid sequence of METS2 protein is shown in SEQ ID NO. 2, and the gene encoding METS2 protein is METS2 Gene, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. Use of the METS2 protein as claimed in claim 4 in regulating rice grain shape.

6. A recombinant vector, characterized in that: Containing the claim 1 METS2 Gene, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The recombinant vector according to claim 6, characterized in that: The recombinant vector is a plant overexpression vector.

8. Use of the recombinant vector according to claim 6 or 7 in regulating rice grain shape.

9. A method for cultivating short-grain transgenic rice, characterized by: Will METS2 The gene was overexpressed in rice plants, and short-grain transgenic rice was obtained through screening and cultivation; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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