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Application of rice RAG2 gene in improving quantitative character of rice

A quantitative trait, rice technology, applied in the field of genetic engineering, can solve the problem of unclear biological function of RAG2, and achieve the effect of alleviating food shortage, increasing seed size, and improving quality

Inactive Publication Date: 2017-09-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

By comparing the protein sequence of RAG2, it was found that it also has homology with plant lipid transporter (Alvarez et al., Classification of rice allergic protein cDNAs belonging to the alpha-amylase / trypsin inhibitor gene family, Biochim Biophys Acta, 1995) , but the biological function of RAG2 in rice seed formation remains unclear

Method used

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  • Application of rice RAG2 gene in improving quantitative character of rice
  • Application of rice RAG2 gene in improving quantitative character of rice
  • Application of rice RAG2 gene in improving quantitative character of rice

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: Amplification of RAG2 gene full-length cDNA

[0023] For the gene RAG2 (gene accession number LOC_Os07g11380) required by the present invention, the full-length sequence of the RAG2 gene is mainly amplified by RT-PCR method. The specific operation is as follows:

[0024] 1) Extract RNA from the seeds of the japonica rice variety Zhonghua 11 (ZH11) (a gift from researcher Li Meifang, Institute of Crop Science, Chinese Academy of Agricultural Sciences, which is a rice variety publicly used in China) on the 14th day after flowering. The reagent used for RNA extraction is Invitrogen The company's Trizol extraction kit (see the kit's instruction manual for specific steps);

[0025]2) The steps to synthesize the first strand of cDNA by reverse transcription in RT-PCR are as follows: ① Prepare mixed solution 1: 4 μg of total RNA, 2U of DNaseI, 1 μl of 10xDNaseI buffer, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase ) Treat water (0.01% DEPC) to 10...

Embodiment 2

[0032] Embodiment 2: Construction of RAG2 overexpression vector

[0033] The gene required by the present invention is amplified by the RT-PCR method to obtain the full-length sequence of RAG2. For the long cDNA sequence, primers including restriction sites were designed, and the full-length cDNA of the RAG2 gene (from Example 1) was used as a template to amplify the full-length fragment of the gene by PCR. The amplified product was connected to pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., ie Promega, USA) by T / A cloning for sequencing verification.

[0034] The primers used to clone the full-length fragment of RAG2 containing restriction sites are as follows:

[0035] puRAG2-F:5'-GGGGGTACCATGGCTTCCAACAAGGTAGT-3';

[0036] puRAG2-R:5'-GGGGGATCCGTGACCAGTTCTCGGGGTCCTA-3'

[0037] A 498bp DNA fragment was amplified, and the fragment included the nucleotide sequence shown in the sequence table SEQ ID NO: 1, which is the coding region (CDS) at position...

Embodiment 3

[0042] Example 3: Transformation of plasmid vectors and detection of positive and expression levels of overexpressed transgenic plants

[0043] 1) The newly constructed overexpression vector pU2301-RAG2-cFLAG (from Example 2) was introduced into Agrobacterium EHA105 (purchased From the CAMBIA laboratory in Australia, which is a conventional commercial strain) strain. The transformed strain was named OX-RAG2.

[0044] 2) Transform the OX-RAG2 obtained in the previous step into the japonica rice variety Zhonghua 11 (ZH11), and the transformation method refers to the method reported by Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence Analysis of the boundaries of the T-DNA. Plant J, 1994, 6:271-282.) and the standard method of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (Patent No. ZL 200710053552.9). Obtain transgenic plants of the T0 generation.

[0045] 3) Total DNA...

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Abstract

The invention discloses application of rice RAG2 gene in improving quantitative character of rice. The rice allergen protein RAG2 is one type of soluble protein in rice seed storage protein; the applicant constructs the over-expression carrier of the gene and converts the japonica rice variety Zhonghua No.11 (ZH11) of rice, the seed of the obtained over-expression transgenic positive plant is increased, and the output is obviously higher than the output of the wild type; the contents of protein and fat in the rice are obviously increased, the content of starch is reduced, and the quality of the rice is improved. The gene has new application in culturing the good plant variety with high output and high quality, so as to provide new possibility for culturing the new crop variety with increased output and improved quality.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering application, and specifically relates to the application of rice RAG2 gene in improving rice quantitative traits, and the quantitative traits refer to rice yield or rice quality. In the present invention, by constructing transgenic rice plants with enhanced RAG2 gene expression, it is found that in the RAG2 overexpressed transgenic T2 generation plants, the seeds of the positive transgenic plants are increased, the yield is significantly higher than that of the wild type, and the rice protein and fat content are also significantly increased. Improve the nutritional quality of rice. Therefore, the gene has a new application in cultivating excellent high-yield and high-quality plant varieties, thereby providing the possibility of cultivating new high-yield and high-quality crop varieties. Background technique [0002] Rice is not only one of the three most important food crops (rice, wh...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00
CPCC07K14/8107C12N15/8245C12N15/8247C12N15/8251C12N15/8261
Inventor 姚家玲周伟
Owner HUAZHONG AGRI UNIV
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