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Method for efficiently extracting exogenous gene expression protein of insect-resisting rice in soil

A technology of exogenous gene and extraction method, which is applied in the field of efficient extraction of exogenous gene expression protein of insect-resistant rice in soil, achieving the effect of simple steps, high extraction efficiency and easy operation

Inactive Publication Date: 2010-12-22
ZHEJIANG UNIV
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Problems solved by technology

However, domestic studies on the residues of exogenous proteins in transgenic Bt rice in soil have rarely been reported. Therefore, it is necessary to establish a method for efficiently extracting exogenous gene-expressed proteins from insect-resistant transgenic rice.

Method used

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Embodiment 1

[0015] Reagents and medicines required by the present invention include Na 2 CO 3 , NaHCO 3 , EDTA, Na 4 P 2 o 7 10H 2 O, DTT, Triton X-100, etc., are all chemically pure.

[0016] The selected material is a transgenic rice line with high resistance to Lepidoptera pests cultivated by Huazhong Agricultural University——TT51. Its exogenous gene is a crylAb / crylAc fusion gene artificially transformed and synthesized by Chinese scientists. Minghui 63". The transgenic rice has a stable insect resistance of more than 80% against Lepidopteran pests such as rice leaf roller, rice stem borer, rice stem borer and rice borer, which has the advantages of saving input costs, reducing labor intensity and poisoning risk, and greatly reducing the amount of pesticides used , Reduce environmental pollution, maintain the dynamic balance of rice field biological populations and other advantages. During the rice filling stage, take TT51 straw, pick out other plant tissue residues, wash them...

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Abstract

The invention provides a method efficiently extracting exogenous gene expression protein of insect-resisting rice in soil, which comprises the steps of: taking a soil sample around the insect-rice, adding a protein extract solution according to the addition of 0.5-10mL / g soil, vibrating and extracting for 0.5-2h at a room temperature, centrifuging, taking supernatant for repeatedly extracting 1-3 times, merging the supernatants to obtain a solution containing the exogenous gene expression protein of the insect-resisting rice, wherein the protein extraction solution comprises 50-100mM of Na2CO3, 50-100mM of NaHCO3, 1.0-5.0mM of EDTA (Ethylene Diamine Tetraacetic Acid), 20-50mM of Na4P2O7.10H2O, 5-10Mm of DTT (dithiothreitol) and 0.01-0.1 percent of Triton X-100, the solvent is water and pH value is 7-10. The method has the advantages of simple steps, convenient operation and high extraction efficiency, provides the basis for environmental behavior and fate of protein in the soil and provides a reference technical method for the similar research.

Description

(1) Technical field [0001] The invention relates to a method for efficiently extracting exogenous gene expression proteins of insect-resistant rice in soil, in particular to a method for extracting CrylAb / CrylAc insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs) in soil. (2) Background technology [0002] According to the statistics of ISAAA Chairman Clive James, in 2009, 14 million farmers in 25 countries around the world planted 134 million hectares of genetically modified crops, and the actual area has reached 180 million hectares. my country is at the advanced level in the world in the selection and breeding of Bt genetically modified rice, and is likely to become one of the first countries in the world to commercially plant transgenic rice. For example, the Institute of Nuclear Agriculture, Zhejiang University, in cooperation with the University of Ottawa, Canada, successfully introduced the Bt gene into japonica rice Xiushui 11 and other varieties. In ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/14
Inventor 叶庆富张燕飞汪海燕
Owner ZHEJIANG UNIV
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