Method for detecting drug resistance of chilo suppressalis to Cry1Ac toxin

A technology for stem borer and drug resistance, applied in the fields of biotechnology and crop pest control, to achieve the effect of good accuracy, short cycle and high sensitivity

Inactive Publication Date: 2013-02-06
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A molecular detection method is very important when the demand for rapid detection technology of Bt protein in Chilo suppressalis is increasing, and ordinary biological detection methods cannot meet these requirements at all

Method used

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  • Method for detecting drug resistance of chilo suppressalis to Cry1Ac toxin
  • Method for detecting drug resistance of chilo suppressalis to Cry1Ac toxin

Examples

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Embodiment 1

[0029] A method for detecting Cry1Ac toxin resistance of C. chinensis, the steps of the method are:

[0030] 1. Extraction of BBMV from the midgut of S. chinensis

[0031] (1) Take the collected midgut samples out of the ultra-low temperature freezer and thaw them on ice. After the midgut was thawed, carefully remove the midgut with tweezers and place it in a glass homogenizer.

[0032] (2) Add 9 times the midgut volume of homogenization buffer Buffer A (300mM mannitol+5mM EGTA+17mM tris-HCl+1Mm PMSF) and 1 / 25 midgut volume of protease inhibitor (P.I., Roche, Germany) to in a glass homogenizer and homogenize on ice. After the homogenization, it was transferred to a 50ml centrifuge tube and placed on ice.

[0033] (3) Take out from the refrigerator at 4°C, and add 24mM MgCl 9 times the mass and volume of the midgut. 2 .

[0034] (4) Place on ice for 15 minutes.

[0035] (5) Centrifugation (Heal Force, Neofuge 15R), 4°C, 2500 rpm, 15min.

[0036] (6) The supernatant after...

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Abstract

The invention discloses a method for detecting drug resistance of chilo suppressalis to Cry1Ac toxin, which comprises the steps of: extraction of brush border membrane vesicles in the chilo suppressalis, dimensional electrophoretic separation, enzyme-linked immunosorbent assay (ELISA), shooting after developing and performing photshop software analysis and the like. Compared with the prior art, the method for detecting drug resistance of chilo suppressalis to Cry1Ac toxin provided by the invention has the advantages of high flexibility, good accuracy and short detection period and the like, and according to the method, the sensibility change of some chilo suppressalis population to Cry1Ac can be preliminarily detected, so that a direct evidence is provided for drug resistance monitoring, administration and pest integrated control of the chilo suppressalis.

Description

technical field [0001] The invention belongs to the fields of biotechnology and crop pest control, and relates to a method for detecting the drug resistance of Cry1Ac toxin, in particular to a molecular detection method for detecting Cry1Ac toxin resistance of C. Background technique [0002] The Cry toxin produced by Bacillus thuringiensis is the most successfully applied anti-insect gene in transgenic crops. There are two hypotheses for the mechanism by which Cry toxins poison insects. The first hypothesis is that Cry toxin is hydrolyzed and activated by midgut protease and binds to midgut-specific receptors, and the toxin subunits oligomerize to form a hole structure in the membrane, resulting in cell swelling and lysis, and then causing insect death. One hypothesis is that the toxicity of the toxin has nothing to do with oligomerization. After the toxin binds to the midgut-specific receptor protein, Mg is activated. 2+ Dependent signaling pathways interfere with the st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 马伟华陈龙佳邱琳侯蕾蕾张宗磊张志林牛林
Owner HUAZHONG AGRI UNIV
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