中国农业科学

• 植物保护 • 上一篇    下一篇

含有α-胰凝乳蛋白酶作用位点的Cry3A突变体的构建、表达、纯化以及杀虫活性分析

刘京国,赵晓萌,杨爱珍,师光禄   

  1. 北京农学院生物技术学院/农业部都市农业(北方)重点开放实验室,北京 102206
  • 收稿日期:2010-07-30 出版日期:2011-04-02 发布日期:2010-11-11
  • 通讯作者: 师光禄,Tel:010-80799116;E-mail:glshi@126.com
  • 作者简介:刘京国,Tel:010-80795321;E-mail:jgliu2008@yahoo.com.cn
  • 基金资助:
    北京市自然科学基金(5092007、5112009)

Construction, Expression, Purification and Insecticidal Activity Analysis of Cry3A Mutant with α-Chymotrypsin Site

LIU Jing-guo, ZHAO Xiao-meng, YANG Ai-zhen, SHI Guang-lu   

  1. College of Biotechnology, Beijing University of Agriculture/Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 102206
  • Received:2010-07-30 Online:2011-04-02 Published:2010-11-11

摘要: 【目的】构建含有α-胰凝乳蛋白酶作用位点的Cry3A的突变体Cry3Am,以期提高Cry3A的活化效率,增强杀虫活性。【方法】利用重叠PCR技术,构建Cry3A杀虫蛋白的突变体Cry3Am,使之在原核细胞中表达纯化突变体蛋白;将Cry3Am与饲料混合后饲喂黄粉虫,用以评价Cry3Am的杀虫活性。【结果】成功地在Cry3A杀虫蛋白结构域I的α3和α4之间加上1个α-胰凝乳蛋白酶的作用位点,并提取Cry3Am到杀虫蛋白。体外酶切试验结果表明,Cry3Am很容易地被降解为55 kD的杀虫蛋白。生测分析结果表明,Cry3Am对黄粉虫的毒力是Cry3A的2倍以上。【结论】向Cry3A中插入α-胰凝乳蛋白酶作用位点可以使突变体Cry3Am很容易地被α-胰凝乳蛋白酶降解成55 kD的片段,并能提高Cry3A的杀虫活性。

关键词: Cry3A突变体 , α-胰凝乳蛋白酶 , 表达纯化 , 杀虫活性分析

Abstract: 【Objective】The objective of this study is to construct a Cry3A mutant containing a α-chymotrypsin site, and to enhance its insecticidal activity. 【Method】 The Cry3Am was constructed by overlap PCR and the Cry3Am protein was purified. Tenebrio molitor were fed with Cry3Am and rolled oats to evaluate the toxicity of Cry3Am. 【Result】 The Cry3Am with a α- chymotrypsin site at the linker between α3 and α4 of Cry3A domain I was constructed successfully. In vitro digestion result showed that Cry3Am was easier to be digested to 55 kD fragment than Cry3A. In vivo bioassay result indicated that the toxicity of Cry3Am against T. molitor was two times stronger than that of Cry3A. 【Conclusion】 The insertion of α-chymotrypsin site resulted in the Cry3Am digested easily by α-chymotrypsin to 55 kD fragment, and enhanced the toxicity of Cry3Am to T. molitor.

Key words: Cry3A mutant , α-chymotrypsin , expression and purification , insecticidal activity analysis