两种蜘蛛毒素肽与苏云金芽胞杆菌Cry1Ac蛋白的融合表达及杀虫活性

doi:10.16409/j.cnki.2095-039x.2018.06.006

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (6): 838-847.DOI: 10.16409/j.cnki.2095-039x.2018.06.006

两种蜘蛛毒素肽与苏云金芽胞杆菌Cry1Ac蛋白的融合表达及杀虫活性

陈珺君¹, 刘芳², 廖先清², 张志刚², 闵勇², 饶犇², 杨自文², 周荣华², 刘晓艳²

1. 武汉大学生命科学学院, 武汉 430072;
2. 湖北省农业科学院/湖北省生物农药工程研究中心/湖北省农业科技创新中心生物农药分中心, 武汉 430064

收稿日期:2018-09-03 出版日期:2018-12-08 发布日期:2018-12-08
通讯作者: 刘晓艳,博士,研究员,E-mail:xiaoyanliu6613@163.com;周荣华,硕士,副研究员,E-mail:526002407@qq.com
作者简介:陈珺君,硕士研究生,E-mail:1525978070@qq.com;刘芳,助理研究员,E-mail:24807004@qq.com
基金资助:
国家自然科学基金（31500428）；湖北省农科院竞争性项目（2016jzxjh026）；湖北省技术创新专项（2016ABA103、2017ABA160）；国家重点研发计划（2017YFD0201205）；湖北省农科院重大研发成果培育专项（2017CGPY01）；湖北省农业科技创新中心创新团队项目（2016-620-000-001-038）

Fusion Expression and Insecticidal Activity of Two Spider Toxin Peptides with Cry1Ac Protein from Bacillus thuringiensis

CHEN Junjun¹, LIU Fang², LIAO Xianqing², ZHANG Zhigang², MIN Yong², RAO Ben², YANG Ziwen², ZHOU Ronghua², LIU Xiaoyan²

1. College of Life Science, Wuhan University, Wuhan 430072, China;
2. Biopesticide Branch, Hubei Innovation Centre of Agricultural Science and Technology/Hubei Biopesticide Engineering Research Centre/Hubei Academy of Agricultural Sciences, Wuhan 430064, China

Received:2018-09-03 Online:2018-12-08 Published:2018-12-08

摘要/Abstract

摘要： 蜘蛛毒液中含有多种杀虫肽，因而具有较强的杀虫作用，可迅速杀死农林害虫。蜘蛛毒素肽（ω-ACTX-Hv1a，ω-ACTX-Hv2a）是从澳大利亚的多能厚爪蛛Hadronyche versuta的毒液中分离获得，对昆虫有毒但对哺乳动物无毒。本文通过将人工合成的两种蜘蛛肽基因（hv1a、hv2a）与来源于对棉铃虫具有高活性的苏云金芽胞杆菌（Bacillus thuringiensis，Bt）野生菌株NBIC380中的杀虫基因cry1Ac进行融合并在NBIC380中表达，获得了7种不同的重组菌株BtBM-Ve（含有空表达载体）、BtBM-Ac（含有cry1Ac基因）、BtBM-1a（含有hv1a基因）、BtBM-2a（含有hv2a基因）、BtA1a（含有cry1Ac+hv1a融合基因）、BtA2a（含有cry1Ac+hv2a融合基因）和BtA（1+2）a（含有cry1Ac+hv1a+hv2a融合基因）。重组菌分别进行了棉铃虫Helicoverpa armigera 2龄幼虫、秀丽隐杆线虫Caenorhabditis elegans和朱砂叶螨Tetranychus cinnabarinus的生物活性测定，结果显示BtBM-Ve无杀虫增效作用，BtBM-Ac、BtBM-1a、BtBM-2a、BtA1a、BtA2a和BtA（1+2）a针对不同的虫源具有不同的杀虫增效活性，对3种害虫杀虫增效最多的重组菌是BtA（1+2）a，对棉铃虫2龄幼虫杀虫增效百分比为93.43%，对秀丽隐杆线虫为34.48%，对朱砂叶螨为13.46%。本文构建的基因工程菌对防治鳞翅目害虫、螨虫和线虫具有重要的理论意义和应用前景。

关键词: 蜘蛛毒素肽, 苏云金芽胞杆菌, 融合蛋白, 杀虫活性

Abstract: Spider venom contains many insecticidal peptides, thus showing strong insecticidal effect and able to kill agricultural and forestry pests quickly. The spider toxin peptides (ω-ACTX-Hv1a and ω-ACTX-Hv2a) were isolated from the venom of Hadronyche versuta, which are toxic to insects but not to mammals. In this study, the insecticidal gene cry1Ac from Bacillus thuringiensis (Bt) wild type strain NBIC380 was fused with the two synthetic virulent peptide genes (hv1a and hv2a) and the fused gene and the individual genes were expressed in BCIC380. Seven recombinant strains were obtained, BtBM-Ve (empty plasmid control), BtBM-Ac (carrying cry1Ac gene), BtBM-1a (carrying hv1a gene), BtBM-2a (carrying hv2a gene), BtA1a (carrying fusion gene cry1Ac-hv1a), BtA2a (carrying fusion gene cry1Ac-hv2a), BtA(1+2)a (carrying fusion gene cry1Ac-hv1a-hv2a). The insecticidal activities of the recombinant strains were tested against the 2^nd instar larvae of Helicoverpa armigera, Caenorhabditis elegans and Tetranychus cinnabarinus. The results showed that BtBM-Ve had no insecticidal and synergistic effects, BtBM-Ac, BtBM-1a, BtBM-2a, BtA1a, BtA2a and BtA(1+2)a had varying insecticidal and synergistic activities against different pests. BtA(1+2)a was the most effective recombinant against the three pests, with an increase in insecticidal efficiency of 93.43% against the H. armigera 2^nd instars, 34.48% against C. elegans, and 13.46% against T. cinnabarinus. The genetically engineered strains constructed in this study are of significant potential in control of Lepidopteran, mite and nematode pests.

Key words: spider toxin peptide, Bacillus thuringiensis, fusion protein, insecticidal activity

中图分类号:

Q939.96

陈珺君, 刘芳, 廖先清, 张志刚, 闵勇, 饶犇, 杨自文, 周荣华, 刘晓艳. 两种蜘蛛毒素肽与苏云金芽胞杆菌Cry1Ac蛋白的融合表达及杀虫活性[J]. 中国生物防治学报, 2018, 34(6): 838-847.

CHEN Junjun, LIU Fang, LIAO Xianqing, ZHANG Zhigang, MIN Yong, RAO Ben, YANG Ziwen, ZHOU Ronghua, LIU Xiaoyan. Fusion Expression and Insecticidal Activity of Two Spider Toxin Peptides with Cry1Ac Protein from Bacillus thuringiensis[J]. journal1, 2018, 34(6): 838-847.

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两种蜘蛛毒素肽与苏云金芽胞杆菌Cry1Ac蛋白的融合表达及杀虫活性

Fusion Expression and Insecticidal Activity of Two Spider Toxin Peptides with Cry1Ac Protein from Bacillus thuringiensis

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