球形芽胞杆菌Cry48Aa/Cry49Aa毒素在苏云金芽胞杆菌中的共表达及杀蚊活性

doi:10.16409/j.cnki.2095-039x.2015.06.019

中国生物防治学报 ›› 2015, Vol. 31 ›› Issue (6): 950-955.DOI: 10.16409/j.cnki.2095-039x.2015.06.019

球形芽胞杆菌Cry48Aa/Cry49Aa毒素在苏云金芽胞杆菌中的共表达及杀蚊活性

郭青云¹, 蔡全信², 胡晓敏², 闫建平², 袁志明²

1. 赣南师范学院生命与环境科学学院, 赣州 341000;
2. 中国科学院武汉病毒研究所, 武汉 430071

收稿日期:2014-12-24 出版日期:2015-12-08 发布日期:2015-12-17
通讯作者: 袁志明
作者简介:郭青云,女,博士,讲师,E-mial：qingyun612@163.com
基金资助:
国家自然科学基金(31272384)

Coexpression of Cry48Aa/Cry49Aa Toxin from Lysinibacillus sphaericus in Crystal-minus B. thuringiensis and Activity against Mosquito Larvae

GUO Qingyun¹, CAI Quanxin², HU Xiaomin², YAN Jianping², YUAN Zhiming²

1. College of Life and Environmental Sciences, Gannan Normal University, Ganzhou 341000, China;
2. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

Received:2014-12-24 Online:2015-12-08 Published:2015-12-17

摘要/Abstract

摘要： 球形芽胞杆菌Cry48Aa/Cry49Aa复合杀蚊毒素对库蚊有高毒力,并能杀死二元毒素BinA/BinB抗性蚊虫,是一种比较有潜力的新型杀蚊毒素蛋白,但Cry48Aa在野生菌株中表达量较低限制了该毒素的开发与应用。本文将cry48Aa基因启动子置换,并将cry48Aa和cry49Aa在苏云金芽胞杆菌中进行共表达。SDS-PAGE检测重组蛋白表达,可见120 kD左右的Cry48Aa蛋白带和53 kD的Cry49Aa蛋白带,cry48Aa在自身启动子下表达量偏低,在置换强启动子Pcry1Aa后表达量明显提高,但Cry48Aa仍低于Cry49Aa的表达量。重组菌株BMB171-pBU-cry48Aa-cry49Aa和BMB171-pBU-Pcry1Aa-cry48Aa-cry49Aa产生典型的双锥型和方形2种晶体。两菌株发酵液对实验室饲养的二元毒素敏感(CqSL)和抗性致倦库蚊(CqRL/C3-41)品系4龄幼虫都表现出高毒力,LC₅₀分别为3.05和3.45以及0.65和0.82 μL/mL。

关键词: 球形芽胞杆菌, Cry48Aa/Cry49Aa, 表达, 致倦库蚊, 杀蚊毒力

Abstract: The Cry48Aa/Cry49Aa, two-component toxin, shows high toxicity against Culex species, including binary toxin BinA/BinB-resistant Culex larvae. Being a potential novel mosquitocidal toxin, however, the low expression of Cry48Aa in the wild strain limits its development and application. In this work, the promoter of cry48Aa was replaced and cry48Aa and cry49Aa were coexpressed in Bacillus thuringiensis. The expression of recombinant proteins were detected by SDS-PAGE, visualized as a 120 (Cry48Aa) and a 53 kDa (Cry49Aa) bands. The expression amount of cry48Aa was low in its own promoter, which however, was significantly increased after the replacement of its promoter with the strong promoter Pcry1Aa but was still lower than the expression of Cry49Aa. Both the recombinant strains BMB171-pBU-cry48Aa-cry49Aa and BMB171-pBU-Pcry1Aa-cry48Aa- cry49Aa produced bipyramidal and squared crystals. The bioassay result showed that the two strains were effective against the 4th instar larvae of Bin-susceptible and -resistant C. quinquefasciatus, with the LC₅₀ of 3.05 and 3.45, and 0.65 and 0.82 μL/mL, respectively.

Key words: Lysinibacillus sphaericus, Cry48Aa/Cry49Aa, expression, Culex quinquefasciatus, mosquitocidal activity

中图分类号:

S476.11

郭青云, 蔡全信, 胡晓敏, 闫建平, 袁志明. 球形芽胞杆菌Cry48Aa/Cry49Aa毒素在苏云金芽胞杆菌中的共表达及杀蚊活性[J]. 中国生物防治学报, 2015, 31(6): 950-955.

GUO Qingyun, CAI Quanxin, HU Xiaomin, YAN Jianping, YUAN Zhiming. Coexpression of Cry48Aa/Cry49Aa Toxin from Lysinibacillus sphaericus in Crystal-minus B. thuringiensis and Activity against Mosquito Larvae[J]. journal1, 2015, 31(6): 950-955.

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球形芽胞杆菌Cry48Aa/Cry49Aa毒素在苏云金芽胞杆菌中的共表达及杀蚊活性

Coexpression of Cry48Aa/Cry49Aa Toxin from Lysinibacillus sphaericus in Crystal-minus B. thuringiensis and Activity against Mosquito Larvae

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