摘要
【目的】Ⅲ型海葵毒素(Av3)对昆虫具有显著的选择性毒性,对其作用机制的研究对新型高选择性毒性杀虫剂的设计研究具有重要意义。【方法】反向高效液相色谱及电喷雾质谱用于鉴定化学合成的Av3野生型(Av3 wild type,Av3-WT)及其突变体的纯度和分子量;生物活性测定检测Av3-WT及其突变体对德国小蠊Blattella germanica成虫的毒力;双电极电压钳技术检测Av3-WT及其突变体对德国小蠊钠通道BgNav1-1a失活的抑制作用。构建基于BgNav1-1a与大鼠钠通道rNav1.2a的重组嵌合体,通过双电极电压钳技术确定BgNav1-1a上参与Av3-WT选择性毒性的关键区域。【结果】芳香族氨基酸Y7,W8和Y18分别突变后形成的Av3-WT突变体Y7A,W8A和Y18A对德国小蠊成虫的毒力显著降低,半数击倒剂量(KD_(50))与Av3-WT相比均增加了超过10倍;与Av3-WT对通道失活62%的抑制率相比,250 nmol/L的毒素突变体Y7A,W8A和Y18A对BgNav1-1a失活的抑制作用也显著降低,通道失活的抑制率分别降低到12%,23%和8%;以rNav1.2a胞外环DI/SS5-S6替换BgNav1-1a的相应序列获得的重组嵌合体钠通道对Av3-WT毒素的敏感性几乎丧失,仅3.6%的通道在1μmol/L Av3-WT作用下失活被抑制。BgNav1-1a胞外环DI/SS2-S6上的His404突变为Tyr后几乎丧失对毒素的敏感性,仅6%的通道在1μmol/L Av3-WT作用下失活被抑制。【结论】芳香族氨基酸Tyr7,Trp8和Tyr18参与到构成Av3-WT分子的生物活性表面;钠离子通道胞外环DI/SS2-S6是影响Av3-WT毒素发挥选择性的关键结合区域,BgNav1-1a的DI/SS2-S6上的His404则是影响Av3-WT选择性毒性的关键氨基酸。
【Aim】Investigation of the action mechanisms of selective toxicity of type III sea anemone toxin(Av3)on insects would be highly valuable for future design of highly selective anti-insect compounds.【Methods】Reversed-phase high performance liquid chromatogram(RP-HPLC)and electrosprayionization mass spectrum(ESI-MS)were used to identify the purity and molecular weight of chemically synthesized Av3 wild type(Av3-WT)and its mutants.The toxicities of Av3-WT and its mutants to Blattella germanica adults and their inhibition effect on the inactivation of sodium channel BgNav1-1a were tested by bioassay and double voltage clamp,respectively.Systematically replacing the extracellular loops of BgNav1-1a with those of rat sodium channel rNav1.2a was executed to determine the key region for Av3-WT selective toxicity by double voltage clamp.【Results】The toxicity of three Av3-WT mutants Y7A,W8A and Y18A generated via mutation of aromatic amino acids Y7,W8 and Y18 to B.germanica adults was significantly reduced,and the median knockdown dose(KD_(50))values of these three mutants increased by more than 10-fold compared with that of Av3 wild-type toxin(Av3-WT).As compared to the Av3-WT,the inhibition of the mutants Y7A,W8A and Y18A at the dose of 250 mmol/L on the inactivation of BgNav1-1a significantly decreased.The inhibition rates of Y7A,W8A and Y18A on channel inactivation were 12%,23%and 8%,respectively,while that of Av3-WT was 62%.The chimera bearing DI/SS2-S6 of the rat sodium channel in BgNav1-1a almost lost sensitivity to Av3-WT,the inactivation of only 3.6%channels was inhibited.Substitution His404Y in DI/SS2-S6 completely abolished the inhibition effect of toxin on channel inactivation,and the inactivation of only 6%channels was inhibited by 1μmol/L Av3-WT.【Conclusion】Three aromatic residues Tyr7,Trp8 and Tyr18 are involved in the constitution of bioactive surface of Av3-WT,DI/SS2-S6 is the key region affecting the specificity action of Av3-WT,and His404 in DI/SS2-S6 is critical for Av3-WT selective toxic
作者
朱庆
高蓉
ZHU Qing;GAO Rong(Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;Department of Hygienic Analysis, School of Public Health, Nanjing Medical University, Nanjing 211166, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2021年第11期1283-1292,共10页
Acta Entomologica Sinica
基金
江苏省自然科学基金项目(BK20170611)
国家自然科学基金项目(31801767)。
关键词
德国小蠊
杀虫剂
海葵毒素
生物活性表面
钠通道
双电极电压钳
Blattella germanica
insecticide
sea anemone toxin
bioactive surface
sodium channel
double voltage clamp
