摘要
目前对转苏云金芽胞杆菌Bacillus thuringiensis(Bt)的研究发现,液泡型ATP酶(Vacuolar-type proton ATPase,V-ATPase)可能是Bt的一类新型受体。我们前期通过构建棉铃虫的中肠酵母文库筛选Cry1Ac的结合蛋白发现棉铃虫V-ATPase亚基B(V-ATPase B)可以与Cry1Ac结合。为明确V-ATPase B在Cry1Ac毒力和昆虫对Cry1Ac抗性机制中的作用,本研究首先采用实时荧光定量PCR技术分析了该基因在抗感Cry1Ac棉铃虫幼虫中及其受到Cry1Ac诱导时的基因表达情况;通过Ligandblot进一步地证实了其与Cry1Ac的结合特性;并通过在Sf9细胞中表达V-ATPase B的试验验证了其功能。结果表明V-ATPase B在抗性品系及受到Cry1Ac诱导时均下调表达,Ligand blot证实了V-ATPase B与Cry1Ac特异性结合;并且在昆虫细胞内过表达该基因,会增强Cry1Ac的细胞毒力。研究结果表明棉铃虫V-ATPase B是Cry1Ac的功能受体,并有可能通过降低基因表达来参与棉铃虫对Cry1Ac的抗性形成。
Current studies on the mode of action of Bacillus thuringiensis(Bt) have found that vacuolar-type proton ATPase(V-ATPase) may be a new receptor of Bt proteins.In our previous study of screening for Cry1Ac binding protein from the midgut yeast library of cotton bollworm,we found that V-ATPase subunit B(V-ATPase B) was a Cry1Ac binding receptor.To clarify the role of V-ATPase B in Cry1Ac toxicity and insect resistance to Cry1Ac,we measured the expression levels of V-ATPase B in Cry1Ac-susceptible and-resistance strains and in Cry1Ac induced larvae using real-time quantitative PCR(RT-PCR),characterized the binding of V-ATPase B with Cry1Ac protein using Ligand blot,and tested the functions of V-ATPase B in the toxicity of Cry1Ac by expressing V-ATPase B in Sf9 cells.The results showed significantly reduced expression levels of V-ATPase B in Cry1Ac-resistant and Cry1Ac-feeding H.armigera larvae.Ligand blot experiments verified the interaction between V-ATPase B and Cry1Ac protein.Importantly,overexpressing V-ATPase B in cells caused higher cytotoxicity of Cry1Ac.These results suggest that V-ATPase subunit B is a functional receptor of Cry1Ac and may be involved in resistance evolution of cotton bollworm to Cry1Ac through reducing its expression.
作者
段云鹏
姚雪
李品
王萌涵
胡守印
胡恩静
刘永刚
杨淑芳
魏纪珍
DUAN Yunpeng;YAO Xue;LI Pin;WANG Menghan;HU Shouyin;HU Enjing;LIU Yonggang;YANG Shufang;WEI Jizhen(College of Plant Protection,Henan Agricultural University/Henan International Laboratory for Green Pest Control,Zhengzhou 450002,China)
出处
《中国生物防治学报》
CSCD
北大核心
2022年第5期1094-1102,共9页
Chinese Journal of Biological Control
基金
国家自然科学基金(32172401)
河南省农业领域科技攻关项目(212102110137)
河南省大学生创业训练项目(S202110466023)。
