意大利蜜蜂α1亚型烟碱型乙酰胆碱受体功能表达和分析

Functional expression and analysis of Apis melliferaα1 nicotinic acetylcholine receptor

尽管作用于昆虫烟碱型乙酰胆碱受体(nicotinic acetylcholine receptors,nAChRs)的新烟碱类杀虫剂是农作物害虫防治的常用工具,但它们对意大利蜜蜂等传粉昆虫产生严重的负面影响。本研究克隆意大利蜜蜂nAChRα1亚基基因,对其进行系统发育分析,并利用双电极电压钳技术研究α1亚型nAChR与新烟碱类杀虫剂(吡虫啉、噻虫胺和呋虫胺)的相互作用机制。序列比对和系统进化树分析显示,意大利蜜蜂nAChRα1亚基具有典型的nAChRs亚基家族基因结构域特征,与半翅目昆虫的α1亚基关系最远,与同属膜翅目蜜蜂的α1亚基关系最近。将意大利蜜蜂nAChRα1亚基与大鼠nAChRβ2亚基在非洲爪蟾卵母细胞中共表达,电生理结果显示吡虫啉对Amα1/rβ2 nAChR的激动效率最高,噻虫胺最低,而Amα1/rβ2 nAChR对呋虫胺的激动亲和力最高,对吡虫啉最低。这表明意大利蜜蜂nAChRα1亚基是新烟碱类杀虫剂的作用靶标之一,并且Amα1/rβ2 nAChR对不同新烟碱类杀虫剂敏感性存在差异。本研究结果对于开发作用于害虫的新型高选择性杀虫剂具有重要理论指导意义。

Neonicotinoids that act on insect nicotinic acetylcholine receptors(nAChRs)are common tools for crop pest control,but they have serious negative effects on pollinators such as Apis mellifera.In this study,the nAChRα1 subunit gene was cloned from A.mellifera and systematically analyzed.The interaction mechanism between the nAChRα1 subtype and neonicotinoids(imidacloprid,clothianidin,and dinotefuran)was investigated by using the two-electrode voltage clamp technique.Sequence alignment and phylogenetic tree analysis showed that the nAChRα1 subunit of A.mellifera had typical domain characteristics in the nAChR subunit family genes,and was most distantly related to theα1 subunit of Hemiptera insects and most closely related to theα1 subunit of bees from Hymenoptera.The combination of A.mellifera nAChRα1 subunit and Rattus norvegicus nAChRβ2 subunit was co-expressed in Xenopus laevis oocytes.Electrophysiological results showed that imidacloprid had the highest agonistic efficiency and lowest agonistic affinity,that clothianidin had the lowest agonistic efficiency and that dinotefuran had the highest agonistic affinity for Amα1/rβ2 nAChR,indicating that theα1 subunit of A.mellifera nAChRs is one of the targets of neonicotinoids and the sensitivity of Amα1/rβ2 nAChR to different neonicotinoids is different.All these results have important theoretical significance for the development of novel highly selective insecticides for target insects.