14-3-3ε蛋白在蜜蜂响应亚致死剂量吡虫啉毒害过程中的作用及其靶蛋白的鉴定

Imidacloprid, a representative product of neonicotinoid insecticides, seriously harms the bees' living environment. Imidacloprid mainly impacts on the nicotinic acetylcholine receptors (nAChR) of nervous system, acting as agonists by binding to the alpha subunit of nAChR to disturb the normal neural signal transmission of insect. It has been reported that 14-3-3 can directly bind to the nAChR-α4 subunit, thereby enhancing the stability of AChR. Our previous results have found that the down-regulated expression of 14-3-3ε in adult honey bees (Apis mellifera L.) was somehow associated with sublethal doses of imidacloprid. Based on the previous results we speculate that 14-3-3 proteins may play a key role in resistance to the poison effect by sublethal doses of imidacloprid in honey bees. In this research, we will identify the 14-3-3ε interactome in A. mellifera by using the affinity-purification mass spectrometry (AP-MS) strategy. To further assess the effects of down-regulated expression of 14-3-3ε on downstream target proteins and the related signal transduction pathways in responding to the sublethal doses of imidacloprid toxicity, cluster analysis, signal pathway and protein-protein interaction network will be conducted according to the semi-quantitative interactome data, further, choose a representative targets of 14-3-3 in key interactome and confirm its interaction with 14-3-3 in vitro and in vivo. These results will provide theoretical and experimental bases for further clarifying the molecularly biological mechanisms underlying honey bee responding to sublethal doses of imidacloprid.

吡虫啉作为新烟碱类杀虫剂的代表，严重危害蜜蜂的生存环境。吡虫啉与乙酰胆碱竞争昆虫神经系统的烟碱型乙酰胆碱受体(nAChR) α亚基上的结合部位，从而阻断神经信号的传递。以往研究发现14-3-3蛋白能够直接结合nAChR的α4亚基并提高AChR的稳定性。本课题组在前期工作中发现意大利蜜蜂(Apis mellifera L .) 经亚致死剂量吡虫啉处理后14-3-3ε基因表达下调。本研究拟选取14-3-3ε作为目标蛋白，利用靶向蛋白质组技术鉴定意蜂14-3-3ε的相互作用组；通过聚类分析、信号通路和蛋白相互作用分析等初步探讨亚致死剂量吡虫啉毒害过程中14-3-3ε表达下调对其下游靶蛋白及其相关信号转导通路的影响；筛选典型的差异性14-3-3ε靶蛋白，通过体外、体内实验验证其与14-3-3ε的相互作用，为进一步探讨蜜蜂响应亚致死剂量吡虫啉毒害影响的分子生物学机制奠定理论与实验基础。

14-3-3蛋白是一类高度保守的酸性蛋白家族，通过调控其特异性结合靶标蛋白参与各种细胞生理反应过程。本课题组在前期工作中发现意大利蜜蜂(Apis mellifera L .) 经亚致死剂量吡虫.啉处理后14-3-3ε基因表达下调。本课题在前期工作的基础上，选取14-3-3ε为目标蛋白，通过AP-MS (Affinity-purification mass spectrometry) 实验策略鉴定了14-3-3的结合靶蛋白。我们分别在亲和串联在在对照组（CK）和吡虫啉处理组（IMI）中获得了172和304个14-3-3ε结合蛋白， GO功能聚类及KEGG代谢途径分析表明大多数蛋白质与“碳水化合物和能量代谢”、“氨基酸代谢”、“蛋白质折叠、分类和降解”以及“运输和分解代谢”有关。我们进一步发现，其中62个蛋白与14-3-3ε的结合受到吡虫啉处理影响。大部分与能量代谢及防御机制相关的蛋白与14-3-3ε的结合均上调。其中，14-3-3蛋白与E3 ubiquitin transferase、Electron transfer flavoprotein、glycerol-3-phosphate dehydrogenase的结合变化最为显著，需要进一步验证其互作机制。本课题按照计划已基本完成了相关的科研工作，并在此基础上有了创新性发现，为进一步研究14-3-3蛋白下游通路的分子机制提供了理论和实验依据。