二化螟神经肽Allatostatin C 受体作为新型杀虫剂靶标的药理学研究

At present, there are a few insecticides to control rice pests, and it is urgent to develop environmentally friendly high selective insecticides to control rice pests. Based on insect neuropeptide signaling pathway, a novel insecticidal target may be explored. Four Allatostatin (AS-A, AS-B, AS-C and AS-CC) of insect has been found by bioinformatics analysis, phylogenetic tree showed that AS-C is the latest evolution, and the classification according to AS-C gene sequence in phylogenetic tree is almost the same as insect morphological classification. We speculated that AS-C may closely related to insect metamorphosis. On the basis of previous research, striped rice borer (Chilo suppressalis) has been used as research object in this project, based on the cloning of AS-C and its receptors of Chilo suppressalis, the function of AS-C and its receptor in the process of growth and metamorphosis has been researched by RNAi technology and the binding assay of receptor and ligand. In order to evaluate the selectivity and safety of insecticides with AS-C receptor as its insecticidal target, the effect of AS-C in Chilo suppressalis on growth and development of different insects has been studied. The potention of AS-C receptor of Chilo suppressalis as insecticidal target has been comprehensively evaluated, and a cell level model for the screening of new insecticides with the AS-C receptor as its insecticidal target was established. This project provide a theoretical basis for development of new pesticides, and has a broad application prospect.

目前防治水稻害虫的高效杀虫剂品种比较少，开发出对环境友好的高选择性杀虫剂迫在眉睫。申请者前期对昆虫神经肽Allatostatin四种类型（AS-A、AS-B、AS-C和AS-CC）进行生物信息学分析，发现AS-C基因在进化树上排列几乎与昆虫形态学分类一致，推测AS-C调控系统可能与昆虫变态密切相关。若要快速筛选出靶向昆虫神经肽AS-C受体的高选择性杀虫剂，需要先对该靶标受体的分子特性及其调控功能进行详细的诠释。本项目拟以二化螟为研究对象，克隆二化螟AS-C及AS受体基因；构建受体的CHO细胞表达体系，通过受体—配体结合试验鉴定出二化螟AS-C对应的受体；采用RNAi和人工注射多肽试验阐明二化螟AS-C及其受体的调控功能；利用计算机分子模拟技术、受体-配体结合试验研究二化螟AS-C与其受体的互作机理；综合评价二化螟AS-C受体作为新型杀虫剂靶标的潜能及其药理学特性，为新农药创制提供理论依据。

本项目研究目的主要是研究昆虫神经肽AS-C的调控功能和杀虫靶标潜能。首先采用生物信息学分析昆虫AS-C神经肽的序列特性和进化特征。昆虫C型AS家族分为三类（AS-C、AS-CC和AS-CCC），AS-CC普遍存在各个类群昆虫中。AS-C只在完全变态类（鳞翅目、鞘翅目和双翅目）中发现，而AS-CCC在不完全变态类和膜翅目昆虫中发现。AS-C神经肽在完全变态类昆虫和不完全变态类昆虫之间差异很大。然而RNAi实验和多肽注射实验结果表明二化螟死亡率并没有受到显著影响，这说明直接靶向昆虫AS-C受体开发防虫靶标的潜能不大。采用RNAi后转录组差异表达谱分析AS-C及其受体基因RNAi对下游信号通路基因表达的影响。结果表明AS-C及其受体基因RNAi后明显影响了柠檬酸合成通路、脂肪酸合成通路、卵黄蛋白、免疫相关基因以及一些神经肽信号基因等的表达水平。特别值得关注的是AS-CR基因干扰后卵黄原蛋白表达水平显著上调，推测AS-CR可能通过控制柠檬酸转运从而调节胞内乙酰辅酶和保幼激素的水平来达到调控卵黄原蛋白基因的合成。本项目研究结果丰富了我们对昆虫AS-C神经肽生理调控功能的认识。