小地老虎触角miRNAs的鉴定及其对信息素结合蛋白PBP2的调控分析

Pheromone binding proteins (PBPs) of the Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) can specially recognize the sex pheromone components, and insect miRNAs can regulate multiple vital life activities. The functional role(s) of insect antennal miRNAs in regulating the perception process of sex pheromone components, however, is still unknown. We speculate insect antennal miRNAs can change the recognization of male moth to the sex pheromones through regulating the protein expression of PBPs, five different experiments will be carried out to verify above speculation: (1) Identification of miRNAs in A. ipsilon antennae; (2) The co-expression of antennal miRNAs with PBP2 in the sensilla trichoid in male antennae; (3) Investigating the regulation region and binding sites of antennal miRNAs with PBP2 at CDS or 3'-UTR, by using heterologous expression system in HeLa and HEK293 cells with the Dual-luciferase assays; (4) Testing the impacts of protein expression of PBP2 after in vivo injecting of antennal miRNAs at protein level; (5) Investigating the electrophysiological and behavioral changes of male adult moths to the sex pheromone components after in vivo injecting of antennal miRNAs. Accomplishment of this project will enhance our knowledge of the regulation basis of miRNAs in insect sex pheromone communication, more importantly, can provide us novel methods, such as using miRNAs, to control insect pests through disrupting the male's perception of female sex pheromone.

小地老虎信息素结合蛋白PBPs能够特异识别性信息素分子，昆虫miRNAs调控诸多生命活动，但触角miRNAs在昆虫性信息素识别过程中的功能还未知。我们推测触角miRNAs可通过调控PBPs蛋白的表达，进而影响雄蛾对性信息素的识别反应。为了验证上述假设，我们拟从5个方面进行实验：(1)小地老虎触角miRNAs的鉴定；(2)触角miRNAs同PBP2在毛形感器中的共表达；(3)HeLa和HEK293细胞表达结合双荧光素酶活测定，明确miRNA作用在PBP2-CDS区还是3'-UTR以及结合位点；(4)蛋白水平明确活体注射miRNAs后对PBP2蛋白表达的影响；(5)电生理和行为学水平明确活体注射miRNAs后对雄蛾识别性信息素的影响。本项目的完成有利于阐明昆虫触角miRNAs对性信息素识别的调控机制。更重要的是，可为我们提供新的策略如通过miRNAs干扰害虫性信息素识别进而达到有效防治的目的。

小地老虎信息素结合蛋白PBP能够特异识别性信息素分子，昆虫miRNAs调控诸多生命活动，触角miRNAs在昆虫性信息素识别过程中的功能还未知，我们推测触角miRNAs可以通过调控PBP蛋白的表达，进而影响雄蛾对性信息素的识别反应。为了验证上述假设，我们进行了5个方面的实验验证： (1) 小地老虎触角miRNA的鉴定；(2)触角miRNAs同PBP2在毛形感器中的共表达；(3) HeLa和HEK293细胞表达结合双荧光素酶活测定，明确miRNA作用在PBP2 CDS还是3'-UTR以及结合位点；(4) 蛋白水平明确活体注射miRNA后对PBP2蛋白表达的影响；(5) 电生理和行为学水平明确活体注射miRNA后对雄蛾识别性信息素的影响。研究结果表明，小地老虎触角中一共存在115个conserved miRNAs和8个小地老虎特异的novel miRNA；原位杂交实验表明aip-miR-3477-5p和PBP2在雄蛾触角中的毛形感器中共表达；双荧光素酶活测定实验明确了aip-miR-3477-5p能够通过结合PBP2 3'-UTR区域的种子序列TGAGATT进而靶向抑制PBP2的表达；通过活体注射aip-miR-3477-5p agomir后，PBP2的表达受到抑制，注射aip-miR-3477-5p agomir的雄蛾对性信息素的EAG反应降低了67.5%，雄蛾能够成功飞到信息素源的个体数量下降了53.1%。上述结果阐明了触角miRNA aip-miR-3477-5p通过负向调控PBP2的表达进而影响小地老虎性信息素识别的分子和细胞机制。本项目中的数据有利于阐明昆虫触角miRNAs对性信息素识别的调控机制，更重要的是，可以为我们提供新的策略如通过miRNA干扰害虫性信息素识别进而达到有效防治的目的。