柑橘大实蝇对寄主植物气味柠檬烯等的外周嗅觉编码机制

The Chinese citrus fruit fly (Bactrocera minax) is an important destructive pest of citrus orchards in China. It prefers to feed on a variety of different citrus species, all having different host-plant volatile profiles. These host volatiles seem to play an important role in the attraction of B. minax adults to the trees and fruit, and in the stimulation of oviposition by females. However, how the host odorants are encoded in the peripheral olfactory system is unclear. Based on our previous research on sensillum types of antenna, behavioral responses to limonene and other host-plant odorants, and odorant-binding proteins (Bmin OPB), the present project will strive to verify some of the peripheral neural mechanisms of olfactory signal transduction that may influence B. minax host plant selection behavior. These efforts will be aimed at: 1) studying the electrophysiological responses of the olfactory sensory neurons (OSNs) in antennal olfactory sensilla, by using single cell recording techniques to define the OSNs’ odorant response spectra; 2) identifying specific odorant binding proteins in target B. minax olfactory sensillar hemolymph, using transcriptome and proteomics techniques in which the target odor binding proteins will be screened, and the specific OBPs will be isolated and identified, and the structure, functions and receptive ranges to ligands will be analyzed. 3) determining the location, in different sensilla, of the binding proteins’ biochemical expression by immunoelectron microscope (IEM) techniques. The output of this project will enhance our understanding of the peripheral neural mechanisms of olfactory signal transduction and the discrete types of different sets of odorant molecules that are involved in Bactrocera minax host-plant preference, which in turn will facilitate designing and optimizing the olfactory-based "insect behavior regulation agent" strategies against B. minax.

柑橘大实蝇Bactrocera minax是我国柑橘重要毁灭性害虫，成虫产卵于柑橘果实内，气味在成虫寄主植物定位、求偶交配和刺激雌虫产卵方面起重要作用，但其嗅觉识别机制尚不清楚。本项目在弄清其触角感器类型、对柠檬烯等寄主植物气味嗅觉行为响应和气味结合蛋白（Bmin OBP）初步研究的基础上，试图进一步揭示其对寄主气味识别的嗅觉机制：用单细胞记录研究触角嗅觉感器的微观电生理反应，探明嗅觉感器对寄主植物气味敏感嗅觉感受神经元及其神经脉冲特征和气味谱；采用基因克隆和蛋白质组学技术分析嗅觉感器淋巴液中靶气味结合蛋白(OBPs)的生化特性和结合特症、配基谱及与配基间的互作；应用免疫电镜进行OBPs的感器定位。该项目对于揭示昆虫对寄主的生态适应和嗅觉进化，丰富实蝇类嗅觉外缘神经功能研究和OBPs家族生态类群，以及设计和优化基于嗅觉的昆虫行为调控剂应用于柑橘大实蝇防治，具有理论和实践意义。

昆虫依靠其灵敏特异的嗅觉系统识别周围环境中复杂气味分子，寄主植物气味物质在昆虫的寄主选择中起重要作用。柑橘大实蝇（Bactrocera minax）是我国柑橘类水果的重要毁灭性害虫，由于其卵、幼虫和蛹期生活隐匿，研究成虫通过柑橘气味定位寄主的研究显得十分重要。本项目主要研究结果如下：（1）通过触角转录组生物信息学分析，共鉴定柑橘大实蝇嗅觉相关基因122个，包括59个BminORs、39个BminOBPs、20个BminIRs、5个BminCSPs和3个BminSNMPs。（2）通过FPKM值评估柑橘大实蝇的所有嗅觉基因，发现绝大部分嗅觉基因均在雌雄触角高表达；半定量RT-PCR对 BminORs和BminOBPs在雌雄触角和足中的表达模式进行分析，发现BminORs基因在雌雄触角均表达，各BminOBPs基因在触角和足的表达模式不一。（3）结合进化树分析结果，克隆并表达了多个普通气味受体基因，并对其中的4 个气味受体基因（BminOR3，BminOR12，BminOR16和BminOR24）进行爪蟾卵母细胞的异源表达和对寄主植物44种气味物质的配体谱研究，结果发现，BminOR3对1-辛烯-3-醇有强烈的电生理反应；BminOR12对苯甲醇等八种气味物质有电生理反应； BminOR16和BminOR24分别对十一醇和芳樟醇有电生理反应。（4）克隆、表达并纯化BminCSP3，荧光竞争实验分析了其与寄主植物气味的结合谱，发现BminCSP3可以结合柠檬醛等多种气味物质。（5）研究了BminOBP25在成虫不同部位的差异表达，发现触角、头(去除触角)、足和产卵器中表达量较高，胸、腹和翅等部位表达量较低，说明该蛋白可能主要参与柑橘大实蝇的嗅觉，并具有其他生理功能。（6）研究了成虫触角感器的超微结构及对柠檬烯等气味物质的SSR反应，明确了各感器内感觉神经元的特征，发现其锥形感器和毛形感器能对部分寄主植物气味物质产生电位反应。上述研究结果提示，柑橘大实蝇触角嗅觉相关基因、以及锥形和毛形感器参与了其对寄主植物气味物质的嗅觉外周神经编码。本项目首次揭示了柑橘大实蝇对寄主植物气味识别相关嗅觉蛋白的结构与功能，为探明成虫识别柑橘气味的嗅觉编码机制，及其对寄主的生态适应性，设计和优化行为调控剂防控柑橘大实蝇提供了有利的支撑。