丽蚜小蜂对烟粉虱寄生选择差异的嗅觉机制研究

The sweetpotato whitefly Bemisia tabaci is an important agricultural pest worldwide and Encarsia formosa, a solitary endoparasitoid, is widely applied with success for the control of whiteflies. In our previous studies, tomato infested with begomovirus tomato yellow leaf curl virus (TYLCV) has been proved to influence the parasitic characteristics and host selection of E. formosa on B. tabaci. However, the olfactory mechanism is still unclear for the host selection. Based on this scientific issue, the important odorant binding protein (OBPs) and chemosensory protein (CSPs) will be obtained from the transcriptome data of E. formosa and their relative expressions in different stages, olfactory organs, and induced conditions are to be analyzed through real-time qPCR technique. The olfactory candidate genes of E. formosa are silenced using RNAi technique and confirmed using qPCR in combination with behavioral response of E. formosa for host whiteflies/plants. Finally, the chemical volatile substances having strong binding capacity with the key OBP/CSP proteins of E. formosa are screened using fluorescence competitive binding assays and field trials experiments. The results would be helpful for illustrating the olfactory mechanism of the host selection by E. formosa, and also providing scientific basis for the behavioral regulation of parasitoids in controlling the whiteflies.

丽蚜小蜂Encarsia formosa是世界农业害虫烟粉虱Bemisia tabaci的重要寄生性天敌之一。前期研究表明，番茄感染双生病毒TYLCV后影响丽蚜小蜂对B和Q烟粉虱的寄生特性及寄主选择性，但这种选择性差异的嗅觉机制尚不明确。针对这个科学问题，本项目拟以丽蚜小蜂为研究对象，从转录组数据中获得其重要气味结合蛋白OBPs和化学感受蛋白CSPs，采用实时荧光定量PCR技术研究候选基因在不同时期、感器及诱导条件下的相对表达谱，确定候选基因；运用RNAi技术沉默候选基因，通过qRT-PCR方法检验其沉默效果，同时结合行为反应检测基因沉默对丽蚜小蜂感受烟粉虱/植物化学物质的影响；最后采用竞争结合试验确证可与丽蚜小蜂嗅觉识别蛋白OBP/CSP具有强结合能力的植物挥发性组分。研究结果有助于阐明丽蚜小蜂的寄生和寄主选择性差异的内在机制，也可为研发设计基于寄生蜂行为调控的控害技术提供科学依据。

烟粉虱Bemisia tabaci是世界性农业害虫，B和Q是目前在我国为害最严重的两个生物型（隐种）。丽蚜小蜂是烟粉虱的重要寄生性天敌，对烟粉虱B和Q存在寄主选择性差异，但丽蚜小蜂寄生选择烟粉虱寄主的嗅觉机制尚不明确。本项目构建了丽蚜小蜂的转录组数据库，并获得66,632个unigene，58.12%的unigenes与丽蝇蛹集金小蜂Nasonia vitripennis具有最高的相似度；鉴定获得丽蚜小蜂的39条嗅觉结合蛋白OBPs（其中31个典型OBPs和8个Minus-C OBPs）和10条化学感受蛋白基因CSPs，上述基因全部克隆获得全长序列并提交Genbank数据库；研究OBP和CSP基因在丽蚜小蜂不同发育阶段、不同组织部位及植物诱导下的表达量，发现OBP10、OBP11、OBP21、OBP22、OBP26、OBP27、OBP29和OBP31等8个嗅觉结合蛋白可能在丽蚜小蜂定位和寄生烟粉虱行为中发挥重要作用；研究发现丽蚜小蜂两个化学感受蛋白CSP3和CSP5在成虫期和触角中均表现显著高表达，表明可能参与了丽蚜小蜂的寄主定位，进一步采用荧光竞争结合试验研究丽蚜小蜂两个CSPs蛋白对植物萜烯类等20种挥发性物质的结合特性，发现二者均可不同程度的结合植物挥发性物质，特别是EforCSP3与邻苯二甲酸二丁酯、1-辛烯、β-榄香烯和十三烷的结合能力强，结合常数均小于5 μM；与EforCSP3相比，EforCSP5与气味分子的整体结合能力稍弱；最后，采用三维建模及分子对接进行了结合特性验证。研究结果筛选获得了丽蚜小蜂定位寄生烟粉虱的关键候选嗅觉结合蛋白及化学感受蛋白，这些嗅觉基因与植物挥发物的结合可能是引导丽蚜小蜂成功定位寄主烟粉虱的关键内在机制之一，这也为研发基于寄生蜂行为调控的天敌控害技术提供科学依据。