烟粉虱对偏嗜寄主植物挥发物选择的嗅觉感受和识别机理

Tabaco whitefly Bemisia tabaci (Gennadius) is an important agricultural worldwide invasive insect pest. B. tabaci has a wide range host plants, but was highly selective with plants. Many documents showed that the odorant binding proteins (OBPs) in insect antennae selectively bind with volatiles of plants, and play an important role for host plant selection. In this project, B. tabaci was chose as test targets, and antennal transcriptomes will be sequenced firstly. The OBPs gene will be identified by bioinformatics prediction and quantitative real-time PCR. The antennal specific expressed OBPs are cloned and sequenced. The OBPs are prepared by expressing in E.coli cell, and then the subcellular localization of OBPs will be detected by immunofluorescence. Chemical composition of volatiles of host plant will be collect and identified by SPME-GC-MS，and the binding affinity between OBPs and volatiles of plant are investigated by homology model, molecular docking, Single-factor and Multi-factor binding experiments. The function of the candidate OBPs gene will be checked by RNAi technique. Through feeding whitefly on plants with synthesized dsRNA of target OBPs, gene expression will be checked by qRT-PCR, and the effect of RNAi will be checked by olfactory behavior of whitefly using Four-armed olfactometer. The volatile ingredients and mixture ratio of host plants, which are beneficial to whitefly, will be discrimnated. The results will reveal the host selection and molecular recognition mechanism based on olfactory system, and push the novel, environment friendly pest management techniques, and also provide important theory for B. tabaci population invasion and expansion mechanism.

烟粉虱Bemisia tabaci (Gennadius)是世界范围内的重要农林入侵害虫，虽然寄主广泛，但对寄主植物有明显的选择性。据报道昆虫触角中气味结合蛋白（OBPs）能选择性结合气味分子，对寄主选择起着重要作用。本项目选择烟粉虱为研究对象，通过触角转录组测序与生物信息学预测，获得嗅觉相关的OBPs基因，采用基因原核表达和免疫荧光定位技术，确定特定OBPs在触角感器中的位置；利用SPME-GC-MS收集和鉴定其寄主挥发物的化学成分，通过同源建模、分子对接和单多因子结合实验，发现与配体结合能力强的OBPs；采用RNAi技术，通过饲喂烟粉虱，检测烟粉虱选择行为变化及靶标基因抑制效果，确定对烟粉虱有较强引诱作用的化学成份或混合物配比。研究结果对揭示烟粉虱对寄主植物的嗅觉分子识别机制和认识其种群入侵和扩张机理提供理论依据，对害虫行为调控策略也具有重要借鉴作用。

烟粉虱Bemisia tabaci (Gennadius)是重要的农林入侵害虫，虽然寄主广泛，但对寄主植物有明显的选择性，其中气味结合蛋白（OBPs）在寄主植物的识别中起着重要作用。本项目主要研究内容和重要结果如下：. 利用扫描电镜比较了烟粉虱隐种MED和MEAM的触角的感器结构及数量分布，发现两种生物型之间感器数量无差别，但雌雄虫刺型感器数量及分布不同；完成了烟粉虱头部转录组测序及生物信息学分析，获得了嗅觉相关的8个OBPs基因；荧光定量实验对烟粉虱OBPs基因的组织表达谱研究发现7个OBPs在触角上特异高表达，BtabOBP3基因在烟粉虱足上特异高表达；烟粉虱MED隐种对13种植物挥发性气味物质的趋向行为反应显示，芳樟醇、反-2-己烯醛和桉树脑在不同浓度下对烟粉虱MED隐种雌、雄虫具显著吸引效果。. 成功构建了烟粉虱气味结合蛋白OBP2和OBP3的原核表达载体，并实现了重组蛋白的诱导和纯化。使用荧光探针Bis-ANS，通过荧光竞争结合实验的方法，测定Bta-OBP2和Bta-OBP3与30种气味物质的结合能力，发现上述两个烟粉虱OBP基因与β-紫罗兰酮有较强的结合能力（Ki=15.3µM/L，18.56µM/L），与其他物质的结合能力比较弱。烟粉虱基因BtabOBP8的功能研究结果表明β-紫罗兰酮、月桂烯、柠檬烯和P-伞花烃将1-NPN的相对荧光强度降低到50 %以下，其中β-紫罗兰酮和月桂烯与BtabOBP8的结合能力最强，解离常数分别为13.32 µmol/L和21.53 µmol/L。表明β-紫罗兰酮和月桂烯与BtabOBP8具有良好的亲和力，推测BtabOBP8对β-紫罗兰酮和月桂烯的识别可能参与了烟粉虱对绿色开花植物的吸引和选择作用。. 利用薄膜饲喂法对烟粉虱OBP3基因进行RNA干扰。随机挑选干扰48小时后的烟粉虱成虫作为试虫，利用“Y”型嗅觉仪，以正己烷作为对照，β-紫罗兰酮进行气味选择的为学试验，结果显示对烟粉虱OBP3的RNAi 显著降低了烟粉虱成虫对显著降低了烟粉虱成虫对β-紫罗兰酮的选择次数。. 本项目的研究结果解析了烟粉虱对不同寄主植物识别的嗅觉机制，必将促进基于烟粉虱行为反应的生态调控技术的应用，有助于揭示重大入侵生物在其种群定殖和扩张的寄主选择机制。