川滇桤木气味干扰云南切梢小蠹寄主定位的嗅觉机制

In insects, binding of odorant to odorant binding protein (OBP) is the initial step in mating,host skeeking, oviposition site location, and evasion from unsuitable and dangerous environment. An increasing investigations evidenced that odors emitted from non-hosts can mask or suppress the olfactory cues from the host. However, mechanisms underlying these are not well understood from the aspect of interactions between non-host odor and OBP. The pine shoot beetle, Tomicus yunnanensis, is an importantly economical pest, which damages seriously on Pinus yunnanensis in Yunnan province. Our previously investigations found that the non-host odors can disrupt host selection of this pest. Based on these backgrounds, this project aims to: 1) elucidating changes of the seasonal rhythm of the volatiles in host and nonhost and the electroantennogram (EAG) responses and OBP gene expressions of the beetle to the volatile changes by GC/EAD and RT-qPCR; 2) adaption that can effectively afect the host selection of this pest by olfactory experiments and GC-EAG;3) clone and express the OBP genes of this pest; 4) construct the 3D structures of OBPs by homology modeling and reveal the binding between OBP and active non-host volatiles by molecule docking; and 5) verify the binding characteristics between OBP and active non-host volatiles by fluorescence assay. The results will elucidate the mechanisms of non-host disrupting olfactory behaviors of T. yunnanesis, provide basis for utilize the volatiles for the control of this pest, and helpful for uncovering the mechanisms of pest control by biodiversity from the view of olfactory reorganization and odor masking.

气味结合蛋白与气味分子的结合是昆虫交配，搜寻寄主、产卵场所，逃离不适宜环境和危险生物的第一步。越来越多的实验证实，非寄主散发的气味物质能够掩盖或抑制昆虫对寄主的嗅觉识别，但是有关非寄主气味如何通过与气味结合蛋白结合而掩盖昆虫对寄主的识别至今尚无深入研究。本项目在前期研究基础上，以新命名的云南松重大害虫云南切梢小蠹为研究对象，采用GC/EAD、RT-qPCR分析不同季节寄主和非寄主气味的变化及小蠹虫OBP基因表达与气味变化的适应性；采用同源模建技术分析气味结合蛋白三维结构及气味结合蛋白与气味分子结合模式, 并通过荧光结合实验验证其相互结合特性。最终，阐明非寄主干扰云南切梢小蠹识别寄主气味的机制。为利用气味物质控制云南切梢小蠹提供理论依据和物质基础，同时从气味掩盖角度阐明生物多样性控制害虫的机理。

本项目通过顶空动态法、GC-MS、GC-EAD从非寄主川滇桤木气味（Alnus ferdinandi coburgii）成分中筛选出能有效干扰云南切梢小蠹（Tomicus yunnanensis）寄主定位的化合物5个，分别是（Z）-4-己烯-1-醇、（E）-2-己烯-1醇、4-烯丙基苯甲醚、β-蒎烯、对乙基苯乙酮。基于构建的转录组数据库，从中鉴定到267个嗅觉基因，其中45个OBP基因、12个CSP基因、36个OR基因、35个IR基因、19个GR基因、8个SNMP基因、50个CYP基因、29个CXE基因、33个GST基因。将筛选出的寄主α-派烯和川滇桤木对乙基苯乙酮小分子活性物质与嗅觉蛋白进行分子对接实验，OBP6与α派烯进行分子对接不形成氢键，结合能为-4.03△G，与对乙基苯乙酮进行分子对接赖氨酸4与配体形成氢键，结合能为-3.83△G，结果表明非寄主川滇桤木化合物能与寄主化合物竞争嗅觉蛋白的结合位点，其与蛋白质形成的结构更稳定。川滇桤木气味能诱导解毒基因CYP的表达，处理24h后表达量是对照的200%左右，从而降解寄主气味，干扰云南切梢小蠹的嗅觉定位。用川滇桤木气味处理60d后，云南切梢小蠹卵黄蛋白含量与对照组相比低了42.3%。川滇桤木气味物质还能干扰云南切梢小蠹蛀干和幼虫的生长。研究结果表明，川滇桤木气味物质作为胁迫因子影响云南切梢小蠹寄主定位、蛀干、产卵、幼虫生长发育及雌虫卵黄蛋白含量的影响。本项目从害虫寄主定位、生长发育和繁殖角度揭示非寄主调控害虫种群的机理，为混交林控制害虫的机理研究提供新思路，为害虫调控提供新技术和新方法。

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