假眼小绿叶蝉气味结合蛋白在芳香植物行为驱避作用中的功能研究

Volatile terpenoid compounds derived from aromatic plants exhibit significantly repellant activity against insects, but the olfactory mechanisms of insect perception of these semiochemicals is still unknowed. Study of functional characterization of odorant binding proteins (OBPs) in olfactory receptor of insects in its specific recognition of volatiles of aromatic plants can clarify the repellant mechanisms of aromatic plant volatiles against insects based on the molecular mechanism of insect olfaction, and screen the semiochemicals with high repellant activity for insect behavior manipulation. Previous study showed that lavender volatiles have significantly repellent activity against Empoasca vitis. In this project, the volatiles of lavender are firstly collected and analyzed, and then the compounds with repellant activity from volatiles of lavender are analyzed by using the electrophysiological and behaviorical test. Based on the constructed antennal transcriptome of E. vitis early, the highly expressed OBPs genes in antennal sensilla of leafhoppers will be identified by bioinformatics methods. Then, the recombinant OBPs proteins will be located in antennal sensilla using an immunocytochemical localization method. The binding properties of OBPs with odorants were measured by using the fluorescence competitive binding method. The result of this project will clarify olfactory mechanism of terpenoid compounds from the volatiles of lavender repelling against E. vitis. The project could provide theoretical basis for using aromatic plant volatiles for ecological control of targeted insects, and new ideas for designing insect behavior-regulating products by using OBPs in insect as a target.

芳香植物挥发物对昆虫有驱避作用，萜烯类化合物是其主要组分，但昆虫识别芳香植物挥发物的嗅觉机制尚不清楚。研究昆虫嗅觉感器中气味结合蛋白（OBPs）在识别芳香植物挥发物中萜烯类化合物中的功能，可以初步阐明芳香植物行为驱避作用的嗅觉分子机制，并针对性地筛选用于害虫行为调控的驱避活性化合物。前期研究表明薰衣草挥发物能显著驱避假眼小绿叶蝉。本项目拟首先收集并鉴定薰衣草植物挥发物组分，结合电生理和行为试验筛选对假眼小绿叶蝉具有行为驱避活性的萜烯类化合物；然后通过生物信息学分析鉴定假眼小绿叶蝉触角中高表达的OBPs基因，利用原核杂交技术和免疫组化方法研究OBPs基因组织表达定位；通过荧光竞争结合试验解析假眼小绿叶蝉OBPs对萜烯类化合物的结合特性，揭示萜烯类化合物驱避假眼小绿叶蝉嗅觉机制。本项目结果可为应用芳香植物挥发物生态调控靶标害虫奠定理论基础，并为OBPs靶向筛选、设计昆虫行为调控剂提供新思路。

芳香植物薰衣草释放的萜烯类化合物对小贯小绿叶蝉表现出显著的趋避效应，嗅觉在叶蝉感知环境气味中发挥着重要的作用。然而，气味结合蛋白（OBPs）在叶蝉感知芳香植物挥发物中的功能尚不清楚。本项目采用GC-MS测定了薰衣草植物的挥发性化合物组分，明确了10种主要组分的相对含量，利用“Y”型嗅觉仪测定了小贯小绿叶蝉对薰衣草4种主要挥发物的行为反应，发现柠檬烯和桉叶油醇对叶蝉表现出一定的驱避作用。利用RNA-seq分别对小贯小绿叶蝉触角及身体（不含触角）进行转录组测序，共得到79278个unigenes，平均长度为879 bp。通过注释和嗅觉相关蛋白的特征分析，分别鉴定到33个OBPs和24个化学感受蛋白（CSPs）基因，其中9个OBPs和17个CSPs为新鉴定的基因。通过构建系统发育树比较分析了叶蝉与其他半翅目昆虫OBPs和CSPs的进化关系。利用半定量和荧光定量PCR明确了OBPs和CSPs的组织表达谱，其中24个EonuOBPs和13个EonuCSPs为触角特异性表达。另外，1个EonuOBP和4个EonuCSPs为身体高表达。成功构建了触角高表达EonuOBP12的原核表达载体，并对EounOBP12蛋白进行原核表达及纯化，得到纯度较高的EonuOBP12蛋白。利用荧光竞争结合试验分析了EonuOBP12与19种薰衣草及寄主植物挥发物的结合特性，结果表明EonuOBP12与薰衣草及茶树共有挥发物β-月桂烯、β-石竹烯、α-法呢烯和β-罗勒烯有较强的结合能力；此外，EonuOBP12与茶树挥发物壬醇、顺-丁酸-3-己烯酯、十四烷和正十六烷具有较强的结合能力，表明EonuOBP12在识别薰衣草及寄主植物挥发物中发挥着重要的作用。本项目的研究结果有助于从理论上阐明小贯小绿叶蝉嗅觉识别芳香植物及寄主挥发物的分子机制，对小贯小绿叶蝉的防控具有重要意义。