梨小食心虫在桃、梨之间季节性转移危害的挥发物诱导与嗅觉识别机制研究

The unique phenomenon, seasonal transfer of hazards, in life history of oriental fruit moth, is one of the key factors which bring about rampant disaster in mixed peach and pear orchard. Studies have reported that the volatiles generated by host plants played an important role in inducing and directing the pests transfer or directional selection. In this project, seasonal volatiles generated by peach and pear would be systematically analyzed and behavioral and electrophysiological responses of oriental fruit moth caused by the volatiles would be observed. In addition, field trials would be supplied in order to clarify the volatiles constituents which critically induce the seasonal transfer of oriental fruit moth. On this basis, molecular biological methods would be supplied in order to construct the cDNA library of antenna and obtain the full-length gene sequences of odorant binding protein. According to the main inducing constituents, RNAi and fluorescence combined experiment would proceed to screen the odorant genes.Expression characteristics and immune positioning of the object protein would be also studied, preliminarily reveal the induction and smell identification mechanism about how the plants volatile result in the seasonal transfer between peach and pear of oriental fruit moth. The results not only make important innovations in comprehensively explaining the host selection and seasonal transfer theory, but also cause great practical significance to develop new attractants to oriental fruit moth.

梨小食心虫年生活史中所特有的季节性转移危害现象是引起桃、梨混栽区梨小食心虫猖獗成灾的关键因子之一。大量研究表明，寄主植物所产生的挥发物在害虫的寄主转移或定向选择中起着重要的诱导或指示作用。本项目拟通过对桃、梨挥发物组分及其季节变化的系统分析比较，梨小食心虫对桃、梨挥发物的行为反应、电生理反应观察和田间试验验证，弄清对梨小食心虫季节性转移危害起关键诱导作用的挥发物组分。在此基础上，采用分子生物学方法，构建梨小食心虫触角cDNA文库，获得梨小食心虫气味结合蛋白全长基因序列，根据确认的主要诱导组分，通过RNAi和荧光结合实验，筛选感受气味基因，研究目的蛋白的表达特点和免疫定位，初步较系统地揭示梨小食心虫在桃、梨之间季节性转移危害的挥发物诱导与嗅觉识别机制。研究结果对更加全面科学地诠释害虫寄主选择与季节性转移危害理论有一定的创新，对梨小食心虫新型引诱剂的研发有重要的实践意义。

梨小食心虫Grapholita molesta（Busck）是一种严重危害桃、梨等果树的世界性重要害虫，其特有的在桃梨之间季节性寄主转移现象是引起桃、梨混栽区猖獗发生的主要原因之一，灵敏的嗅觉系统在梨小食心虫完成季节性寄主转移中起着非常重要的作用。本研究通过对桃、梨挥发物组分及其季节变化的系统测试和分析比较，旨在弄清诱导梨小食心虫在桃梨之间季节性转移危害的关键挥发物组分，初步揭示梨小食心虫在桃、梨之间季节性转移危害的挥发物诱导与嗅觉识别机制。取得主要研究结果如下：. 1、通过动态收集桃、梨不同生育期的挥发物，利用GC-MS技术在桃树和梨树上分别鉴定到39种和47种挥发物组分。明确了桃和梨在不同生育期释放的主要组分和特异性组分。. 2、选用4种性信息素和30种桃、梨挥发物进行梨小食心虫的EAG反应和行为，结果表明梨小食心虫对挥发物组分的反应与供试挥发物种类、浓度、成虫性别、交配状况等密切有关，但一些关键挥发物能引起成虫强烈的EAG反应和行为反应。. 3、利用触角转录组测序共鉴定到124种嗅觉相关基因，包括28种OBPs，17种CSPs，48种ORs，4种GRs，24种IRs，2种SNMPs及1种ODE基因。通过半定量PCR研究了OBPs和CSPs基因在不同组织的分布，RT-qPCR测试表明梨小食心虫OBPs基因在触角中的表达与光周期、雌雄性别及交配状态等因素有关。. 4、成功表达和纯化了5种OBP蛋白、5种CSP蛋白和2种性信息素结合蛋白，通过荧光竞争结合试验筛选获得了能够强烈结合性信息素组分或桃、梨寄主植物挥发物的OBPs及与关键OBPs高效结合的气味配体。与OBPs相比，5种CSPs蛋白均不能与性信息素结合，与寄主植物挥发物的结合能力也很弱，表明CSPs识别气味物质的能力有限。. 5、通过全量荧光免疫定位试验发现GmolGOBP2和GmolOBP8主要分布在雌、雄虫触角的锥形感器中。. 6、通过同源建模模拟了GmolGOBP2和GmolOBP8的三维空间结构，利用分子柔性对接、定点突变、配体替代试验验证了Thr9和Trp108分别是GmolGOBP2结合十二烷醇、GmolOBP8结合乙酸-反-8-十二碳烯酯的关键位点。

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