椰甲截脉姬小蜂嗅觉相关基因鉴定及气味结合蛋白功能研究

The olfactory is essential for survival and reproduction of insects. In the olfactory process, olfactory related proteins such as odorant binding proteins play an important role. In this project, on the basis of antennal transcriptome sequencing data that it have been obtained successfully in the early time, a parasitic wasp, Asecodes hispinarum that parasitise significant quarantine pest, coconut leaf beetle, Brontispa longissima will be studied in order to identify the olfactory related genes of A.hispinarum by bioinformatic analysis methods. In this study, full-length odorant binding protein gene will be amplified by RACE, the expression levels of odorant binding protein gene in wasp body will be analysed by quantitative PCR, the binding properties of odorant binding protein with odor molecules will be clarified by prokaryotic expression and fluorescence competitive binding experiments, the electrophysiological responses and behavior responses of wasp to odor molecules that exhibit high binding abilities with odorant binding protein will be measured by EAG and Y-tube olfactometer experiments respectively and the attracting action of odor molecules that effect wasp behavior responses will be verified by field test. This project will help us to clarify the olfactory molecular mechanism of A.hispinarum on the basis of theory. From this project we can also screen out some chemical compounds that show attracting action to wasp. Ultimately these chemical compounds will contribute to the development of slow-release agent to attract A.hispinarum and improve the searching efficiency against insect pests. Thus it will achieve the goal of controlling B.longissima efficiently.

嗅觉对于昆虫的生存繁衍至关重要。在昆虫嗅觉感受过程中，气味结合蛋白等嗅觉相关蛋白发挥着非常重要的功能。本申请以重大检疫害虫椰心叶甲的天敌寄生蜂椰甲截脉姬小蜂为研究对象，在前期成功获得其触角转录组测序数据的基础上，拟对其嗅觉相关基因进行鉴定，并采用RACE技术扩增气味结合蛋白基因全长。利用实时荧光定量PCR分析气味结合蛋白基因组织发育表达谱，通过原核表达和荧光竞争结合试验解析椰甲截脉姬小蜂气味结合蛋白的气味结合谱，利用EAG和Y型嗅觉仪研究与气味结合蛋白结合能力强的气味分子对椰甲截脉姬小蜂的电生理及行为反应，最后采取田间挂板试验验证EAG、Y型嗅觉仪测定结果中能引起反应的气味分子对椰甲截脉姬小蜂的引诱效果。研究结果有助于我们从理论上明确椰甲截脉姬小蜂嗅觉识别的分子机制，指导研制对椰甲截脉姬小蜂有吸引作用的缓释剂应用于提高寄生蜂对目标害虫的搜索效率，达到高效控制椰心叶甲的目标。

椰心叶甲Brontispa longissima是一种严重危害棕榈科植物、国家禁止进境的二类危险性检疫害虫。椰甲截脉姬小蜂Asecodes hispinarum是椰心叶甲幼虫寄生蜂，在控制椰心叶甲为害中起着非常重要的作用。已有研究表明，椰甲截脉姬小蜂寻找寄主时，嗅觉起主要作用。然而，目前椰甲截脉姬小蜂嗅觉机制的研究，国内外还未有报道。基于此，本项目采用转录组测序及生物信息学分析开展了椰甲截脉姬小蜂嗅觉相关基因的鉴定，并采用实时荧光定量PCR技术分析了椰甲截脉姬小蜂气味结合蛋白基因在虫体各组织的表达情况，而且还利用原核表达及荧光竞争结合试验分析了椰甲截脉姬小蜂气味结合蛋白1与17种椰树挥发物的结合特性。此外，还利用Y型管试验测定了与椰甲截脉姬小蜂气味结合蛋白1结合能力强的气味分子对椰甲截脉姬小蜂的行为选择反应。转录组测序共获得2,730,000条clean reads，经Trinity软件拼接组装获得46,363条unigenes。通过blastx比对，21,263条unigenes在nr数据库得到注释。根据注释信息及气味结合蛋白的鉴别特征，鉴定出8个椰甲截脉姬小蜂气味结合蛋白基因，分别命名为AhisOBP1-8。实时荧光定量PCR结果表明，AhisOBP1、AhisOBP2、AhisOBP4、AhisOBP6和AhisOBP7仅在椰甲截脉姬小蜂触角中表达，而AhisOBP3、AhisOBP5和AhisOBP8在椰甲截脉姬小蜂触角、头、胸、腹、足和翅中均有表达。试验结果还表明AhisOBP4和AhisOBP6主要在雌性椰甲截脉姬小蜂触角中表达，其相对表达量分别为98.62%和94.42%，而AhisOBP1、AhisOBP2和AhisOBP7在雌性和雄性椰甲截脉姬小蜂触角中的表达量相当。荧光竞争结合试验结果表明，β-月桂烯，庚醛，异松油烯，壬醛和2-己醇与AhisOBP1结合能力最强，结合常数分别为6.24，7.18，7.47，8.63和 9.96 µmol/L。Y型管试验表明，β-月桂烯和2-己醇对椰甲截脉姬小蜂具有显著的吸引作用，庚醛和异松油烯对椰甲截脉姬小蜂具有显著的趋避作用。本项目研究结果有助于从理论上阐明椰甲截脉姬小蜂嗅觉识别的分子机制，对椰心叶甲的防治具有重要意义。