烟蚜茧蜂嗅觉关联基因鉴定及对EBF特异识别机制的研究

(E)-β-farnesene (EBF) is the key (or only) component of alarm pheromone of most aphid species, also a common volatile of a number of plants. Its traits of repelling aphids and attracting natural enemies arises the role as an important semiochemical among communications of the three trophic levels, plant-aphids-nature enemies. Many documents show binding affinities of insect (aphid and their nature enemies) odorant binding proteins (OBPs) with EBF and other semichemicals. Therefore, to discriminate the particular OBPs for perception odorants or pheromones within nature enemies, can promote the exploitation and development of advanced aphids control strategy through contributing theoretical basis on both nature enemy protection and utilization. In the project, obligate aphid parasitoid Aphidius gifuensis Ashmead is chosen as the test target, its olfactory gene repertoire is to be identified from olfactory organs transcriptome, through “Unigenes” generation and prediction, genes identification and functional annotation. Cross-compare the annotated sequences with antennal transcriptome of Sitobion avenae which is one of the primary hosts of Aphidius gifuensis to screen and clone conserved genes expressed in both species. The recombinant proteins are prepared through prokaryotic expression system (E. Coli) and complex purification procedures. The binding affinities between OBPs and ligands (EBP and other semichemicals) are checked by ligand binding tests and a real-time specific binding with surface plasmon resonance (SPR) technique, and the aphid behavioral responses to ligands are investigated by olfactometers. To the end, the candidate OBPs of good binding affinity with EBF and plant odorants and of positive bioassay reaction are obtained. Meanwhile, the function of the candidate OBPs genes are checked by RNAi technique, through feeding aphid on the artificial diet with synthetized siRNA. The effect of RNAi is checked in the olfactory behavior and target gene expression aspects. The expected results of this project are the obtaining of selective OBPs for EBF perception, or perceptions of other volatiles. Which has a significant value on research and development of environmental friendly and green ecological pest management strategies based on novel nature enemies attractant (aphid repellents), and also enrich the theory of aphid parasitoids’ chemical ecology.

E-β-法呢烯（EBF）是蚜虫报警激素主要或唯一组分，也是一些植物的挥发物组分。可以驱避蚜虫、吸引天敌，是植物-蚜虫-天敌间化学通讯的重要信息物质。蚜虫及其天敌气味结合蛋白（OBPs）可选择性地结合气味分子，研究天敌中与EBF等气味物质特异识别的OBP，将为在新型蚜害防治策略的研发过程中实现保护和利用天敌提供依据。本项目以烟蚜茧蜂(Aphidius gifuensis Ashmead)为研究对象，通过分析雌、雄蜂嗅觉器官等的转录组，获得嗅觉关联基因；对与寄主麦长管蚜共同表达的保守OBP进行克隆及配体结合或SPR检测等实验，筛选特异结合EBF的OBPs蛋白种类；同时，采用RNAi技术，检测保守表达OBP被沉默前后蚜虫选择行为、电生理反应的变化及靶标基因抑制效果。揭示EBF调控烟蚜茧蜂（吸引）行为的分子机理。研究结果对蚜虫绿色防控技术研发，及烟蚜茧蜂化学生态学理论的丰富具有重要意义。

蚜茧蜂是蚜虫寄生性天敌之一，具有寄主专化性强，增殖潜力大，在蚜虫寄主植物上搜索效率高的特点，是自然条件下控制蚜虫种群数量的重要天敌。烟蚜茧蜂对多种蚜虫均有好的生物防治效果，在作物蚜害生物防治方面具有广泛开发和利用潜力及具有重要的经济价值。.本项目执行期内，按计划完成了烟蚜茧蜂处女雌、雄蜂及婚配雌、雄蜂的嗅觉器官——触角的二代转录组测序及上述烟蚜茧蜂的整虫的三代转录组测序。通过联合2+3代测序结果综合分析，获得了理想的烟蚜茧蜂触角全长转录组数据并开展了嗅觉功能基因挖掘和注释工作。同时，结合烟蚜茧蜂嗅觉生理行为调查结果，进一步筛选与烟蚜茧蜂对蚜虫报警信息素EBF和异性挥发物的识别反应关联的嗅觉基因。对该识别过程的分子机制进行了初步的解析。主要结果如下：.1).对烟蚜茧蜂（雌、雄两性）嗅觉器官触角的嗅觉感器进行了细致的超微结构观察分析；.2).开展相关转录组信息并完成了有关嗅觉基因家族的注释分析工作，共获得18个OBP、12个CSPs、67个ORs和40个IRs；.3).对烟蚜茧蜂的气味识别谱的研究证实，烟蚜茧蜂可以识别蚜虫报警信息素EBF和异性蚜茧蜂挥发物并作出反应；.4).联合目标基因差异表达分析和室内嗅觉行为测试，锁定了与对应气味识别可能关联的OBP和CSP蛋白基因类群。.本研究以烟蚜茧蜂为研究对象，结合化学生态学、分子生物学方法，获取嗅觉器官转录组信息，注释鉴定烟蚜茧蜂嗅觉基因、将蚜茧蜂对蚜虫报警信息素EBF及异性蜂嗅觉识别与功能基因相关联，初步解析其嗅觉识别机制。相关工作的开展有助于全面了解烟蚜茧蜂嗅觉感知行为，揭示烟蚜茧蜂寄主搜寻的高效性、对蚜虫报警激素的趋性及利用寄主植物挥发物定位蚜虫的分子机理等。为新型蚜虫行为控制剂的开发、转基因作物基因资源的选择及防治新技术安全性评价提供有关保护和利用天敌方面新的思路和理论依据。

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