橘小实蝇嗅觉受体的注释及其感受甲基丁香酚的分子机制

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a destructive invasive pest in tropical and subtropical areas of the world. Recently, with its expansion to north, B. dorsalis has caused more and more economic loss. In order to have a better control against the dispersal and outbreak of B. dorsalis, it is very urgent to develop novel attractants that possess stronger attractiveness. The core issue for the development of novel attractant is to identify the crucial target. However, although methyl eugenol (ME) has been widely used as the attractant for B. dorsalis for more than 60 years, the target ME acts on as well as the molecular mechanism of its chemical perception in B. dorsalis still remains unknown. This has become the major barrier blocking the process of novel attractant development. Focusing on the olfaction system of B. dorsalis, our group conducted comparative studies between the male and female antennal transcriptomes by RNAseq, as well as the full-length transcriptome by PacBio single molecule sequencing. We obtained the basic sequence information for large number of the genes involved in chemical perception. To further dissect the physiological process of the chemical perception in B. dorsalis and break the bottleneck for novel attractant development, the current project will carry out the genome-wide annotation of olfactory receptors in B. dorsalis. Furthermore, the molecular techniques such as comparative study of antennal transcriptomics upon ME stimulation, co-immunoprecipitation between the phosphorylated ribosome S6 and mRNA of olfactory receptors, calcium imaging, and RNAi will be employed to screen the key olfactory receptors responsible for ME perception. The identified olfactory receptors will serve as the target for the development of novel attractant. Our results will not only reveal the molecular mechanism of ME perception in B. dorsalis, but also provide theoretical evidence contributing to the performance improvement via chemical modification of the species specific attractant.

橘小实蝇是危险性入侵害虫，近年来，其适生范围逐渐北移，危害愈演愈烈。为进一步控制橘小实蝇的扩散和危害，亟需开发新型高效诱剂。诱剂创制的核心是获得关键作用靶标。虽然甲基丁香酚被用作橘小实蝇性诱剂60余年，但其作用靶标及橘小实蝇对其化学感受机制至今仍不清楚，这严重阻碍了新型高效诱剂的研发进程。本课题组前期瞄准橘小实蝇嗅觉系统，通过全长转录组、雌雄成虫触角转录组，获得了大量化学感受相关的基因信息。为了深入研究橘小实蝇化学感受的生理过程、突破诱剂创制的瓶颈，本项目率先利用基因组信息，全面注释橘小实蝇嗅觉受体；结合甲基丁香酚刺激下比较转录组学、核糖体蛋白S6亚基与嗅觉受体mRNA免疫共沉淀、钙离子成像及RNAi技术，鉴定橘小实蝇感受甲基丁香酚的关键嗅觉受体，发掘高效特异诱剂开发的靶标。研究结果不仅将阐明橘小实蝇感受甲基丁香酚的分子机制，还将为通过化学改造提升实蝇类害虫特异性引诱剂的效力提供理论依据。

桔小实蝇是危险性入侵害虫，近年来，其适生范围逐渐北移，危害愈演愈烈。为进一步控制桔小实蝇的扩散和危害，亟需开发新型高效诱剂。诱剂创制的核心是获得关键作用靶标。虽然甲基丁香酚被用作桔小实蝇性诱剂60余年，但其作用靶标及桔小实蝇对其化学感受机制至今仍不清楚，这严重阻碍了新型高效诱剂的研发进程。. 在本项目的资助下，我们利用二代（Illumina-NovaSeq）加三代（Nanopore-PromethION）基因组测序，获得了高质量的桔小实蝇雌虫基因组。基于桔小实蝇基因组、转录组数据库，全面注释桔小实蝇气味结合蛋白和嗅觉受体基因，分别鉴定到49个OBPs基因和73个ORs基因。比较转录组分析了嗅觉基因在不同性别、气味物质诱导下的表达模式，并利用qPCR验证其表达差异。初步筛选了可能参与感受甲基丁香酚的嗅觉基因，为鉴定感受甲基丁香酚的嗅觉基因提供基础数据。. 利用CRISPR/Cas9的基因编辑技术敲除了嗅觉受体共表达受体Orco，表型分析结果显示Orco-/-突变体完全丧失了对甲基丁香酚的触角电位反应及行为上的趋性，证明桔小实蝇主要通过嗅觉系统识别甲基丁香酚。利用微量热涌动技术测定了OBP与甲基丁香酚的离体亲和能力，发现气味结合蛋白OBP56f-2与甲基丁香酚有很强的离体结合能力，随后利用基因编辑技术证实该气味结合蛋白与甲基丁香酚的化学感受密切相关。目前，课题组利用电压钳技术在离体条件下检测了46个嗅觉受体基因与甲基丁香酚的结合能力，但遗憾的是尚未发现结合甲基丁香酚的嗅觉受体。此外，利用基因编辑技术和行为学分析，我们还发现神经肽sNPF信号系统参与桔小实蝇嗅觉行为过程。. 本项目研究不仅揭示了桔小实蝇感受甲基丁香酚的分子基础，还初步构建了嗅觉受体与气味物质互作关系的高通量筛选平台。研究成果不仅为桔小实蝇化学感受相关蛋白功能研究奠定了坚实的基础，还将有助于开发新型诱剂。此外，本研究还将农业害虫嗅觉机制的研究从外周感受机制（气味结合蛋白、嗅觉受体结合的层面）深入到中枢神经系统信号处理的新层次。