昆虫侵害诱导的茶树共性挥发物β-罗勒烯释放及其信号传导的机制研究

In response to herbivore attacks, plants can synthesize and emit many volatile compounds. Herbivore-induced plant volatiles (HIPVs) play important ecological roles in defense against stresses. HIPVs are known to be involved in direct defenses against herbivores and indirect defenses by attracting herbivore enemies, they also play important but relatively unappreciated roles in within-plant or plant-to-plant signaling. In contrast to model plants, there are only limited reports of HIPVs signaling in crops. Tea (Camellia sinensis) is an important crop in China. Our preliminary experiments found that herbivore attacks led to high accumulation and emission of volatile compound β-ocimene from tea plants, which induced defensive metabolite changes by volatile signaling in neighboring plants. Furthermore, jasmonic acid content and its biosynthesis-related key transcript factor MYC2 expression level were also significantly enhanced by the wounding derived from the herbivore attacks. In addition, we obtained the preliminary evidences of involvement of jasmonic acid and MYC2 in up-regulation of β-ocimene synthase, which resulted in biosynthesis of β-ocimene in response to the wounding derived from the herbivore attacks. ABC transporters were proposed to be involved in regulation of emission of β-ocimene from tea plants exposed to herbivore attacks. This research proposal will further elucidate regulation mechanism of β-ocimene synthase by MYC2 based on transient transactivation assay and EMSA assay. Moreover, this research proposal will identify ABC transporters regulating emission of β-ocimene using yeast assay and transport assays with tobacco BY-2 cells, and study how β-ocimene as volatile signaling affects formation of defensive compounds in neighboring plants. This research proposal will provide evidence of occurrence of ABC transporters regulating HIPVs emission in plants for the first time. The results obtained within these studies will advances our understanding of the ecological functions of HIPVs and can be used to develop tea biological control agents against pest insects in the future.

虫害诱导植物挥发性物质（HIPVs）的生态学功能目前大多集中于直接抵御和吸引害虫天敌的研究，而关于其作为信号传导分子研究非常少。前期实验以茶树作为研究对象，已筛选出作为信号分子参与抗虫代谢物形成的挥发性物质β-罗勒烯；虫害可能通过茉莉酸响应路径上关键的转录因子MYC2，进而激活β-罗勒烯合成基因，促使β-罗勒烯合成；ABC转运蛋白可能参与β-罗勒烯的释放；释放的β-罗勒烯可作为信号分子激活临近植株的防御代谢物变化。本项目拟通过拟南芥原生质体瞬时转录激活体系和凝胶迁移实验研究MYC2对β-罗勒烯合成基因的调控；采用酵母和烟草悬浮细胞转运体系功能鉴定调控β-罗勒烯释放的ABC转运蛋白；研究β-罗勒烯处理对茶树中抗虫物质合成路径影响。本研究有望首次鉴定植物中调控HIPVs释放的ABC转运蛋白，获得β-罗勒烯作为植物间信号分子诱导茶树抗虫代谢物形成的直接证据，为今后茶树抗虫的分子改良靶点提供参考。

虫害诱导植物挥发性物质（HIPVs）的相关研究目前主要聚焦在合成和生态学功能方面，关于其如何从植物体内释放出来的机制尚不清楚。本研究围绕着解决茶树共性抗虫挥发性物质β-罗勒烯如何形成与释放的科学问题开展研究。①在合成机制的研究方面：不同添加方式并结合抗虫评价发现β-罗勒烯可以信号传导的方式增强临近健康茶树的抗虫能力；基因表达量分析、异源体内功能验证和亚细胞定位分析表明CsBOS1是参与虫害诱导β-罗勒烯合成的关键基因；基因表达量分析和转录激活实验结果表明CsMYC2a是调控昆虫侵害下CsBOS1表达的关键上游转录因子；代谢物分析和基因表达量分析发现CsBOS1规律性表达可能是引起β-罗勒烯规律性合成的原因之一。②在释放机制的研究方面：外源抑制剂处理结合代谢物分析证实了转运蛋白确实参与β-罗勒烯的释放；基因表达量分析筛选了参与β-罗勒烯规律性释放的候选转运蛋白；利用异源植株过表达和基因沉默体系，筛选并初步建立了挥发性物质转运蛋白的功能验证体系。虫害诱导茶树合成和释放共性挥发性物质β-罗勒烯可作为信号分子，影响临近健康茶树的防御能力。侵害造成的损伤诱导茉莉酸的积累并激活其信号传导途径，进而调控合成基因CsBOS1表达，最终诱导β-罗勒烯在黑暗条件下大量的合成；合成后的β-罗勒烯可在转运蛋白的参与下，在白天中被转运释放到外界环境中。本研究首次鉴定植物中调控HIPVs释放的ABC转运蛋白，获得β-罗勒烯作为植物间信号分子诱导茶树抗虫代谢物形成的直接证据，为今后茶树抗虫的分子改良靶点提供参考。