棉铃虫对质体介导RNAi不应性的分子机制与治理策略研究

With development of pesticide and Bt resistant evolved by cotton bollworm, it becomes very important and urgent to develop new and alternative biotechnology for efficient pest control. Previously, we have established an efficient technology for control Colorado potato beetle by plastid-mediated RNA interference (RNAi). However, no effect was observed when we used the similar technology for controlling of Cotton bollworm. In this proposal, we are aimed to explore the molecular mechanism of refractoriness to RNAi in Helicoverpa armigera. We will identify the key RNAi molecules that confer the resistance to H.armigera and elucidate the role of siRNA methylation in RNAi-mediated plant protection. Impact of midgut dsRNase on the refractoriness to RNAi will also be investigated. Furthermore, "avoidance" and "inhibition" strategies for pest control will be verified by increase the expression levels of insecticidal RNAi molecular and co-silence the expression of dsRNase and target genes. Inhibition of dsRNase activity would enhance the ability of insect resistance by plastid-mediated RNAi. Taken together, the completeness of this project will contribute to elucidate the mechanism of RNAi-mediated crop protection technology for pest control, expand the scope of application of this technology, and provide the basis for engineering resistance against other insect pests.

随着棉铃虫对农药与Bt的抗性进化，开展备选抗虫技术研究将十分迫切和重要，利用RNAi技术来控制害虫有很大的发展潜力。前期，我们利用植物质体高效与稳定表达杀虫RNA的特点与优势，创建了质体介导的RNAi抗虫技术，在对马铃薯甲虫的防治中取得了良好效果，而对棉铃虫的防治中效果却不佳。本项目将探索棉铃虫对质体介导RNAi不应性的分子机制，鉴定植物介导RNAi抗虫所需的关键RNAi分子，研究siRNA甲基化在RNAi抗虫中的作用，探索棉铃虫中肠dsRNA酶对RNAi不应性的影响。进而提出并验证“回避”与“抑制”的抗虫新策略。提高关键抗虫RNAi分子在质体中的表达水平。共沉默dsRNA酶基因和靶基因的表达，抑制dsRNA酶对RNAi不应性的影响，增强质体介导RNAi植物的抗虫性。成果将有助于阐明植物介导RNAi的抗虫机制，扩展该技术的应用范围，为其它害虫的防控提供理论基础与技术手段。

植物介导RNAi抗虫技术是一种非常有潜力的害虫防治策略，虽然目前植物介导RNAi抗虫技术对棉铃虫防控中取得了一些效果，但还存在一些问题需要解决：1）植物介导RNAi抗棉铃虫关键RNAi分子还没有鉴定；2）棉铃虫中肠dsRNA酶如何影响对RNAi不应性还不清楚；3）如何提高质体介导RNAi抗棉铃虫的效果。在该项目的支持下，项目负责人从细胞核转基因植物与质体转基因植物表达RNAi分子不同类型，以及棉铃虫中肠液对不同RNAi分子类型的降解等方面去揭示了植物介导RNAi抗棉铃虫的内在分子机制。结果发现siRNA才是植物介导RNAi抗棉铃虫最有效的分子，同时也证实了棉铃虫中肠中高活性的dsRNase对dsRNA的降解导致了棉铃虫对质体介导RNAi的不应性，进一步通过在质体中表达大量的甲基化siRNA提高了质体介导RNAi抗棉铃虫的效果。研究结果具有创新性，开辟了RNAi防控棉铃虫新策略，进一步可扩展到其它害虫的防控研究中，为植物保护提供新的思路和手段。