飞蝗dsRNA降解酶的生化功能研究及抑制剂筛选体系的建立

The application of RNA interference (RNAi) technology in pest control is a new strategy in plant protection. However, the presence of dsRNase in locusts can rapidly degrade exogenous dsRNA and then result in ineffective RNA interference, which has become a bottleneck in the application of RNAi technology. Analyzing the biochemical functions of these enzymes and screening their inhibitors can promote the application of RNAi technology in pest control. This project aims to conduct as following: (1) obtain LmdsRNase proteins with high purity and activity by eukaryotic expression system, and determine its sequence preferences by in vitro degradation experiments combined with small RNA sequencing; (2) synthesize multiple fluorescent labeled interfering RNA to detect the kinetic parameters of the enzyme degradation of different substrates; (3) construct screening system for dsRNA degradation enzyme activity based on luciferase reporting system. The screening system was used to identify the key amino acid sites of the degrading enzymes, and small molecule inhibitors were screened for these key sites. These results would help researchers to better design dsRNA or mixtures of dsRNA to improve the stability of dsRNA, and thereby improve the efficiency of insect RNAi, and ultimately break through the bottleneck of the application of RNAi technology in entomology and plant protection.

RNAi（RNA interference）技术应用于害虫防治是植保行业的新策略。但在飞蝗体内存在dsRNA降解酶（dsRNase）可快速降解外源dsRNA，导致RNA干扰无效，这成为RNAi技术应用的瓶颈。解析这一类酶的生化功能以及筛选其抑制剂，可以促进RNAi技术应用于害虫防治。本项目拟开展如下工作：（1）用真核表达系统获得高纯度高活性的LmdsRNase蛋白，体外降解实验结合小RNA测序技术明确其切割dsRNA的序列偏好性；（2）合成荧光标记的多种干扰RNA，检测该酶降解不同底物的动力学参数；（3）基于荧光素酶报告系统构建dsRNA降解酶活性筛选体系，鉴定该酶的关键氨基酸位点，针对关键位点筛选小分子抑制剂。研究结果有助于科研工作者更好地设计dsRNA或设计dsRNA混配制剂以提高dsRNA的稳定性，进而提高昆虫RNAi的效率，最终突破RNAi技术在昆虫学及植物保护领域应用的瓶颈。

RNA生物农药被列入《“十四五”全国农药产业发展规划》优先发展名录，但昆虫RNAi效率高低成为其开发的重要技术瓶颈。前期研究发现降低昆虫dsRNA降解酶（dsRNase）的活性可提高昆虫RNAi效率。在飞蝗肠道内存在dsRNA降解酶可快速降解外源dsRNA，导致飞蝗饲喂RNA干扰无效。解析这一类酶的生化功能以及筛选其抑制剂，可以促进RNAi技术应用于害虫防治。本研究共开展了五方面的研究内容，第一，获得了高纯度高活性的LmdsRNase蛋白；第二对其序列偏好性进行了分析，发现LmdsRNase1和2在降解dsRNA的长度丰度、位置以及碱基端均存在差异；第三对其底物选择性进行了分析，发现LmdsRNase可以降解dsDNA；第四，基于荧光素酶报告系统构建了这一类酶的活性筛选体系，并在飞蝗和亚洲玉米螟上进行了验证，该体系可行并成熟；第五，鉴定了飞蝗dsRNA降解酶的多个关键氨基酸位点，并对三种候选抑制剂进行了初步检测。研究结果可为RNA生物农药的开发提供理论指导，并在完善抑制剂的工作后有望开发RNAi的害虫防治产品。