家蚕免疫致敏关键基因鉴定及其在Bt介导的靶向RNAi技术防治鳞翅目害虫中的应用研究

Although the phenomenon of immune priming has been found in many insects, the key genes regulating this immune response are still unknown. Bacillus thuringiensis (Bt) is a widely used biological insecticide. Strong resistance to Bt and its toxin have been developed in many insect pests, and this is the key factor that hinders the application of Bt in the field of biological control, but there is no effective method to solve this problem. Many researches showed that the resistance of insect to Bt is related to the host immune priming response induced by this bacterium. The emergence of RNAi technology has opened up a new direction for biological control of pests. This study intends to combine organically the insect immune system, RNAi technology and Bt, and identified the key genes involved in Bombyx mori immune priming induced by Bt using transcriptome analysis and gene editing. Targeting at the genes regulated immune priming, we will construct Bt engineering bacteria by the techniques of PCR, enzyme digestion and site directed mutagenesis, and so on.Using Bt mediated RNAi technology, new type of engineering bacteria can knockout the key genes involved in immune priming based on insecticidal activity of Bt toxin protein by feeding way. Therefore, the engineered bacteria will have double insecticidal activity to prevent the occurrence of immune priming response. This project promised to solve the problem of resistance generated from Bt which is used as a biological insecticide and provide new ideas for more efficient utilization of this agent in biological control.

免疫致敏现象已在众多昆虫中被发现，而调控免疫致敏反应的关键基因仍未知。苏云金芽孢杆菌(Bt)是目前应用较广泛的生物杀虫剂。有害昆虫对Bt及其毒素产生抗性是阻碍Bt在生物防治领域应用的关键因素，至今仍未见有有效方法解决这一问题。研究表明：害虫对Bt产生抗性与Bt诱发宿主免疫致敏反应有关。RNAi技术的出现为害虫的生物防治开拓了新的方向。本研究拟将昆虫免疫系统—RNAi技术—Bt有机结合，利用转录组及基因编辑技术对Bt诱导的家蚕免疫致敏关键基因进行筛选和鉴定。以调控免疫致敏反应关键基因为靶标，通过PCR、酶切反应和定点突变等技术构建Bt工程菌。新型的工程菌在Bt毒蛋白杀虫作用的基础上，利用Bt介导的RNAi技术，通过饲喂的方式实现免疫致敏反应关键基因的沉默，因此，该工程菌将具备阻止免疫致敏现象发生的双重杀虫活性。本项目为解决Bt作为生物杀虫剂所产生的抗性问题及为其更高效应用于生物防治提供新的思路。

免疫致敏现象已在众多昆虫中被发现，而调控免疫致敏反应的关键基因仍未知。苏云金芽孢杆菌(Bt)是目前应用较广泛的生物杀虫剂。有害昆虫对Bt及其毒素产生抗性是阻碍Bt在生物防治领域应用的关键因素，至今仍未见有有效方法解决这一问题。研究表明：害虫对Bt产生抗性与Bt诱发宿主免疫致敏反应有关。RNAi技术的出现为害虫的生物防治开拓了新的方向。本研究拟将昆虫免疫系统—RNAi技术—Bt有机结合，利用转录组及基因编辑技术对Bt诱导的家蚕免疫致敏关键基因进行筛选和鉴定。研究结果显示: Bt免疫致敏家蚕后，血细胞免疫受体相关基因、抗菌肽基因以及相关吞噬信号通路相关基因在免疫致敏后，表达量上调表达显著；差异基因表达分析发现，大量基因与吞噬体形成有关，并高度富集于吞噬信号通路；Dscam基因、PGRP相关基因以及葡聚糖识别受体相关基因是参与调控免疫致敏的关键；进一步利用基因编辑技术对Dscam、βGRPs和PGRPs进行功能验证，确定了免疫致敏靶标基因。以调控免疫致敏反应关键基因为靶标，通过PCR、酶切反应和定点突变等技术构建获得了一株新的Bt工程菌。新型的工程菌在Bt毒蛋白杀虫作用的基础上，利用Bt介导的RNAi技术，通过饲喂的方式实现免疫致敏反应关键基因的沉默，所获得的工程菌具备阻止免疫致敏现象发生的双重杀虫活性。本项目为解决Bt作为生物杀虫剂所产生的抗性问题及为其更高效应用于生物防治提供新的思路。