鳞翅目昆虫RNAi通路相关基因的功能及其作用机理研究

Lepidopteran insects are the major pests in agriculture, so clarifying its RNAi mechanism will not only beneficial to study the gene function of insects, but also help to use RNAi as a pollution-free technology in insect pest control. By means of transcriptome analysis for dsRNA induced Asian corn borer and various experiments, we discovered seven genes which expression level only can be induced by dsRNA, the known key genes of Ago-2 and Dicer-2 in RNAi pathway were involved in. The preliminary results about the remaining five genes indicated that up56 is a Lepidopteran insect specific gene, and it is also the one of the most dramatic changes after induced by dsRNA. When the expression level of up56 was suppressed, the RNAi efficiency of other genes of Lepidopteran insects can be enhanced significantly. Up26 is the earliest response gene to dsRNA among the seven genes, it may regulate the dsRNA recognition and affect the RNAi pathway. This project will use Asian corn borer, cotton bollworm, diamond back moth, and silkworm as the research objects, focus on the gene function, mechanism and its relationship with RNAi pathway of up56 and up26, and explore the function of other three genes. The final goal is clarifying the RNAi mechanism of Lepidopteran insects. The research results of this project not only have important scientific significance, but also expect to improve the current situation of the low RNAi efficiency of Lepidopteran insects, promote the application of RNAi technology in insect pest control.

鳞翅目昆虫是最主要的农业害虫，弄清其RNAi机理不仅有利于昆虫基因功能研究，而且有助于将RNAi技术早日应用于害虫的无公害治理。通过对dsRNA诱导后玉米螟转录组分析及实验验证，发现了7个仅受dsRNA诱导的基因，其中包括RNAi通路关键基因Ago-2和Dicer-2。对其它5个基因的初步研究表明：up56是鳞翅目昆虫特异的、受dsRNA诱导后变化最剧烈的基因。抑制up56的表达，能够提高其它基因的RNAi效率；up26是对dsRNA响应最早的基因，可能通过调控昆虫对dsRNA的识别而影响RNAi通路。本项目将以亚洲玉米螟、棉铃虫、小菜蛾及家蚕为研究对象，重点研究up56和up26的基因功能、作用机理及其与RNAi通路的关系，探索其它3个基因的功能，解析鳞翅目昆虫的RNAi机理。本研究结果不仅具有重要的科学意义，而且有望改善鳞翅目昆虫RNAi效率较低的现状，促进RNAi在害虫防治中的应用。

化学农药的大量使用，一方面大幅促进了农业的增产增收，另一方面也带来了严重的环境污染。急需开发出高效、低毒、绿色无污染的新型生物农药。. RNAi生物农药的核心成分是dsRNA，其结构和组成与天然有机物中存在的核酸完全一致，能够在自然条件下充分降解，并重新作为动植物的营养成分被循环利用。本项目通过对RNAi机理的研究，为RNAi技术早日应用于害虫的无公害治理提供了重要基础。通过四年的研究取得如下几个方面的进展：. （1）影响RNAi效率的基因分析. 通过内源和外源dsRNA处理，发现了7个受外源dsRNA强烈诱导的基因，我们推测这些基因可能参与了鳞翅目昆虫的RNAi通路，为提高害虫的RNAi效率奠定了基础（见附件1）。. （2）证明了REase基因具有提高鳞翅目昆虫RNAi效率的作用. REase是鳞翅目昆虫特有的基因，抑制该基因的表达，可以显著提高这类昆虫的RNAi效率，同时提高RNAi对昆虫的致死效率（附件2）。. （3）抑制REase基因可以提高害虫对BT的敏感性. 以棉铃虫为研究对象，利用基因编辑技术将其内源的REase基因敲除，结果发现，棉铃虫对Bt毒素的敏感性显著提高。为RNAi和BT在害虫防治中的应用提供了新方案（见附件3）。. （4）发现了鳞翅目昆虫和鞘翅目昆虫对dsRNA体内加工规律的差异. 通过对REase基因的研究发现，dsRNA在昆虫体内的剪切具有序列特异性，与起始位点和长度无关。鳞翅目昆虫的剪切偏好位点是GGU，而鞘翅目昆虫却没有这种偏好性。该研究从一个侧面说明了为什么鞘翅目昆虫的RNAi效率高于鳞翅目昆虫，并进行了科普宣传（见附件4-5）。