重要农业害虫RNA干扰效率差异机制研究

RNA interference (RNAi) has been widely recognized as a novel strategy for insect pest management. However, there are significant differences in RNAi efficiency among different insect taxonomic groups. For example, coleopterans and orthopterans show significantly higher RNAi efficiency than dipterans and lepidopterans. Such a difference has been regarded as one of the major limiting factors which restrict the application of RNAi for controlling different insect pests. However, we know very little about the molecular mechanisms conferring such differences. This proposed research chooses two important agricultural insect pests, Locusta migratoria (orthopteran) and Ostrinia furnacalis (lepidopteran), as models for comparative studies as they show significantly different RNAi efficiencies. We will focus on the key steps limiting the RNAi efficiency by using integrated approaches, including cell biology, biochemistry, molecular biology and plant biotechnology. We propose to: 1) investigate the biological function of different dsRNases to clarify their effect on the stability of dsRNA in the midgut and hemolymph of the two insect species; 2) compare the cellular uptake and endosomal escape of dsRNA between the two insect species; 3)identify the key genes involved in the systemic RNAi pathway, quantify their expression levels, and study their effect on RNAi efficiency; and 4) utilize the transgenic maize expressing dsRNA to elucidatethe silencing mechanism of plant-mediated RNAi in L.migratoria. This project is expected to elucidate the molecular mechanisms causing differential RNAi efficiencies in different insects, formulate new strategies to overcome the obstacle of RNAi, improve the efficiency of RNAi, and facilitate the application of RNAi-based technologies for insect pest management.

RNAi是一种新型的害虫防控策略。但RNAi效率在不同昆虫类群中差异显著，例如鞘翅目和直翅目等昆虫RNAi效率大大高于双翅目和鳞翅目昆虫，这种差异极大地制约了该技术在害虫防治中的适用性，但其差异机制尚缺乏系统研究。本项目拟选择飞蝗和亚洲玉米螟这两种干扰效率差异较大的重要农业害虫为模式，以制约RNAi效率的关键节点为线条，综合运用细胞生物学、生物化学、分子生物学和植物生物技术等多学科的方法开展比较研究：1)揭示双链RNA酶对两种昆虫中肠和血淋巴dsRNA稳定性及RNAi效率的影响；2）比较两种昆虫对外源dsRNA的吸收途径及内涵体逃逸机制；3）鉴定RNAi通路关键因子并研究其对RNAi效率的影响；4）以表达dsRNA的转基因玉米为材料，探讨植物介导表达dsRNA的抗虫机理。本项目将系统解析昆虫RNAi差异的分子机制，探索改进干扰效率的策略，以提高RNAi效率并促进其在农业害虫控制中的应用。

本项目选择飞蝗和亚洲玉米螟这两种重要农业害虫为模式，以制约RNAi效率的关键节点为线条开展了如下研究：1）比较研究了影响dsRNA稳定性的关键因子及对RNAi效率的作用。通过细胞荧光素酶实验及异源表达技术，明确了肠道核酸酶dsRNase2是导致中肠dsRNA稳定性的关键因子；通过体外组织培养及小RNA测序技术，揭示了亚洲玉米螟dsRNA的稳定性和碱基偏好性是导致其RNAi效率低的主要原因。2）比较研究了dsRNA细胞吸收的关键因子及对RNAi效率的影响。采用RNAi of RNAi实验、药理学实验、荧光示踪技术和免疫组化技术等，系统研究了dsRNA内吞关键因子对dsRNA吸收效率的影响，发现载脂蛋白是dsRNA血淋巴的主要结合蛋白；清道夫受体和载脂蛋白受体参与了dsRNA的吸收；网格蛋白依赖的内吞、小窝/脂筏介导的内吞、巨胞饮及SID参与了飞蝗不同组织细胞的dsRNA吸收。3）研究了核心酶系统对RNA干扰效率的影响。采用siRNA免疫沉定、异源表达、荧光素酶实验及小RNA测序技术研究了核心酶通路关键蛋白对飞蝗RNA干扰效率的影响，揭示了飞蝗Loquacious 3个不同的选择性剪切参与了不同的小RNA通路；明确了小RNA通路关键蛋白Dicers和Agos对siRNA介导的RNAi效率的贡献。4）开展了植物介导表达dsRNA的抗虫机理研究。采用小RNA测序技术，发现了转基因玉米剪切的高丰度siRNA与飞蝗剪切的高丰度siRNA序列高度一致，并验证了高丰度siRNA是转基因玉米沉默飞蝗靶标基因的主要因子，揭示了转基因玉米致死昆虫的分子机制。5）建立了飞蝗基因编辑技术平台，成功获得 dsRNase2、Dicer2a、Dicer2b 基因编辑飞蝗。项目系统解析了昆虫RNAi差异的分子机制，明确了制约RNAi效率的关键因子，探索了改进干扰效率的路径，将有力促进其在农业害虫控制中的应用。