Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects

RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.RNA干扰（RNA interference, RNAi）已成为真核生物中研究基因功能的通用反向遗传学工具，目前也正被开发为害虫治理技术。不同生物的RNAi效率存在显著差异，不同昆虫目类的RNAi效率亦各不相同，范围从高效率的鞘翅目昆虫（coleopterans）到极低效率的鳞翅目昆虫（lepidopterans）不等。本研究针对鳞翅目与鞘翅目细胞系之间，以及科罗拉多马铃薯甲虫（Leptinotarsa decemlineata）与烟芽夜蛾（Heliothis virescens）之间的RNAi效率差异原因展开了探究。无论是注射还是饲喂的双链RNA（double-stranded RNA, dsRNA），在烟芽夜蛾体内的降解速度均快于科罗拉多马铃薯甲虫。鳞翅目与鞘翅目的细胞系及组织均能高效摄取双链RNA。值得注意的是，向鞘翅目细胞系和组织中递送的双链RNA可被摄取并加工为小干扰RNA（small interfering RNA, siRNA）；而鳞翅目细胞系和组织虽能摄取双链RNA，但从这些样本中提取的总RNA内未检测到小干扰RNA。本文所呈现的数据表明，双链RNA的降解与胞内运输是导致鳞翅目昆虫RNAi效率低下的主要因素。