Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.
School of Life Science, Shanghai University, Shanghai, China.
Insect Mol Biol. 2020 Aug;29(4):353-362. doi: 10.1111/imb.12639. Epub 2020 Mar 10.
RNA interference (RNAi), one of the strategies that organisms use to defend against invading viruses, is an important tool for functional genomic analysis. In insects, the efficacy of RNAi varies amongst taxa. Lepidopteran insects are, in large part, recalcitrant to RNAi. The overall goal of this study is to overcome such insensitivity in lepidopterans to RNAi. We hypothesize that over-expression of core RNAi machinery enzymes can improve RNAi efficacy in traditionally recalcitrant species. A transgenic Bombyx mori strain, Baculovirus Immediate-Early Gene, ie1, promoter driven expression of silkworm Dicer2 coding sequence (IE1-BmDicer2), which over-expresses BmDicer2, was generated by piggyBac transposon-mediated transgenesis. Two indexes, the ratio of animals that showed a silencing phenotype and the duration of silencing, were used to evaluate silencing efficiency. Significant knockdown of target gene expression was observed at 48 h postinjection at both the transcriptional and translational levels. Furthermore, we coexpressed B. mori Argonaute 2 BmAgo2）and BmDicer 2 and found that 22% of the animals (n = 18) showed an obvious silencing effect even at 72 h, suggesting that coexpression of these two RNAi core machinery enzymes further increased the susceptibility of B. mori to injected double-stranded RNAs. This study offers a new strategy for functional genomics research in RNAi-refractory insect taxa in general and for lepidopterans in particular.
RNA 干扰 (RNAi) 是生物体抵御入侵病毒的策略之一，是功能基因组分析的重要工具。在昆虫中，RNAi 的效果在不同类群之间有所差异。鳞翅目昆虫在很大程度上对 RNAi 不敏感。本研究的总体目标是克服鳞翅目昆虫对 RNAi 的这种不敏感性。我们假设核心 RNAi 机制酶的过表达可以提高传统上不敏感物种的 RNAi 效果。通过转座子介导的转座生成了转 BmDicer2 编码序列（IE1-BmDicer2）的转基因家蚕品系，即杆状病毒立即早期基因 ie1 启动子驱动的表达，该品系过表达了 BmDicer2。两个指标，即表现出沉默表型的动物比例和沉默持续时间，用于评估沉默效率。在转录和翻译水平上，在注射后 48 小时观察到靶基因表达的显著敲低。此外，我们共表达了家蚕 Argonaute 2（BmAgo2）和 BmDicer 2，并发现即使在 72 小时，22%的动物（n=18）表现出明显的沉默效应，表明这两种 RNAi 核心机制酶的共表达进一步增加了家蚕对注射双链 RNA 的敏感性。本研究为 RNAi 抗性昆虫类群的功能基因组学研究提供了一种新策略，特别是对鳞翅目昆虫。
