Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, People's Republic of China.
PLoS One. 2011 Apr 11;6(4):e18644. doi: 10.1371/journal.pone.0018644.
The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.
RNAi 方法成功用于潜在的害虫控制的关键主要取决于精心的靶标选择和方便的输送系统。我们采用第二代测序技术筛选 RNAi 靶标。Illumina 的 RNA-seq 和数字基因表达标签谱（DGE-tag）技术被用于筛选昆虫东方粘虫的最佳 RNAi 靶标。共获得了 14690 个阶段特异性基因，可作为潜在的靶标，其中 47 个通过 qRT-PCR 得到确认。选择 10 个幼虫阶段特异性表达基因进行 RNAi 试验。当将 50ng/µl 的基因 DS10 和 DS28 的 dsRNA 直接喷洒在放置在滤纸上的刚孵化的幼虫上时，幼虫死亡率约为 40∼50%，而当 10 个基因的 dsRNA 与人工饲料一起喷洒在幼虫上时，处理后 5 天死亡率达到 73%至 100%。qRT-PCR 分析验证了幼虫死亡率与靶基因表达下调之间的相关性。局部应用荧光 dsRNA 证实 dsRNA 确实穿透体壁并在体腔中循环。似乎 DGE-tag 与 RNA-seq 的结合是一种快速、高通量、低成本且易于选择 RNAi 候选靶标基因的方法。更重要的是，它表明 dsRNA 能够穿透表皮并导致鳞翅目昆虫幼虫发育迟缓和/或死亡。这一发现大大拓宽了 RNAi 的靶标选择范围，不仅局限于肠道特异性基因，还包括整个昆虫的靶标，可能为设计基于 RNAi 的技术防治昆虫危害提供新策略。
