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Over-expression of RNA interference (RNAi) core machinery improves susceptibility to RNAi in silkworm larvae.
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2020-03-10 , DOI: 10.1111/imb.12639 L You 1 , F Zhang 2 , S Huang 3 , A Merchant 4 , X Zhou 4 , Z Li 1
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2020-03-10 , DOI: 10.1111/imb.12639 L You 1 , F Zhang 2 , S Huang 3 , A Merchant 4 , X Zhou 4 , Z Li 1
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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.
中文翻译：
RNA干扰（RNAi）核心机制的过度表达提高了家蚕幼虫对RNAi的敏感性。
RNA干扰（RNAi）是有机体防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的功效随类群而变化。鳞翅目昆虫在很大程度上对RNAi有害。这项研究的总体目标是克服鳞翅目动物对RNAi的不敏感性。我们假设核心RNAi机械酶的过表达可以提高传统顽固物种的RNAi功效。一种转基因家蚕应变，杆状病毒立即早期基因，蚕Dicer2编码序列的IE1启动子驱动的表达（IE1-BmDicer2），其过表达BmDicer2，通过产生piggyBac转转座子介导的转基因。使用两个指标，即表现出沉默表型的动物比例和沉默时间，来评估沉默效率。在注射后48小时，在转录和翻译水平上均观察到靶基因表达的显着降低。此外，我们共表达了家蚕双歧杆菌Argonaute 2（ BmAgo2）和BmDicer 2，发现22％的动物（n = 18）甚至在72 h时仍表现出明显的沉默效果，这表明这两种RNAi核心机器酶的共表达进一步增加了该蛋白的表达。桑树芽孢杆菌的敏感性注入双链RNA。这项研究为RNAi难治性昆虫类群特别是鳞翅类的功能基因组学研究提供了新的策略。
更新日期：2020-03-10
中文翻译：
RNA干扰（RNAi）核心机制的过度表达提高了家蚕幼虫对RNAi的敏感性。
RNA干扰（RNAi）是有机体防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的功效随类群而变化。鳞翅目昆虫在很大程度上对RNAi有害。这项研究的总体目标是克服鳞翅目动物对RNAi的不敏感性。我们假设核心RNAi机械酶的过表达可以提高传统顽固物种的RNAi功效。一种转基因家蚕应变，杆状病毒立即早期基因，蚕Dicer2编码序列的IE1启动子驱动的表达（IE1-BmDicer2），其过表达BmDicer2，通过产生piggyBac转转座子介导的转基因。使用两个指标，即表现出沉默表型的动物比例和沉默时间，来评估沉默效率。在注射后48小时，在转录和翻译水平上均观察到靶基因表达的显着降低。此外，我们共表达了家蚕双歧杆菌Argonaute 2（ BmAgo2）和BmDicer 2，发现22％的动物（n = 18）甚至在72 h时仍表现出明显的沉默效果，这表明这两种RNAi核心机器酶的共表达进一步增加了该蛋白的表达。桑树芽孢杆菌的敏感性注入双链RNA。这项研究为RNAi难治性昆虫类群特别是鳞翅类的功能基因组学研究提供了新的策略。
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