Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors
RNAi (RNA interference) is a technology for silencing of target genes via sequence-specific manner. RNAi technology has been used for development of anti-pathogenic crops. In 2007, development of transgenic plants resistant to insect herbivore using RNAi technology was first reported, leading to a burst of efforts aimed at exploitation of RNAi mechanism and control strategy against variety of insect species based on this technique. Mythimna separata belongs to noctuidae family of lepidoptera and is posing threat to crops of economic importance. Recently, outbreaks of M. separata severely threatens corn production in Northern China, calling for new control approaches.
Chitinase genes were chosen as the target genes as they were expressed predominantly in the gut tissue and were reported to be ideal silencing targets in several insect species. Interfering sequences against the target genes were cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3) which was defective in dsRNA degradation activity, so preserving the dsRNA from degradation by cellular machinery. The bacteria were mixed with artificial diet and were fed to M. separata. We showed that oral delivery of bacterially expressed dsRNA would lead to RNAi effects in the recipient insect. Quantitative real-time PCR results showed that expression level of target MseChi1 and MseChi2 genes in gut tissue of M. separata were down-regulated after oral delivery of engineered bacteria expressing the corresponding dsRNA. Sequence-specific siRNA (small interfering RNA) was detected in recipient insects, supporting the existence of siRNA-mediated silencing effects in M. separata. Furthermore, knockdown of MseChi1 and MseChi2 resulted in increased mortality and reduced body weight of the feeding larvae.
We reported a simple and low cost experimental procedure to silence M. separata endogenous gene expression. Our research provides both an experimental foundation for using RNAi technology to control M. separata and also a useful research tool for loss-of-function study of important developmental and regulatory genes in this insect species.