Elucidation Of Exogenous DsRNA/siRNA Mediated RNAi Mechanism In Mythimna Separata

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that widely occurred in eukaryotes. RNAi is mediated by endogenous or exogenous long double-stranded RNA, which is digested by an enzyme belongs to RNase Ⅲ family called Dicer into 21-25 nt double-stranded siRNA. Then the siRNA would bind to Argonaute and Dicer to form the RNA induced silencing complex (RISC) which could guide the antisense strand of siRNA to recognize and integrate it with the target mRNA. After that, the target mRNA would be degraded by endonuclease and caused the silencing of target gene.This study used Lepidoptera insect Mythimna separata as a material, and chose β-actin as RNAi target given its high level of expression. We delivered dsRNA and siRNA to Mythimna separata insect via oral feeding to induce RNAi response and investigate the structure and abundance of siRNA by small RNA sequencing. The length ranges of small RNAs detected are 18-35nt. After sorting the data of sequencing and bioinformatical analysis like reference mapping, small RNA coverage and length distribution, we found that:(1) Both sense and antisense strand have a variant degree of small RNA enrichment and there are several small RNA high-abundance hot spot on target mRNA; (2) Small RNA enrichment on antisense strand was only appeared in the dsRNA or siRNA template region; (3) In the sense strand, we have detected the small RNA both within and out of the dsRNA template region. As no RdRp and its homologous genes were found inside the Mythimna separata, that may be attributed to another endopolymerase which have the similar function with RdRp and extend the effect of mRNA degradation to the outside of the dsRNA template region, then the mRNA fragments were enriched in the sense strand.In order to verify the results of small RNA sequencing, we have detected and confirmed the existence of the phenomenon of small RNA hot spot enrichment by Northern blot experiment. In addition, we have done the qPCR experiment to confirm the silencing of β-actin caused by the action of RNAi, as is consistent with the cleavage state of target site in the sequencing.There have many reports about interference of endogenous gene of insect by using RNAi techor ology at present. And it would be a new research issue of insect control by development of transgenic plant using RNAi techonology, but there were few reports about insect RNAi mechanism especially related to small RNA. We have analyzed the RNAi mechanism mediated by dsRNA/siRNA in detail as well as the structure and abundance of small RNA through the small RNA sequencing method for the first time. Then we have disscussed the RNAi model inside the Mythimna separata.