Genome-wide Characterization And Expression Profiling Of The RNAi Pathway Genes In Plutella Xylostella (L.) And Functional Analysis Of PxDcr-2

RNAi (RNA interference), a highly conserved mechanism in eukaryotic organisms, is triggered by the presence of double-stranded RNA (dsRNA), resulting in the degradation-or translation-repression of complementary single-stranded RNA. RNAi can occur through three pathways, endogenous small interfering RNA (siRNA), micro-RNA (miRNA) and PIWI-interacting RNA (piRNA), according to their biogenesis mechanisms and the type of Argonaute proteins with which they are associated. Although the core components of RNAi pathway are usually well conserved among species, the degree of conservation of these proteins are often diverse among insect species. Therefore, we present a genome-wide investigation of the core RNAi pathway genes in Plutella xylostella, a worldwide destructive pest attacking cruciferous food crops based on the published genome and transcriptome by comparing with orthologues from Caenorhabditis elegans, Drosophila melanogaster, Tribolium castaneum and Bombyx mori. The result revealed two Dicer, one Drosha, five Argonaute, one Pasha and one Loquacious genes except R2D2 gene in the P. xylostella genome, and stage-and tissue-specific expression profiling of these ten genes with qPCR.As a core component of RNAi pathway, Dicer-2 protein determines the production of siRNA and is crucial for the efficiency of RNAi and Dicer-1 is decisive for miRNA. In this study, the full-length complementary DNA (cDNA) of P. xylostella Dicer-1 (PxDcr-1) and P. xylostella Dicer-2 (PxDcr-2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of PxDcr-2 gene. The complete PxDcr-1 CDS was 8,457 bp in length with an open reading frame (ORF) of 2,818 amino acids, and the complete PxDcr-2 CDS was 5,091 bp in length with an open reading frame (ORF) of 1,696 amino acids. Phylogenetic and protein domain analysis showed that PxDcr-1 and PxDcr-2 protein were both similar to Lepidoptera insects, especially Danaus plexippus. In the RNAi experiment, the mRNA level of PxDcr-2 was significantly reduced by PxDcr-2 double-stranded RNA (dsDcr-2-2), but had no significant effect on the development of P. xylostella after dsRNA ingestion. For studying the role of PxDcr-2 in RNAi pathway of P. xylostella, dsDcr-2-2 and dsBursa/dsCYP were injeted to P. xylostella at the same time. And then, the mortality and the rate of malformation were decreased, which demonstrated that PxDcr-2 played an important role in RNAi pathway of P. xylostella.