The Effect Of Double-stranded RNA Endonuclease On RNAi Efficiency In Plutella Xylostella

Recent studies have shown that a type of double-stranded RNA endonuclease(ds RNase)is contained in insects,which can reduce the RNAi efficiency of insects by degrading double-stranded RNA(ds RNA).In this study,The c DNA sequences of the Pxds RNases were identified,and their effects on the RNAi efficiency in P.xylostella was checked in vivo and in vitro.The main findings were as follows:Four distinct c DNA fragments putatively encoding Pxds RNase genes(Pxds RNase1,Pxds RNase2,Pxds RNase3,Pxds RNase4)were identified.Additionally,domain analysis showed that all enzymes contained an Endounuclease NS domain and a signal peptide with Pxds RNase4 not having a signal peptide.The four Endounuclease NS domains were compared to figure out that all four proteins contained active sites,substrate binding sites and Mg2+ binding site.Through evolutionary tree analysis,both Pxds RNase2 and Pxds RNase3 had the highest homology with ds RNases in other Lepidoptera,and Pxds RNase4 had the highest homology with ds RNases in Diptera.For stage-specific expression of four Pxds RNases,Pxds RNase1 and Pxds RNase4 were expressed in all stages of P.xylostella with the highest expression at the 4th instar larvae;Pxds RNase2 and Pxds RNase3 were only expressed in the larval stage with the highest expression level at the 4th instar larvae,and there was almost no expression in pupae and adults.For tissue-specific expression,Pxds RNase1 expression was of the highest in the hemolymph,followed by the fat body;Pxds RNase2 and Pxds RNase3 were almost specifically expressed in the midgut;Pxds RNase4 was expressed in all tissues.To study the function of Pxds RNases on the RNAi efficiency,Chitin of P.xylostella(Px Cht)was selected as the reporter gene.the transcription level of Px Cht were detected after injection or feeding the mixture of ds Pxds RNase1/2/3/4(ds RNA of Pxds RNase1/2/3/4)and ds Px Cht(ds RNA of Px Cht).The mixture of ds Px Cht and ds EGFP was used as a control.In the injection group,The transcription of Px Cht in the larvae injecting the mixture of ds Px Cht and ds Pxds RNase1/2/3 reached significantly higher levels than that in the larvae ingesting ds Px Cht and ds EGFP,and Pxds RNase1 had the greatest effect on the RNAi efficiency,but Pxds RNase4 no effect;In the feeding group,the transcription of Px Cht in the larvae ingesting the mixture of ds Px Cht and ds Pxds RNase1/2/4 reached significantly higher levels than that in the larvae ingesting ds Px Cht and ds EGFP,and it was surprising that ds Pxds RNase4 effectively reduced the transcript level of Pxds RNase1,further reducing the RNAi efficiency.The recombinant enzymes,His-Pxds RNase1,His-Pxds RNase2 and His-Pxds RNase3,were successfully expressed in PET-30a(+)vector using the prokaryotic expression system.Incubating ds RNA with recombinant protein,the recombinant protein of Pxds RNase1 could degrade ds RNA rapidly,Pxds RNase3 could cleave ds RNA without complete degradation,and Pxds RNase2 could not degrade ds RNA.These results suggest that the roles of Pxds RNase1,Pxds RNase2,and Pxds RNase3 involved in the RNAi process in P.xylostella might be different.