Synthesis Of DsRNA Of Wax Ester Synthase Gene In Ericerus Pela And Its Expression In Spodoptera Frugiperda Sf9 Cell Line

The white wax is a very important industrial raw material which is secreted by the male scale Ericerus pela Chavannes during the second instar larval stage.It has been widely used in chemical,pharmaceutical,food,and cosmetics industries.Wax synthase is the key synthase in wax biosynthesis,catalyticing long chain fatty alcohols and long chain fatty acyl-coenzyme A to generate wax ester.We amplified the cDNA of ws according to the full-length sequences we obtained preliminary,synthetized dsRNA in vitro with kits then injectioned in female adults of E.Pela and detected the interference effect of ws by qRT-PCR.Laiding the foundation for verifying the physiological function of WS.Besides,we used Bac-to-Bac expression system to constructed pFastBac^TMHT B expression vector of ws,and transforming E.coli DH10 Bac^TM,after resistant and blue-white screening,we obtained the recombinant bacmid rBacmid/EpelWS.Then transfected r Bacmid/EpelWS into Sf9 cells,and expressed WS preliminarily.We used immune fluorescence and western blot to observe and verify the expresstion of WS.The main findings are as follows:?1?dsRNA of WS was transcribed in vitro with kits,Sequencing results showed that dsRNA was successfully synthesized.?2?qRT-PCR showed that the expresstion of ws mRNA was significantly reduced in E.Pela with an injection of ws' s dsRNA compared with the control group which had an injection of gfp's dsRNA after a week,which laid the foundation for the follow-up study of its function.?3?WS expression vector was successfully constructed by Bac-to-Bac expression system,PCR identification and sequencing results showed that the target fragment was inserted into the vector.The recombinant plasmid rBacmid/EpelWS was successfully obtained and after transfecting Sf9 cells,P3 virus was harvested.Western blot showed the presence of target protein in the total protein of Sf9 cells infected by P3 virus,WS was successfully expressed.This study transcribed the ws' s dsRNA of E.Pela in vitro,and successfully expressed WS in insect cells,which laid a foundation for further study of the biochemical properties and physiological function of WS in E.pela.