Activity Analysis Of Far2,the Key Genes Involved In Wax Excretion Of Ericerus Pela,and The Expression Of Ws DsRNA In Vitro

The white wax secreted by the second instar male larvae of Ericerus pela(Chavannes)was a widely used natural wax ester,which is a mixture from various wax monoesters.Resarch had shown that the fatty acid can be reduced to fatty alcohol by FAR,then the wax ester was synthized by fatty acid and fatty alcohol under the action of wax synthase(WS).Although the candidate gene far2 and ws involved in wax secretion had been identified in previous work,far gene function and other characters still need further research.In this study,the prokaryotic expression of FAR2 were obtained after getting the c DNA ORF of E.pela far2 gene,supply the protein to make monoclonal antibody;and FAR2 was successfully expressed in Bm N cells by using Bac-to-Bac baculovirus expression system,The protein FAR2 was collected for enzyme reaction in vitro,and detection of enzyme reaction products by using gas chromatography.And conduct prokaryotic expression to another key enzyme gene of wax ester synthesis.Then a large number of E.pela ds RNAs(double-stranded RNA,ds RNA)were obtained in low cost by prokaryotic expression.The main results was as follows:(1)p ET-30a-e GFP/Epel FAR2,the E.pela FAR2 prokaryotic expression vector was successfully constructed.Then the optimum induction condition was identified by Western blot and analysed by SDS-PAGE.The IPTG adding amount was 0.8mmol·L-1,and the induction time was 8h.(2)Recombinant plasmid p Fast-ie1-FAR2-e GFP of Bac-to-Bac expression system was successfully constructed.Bm N cells were transfected by the recombinant bacmid and lots of fluorescence could be observed under fluorescence inverted microscope.Western blot analysis showed that FAR2 was successfully expressed.Then protein were collected at different time after cells infected by P2 virus strains,and Western blot results showed that 48 h was the best time to collect protein after the cells infected with recombinant bacmid.(3)The enzyme activity was carried out based on atty acyl-Co A with different carbon chain length by using the collected FAR.Then the reaction production was analyzed by GC,and the results showed that C28 fatty acid can be reduced to C28 fatty alcohol by the action of FAR.(4)To prepare a large number of ws ds RNA of E.pela,E.pela L4440/Epel WS interference vector was constructed by using prokaryotic expression system.After transformation,the average amount of ws ds RNA induced by IPTG was 1705ng·m L-1,and the electrophoresis detection showed the ds RNA expression had a good quality whthout tailing phenomenon.Generally,we have performed FAR2 prokaryotic expression,eukaryotic expression,enzyme activity analysis successfully,and obtaining of ws ds RNA.These results laid the foundation for the monoclonal antibody preparation and studies on biochemical characteristics.In addition,the expression of ws ds RNA in vitro has significance for the control of scale insects.