Feeding RNA Interference Technology Of Entomopathogenic Nematode

Genes of entomopathogenic nematode are of highly homologous with those of the model organism Caenorhabditis elegans.However,the researches on genetic function of parasitic nematodes through C.elegans are limited,particularly in clarifying the symbiotic mechanisms between parasite and host.It is urgent to establish technologies for researching genetic function of entomopathogenic nematodes.This study used the nematode Steinernema carpocapsae as a model system to explore the method of feeding RNAi.It provided a new techniques for researching genetic function of entomopathogenic nematodes..The main conclusions obtained in this study were as follows:1.S.carpocapsae was fed with E.coli HT115 strain on the medium witha diameter of 90mm.The best inoculation amount was 500 infective juveniles(IJ)/dish with the GO nematodes recovery of more than 80%,the highest production of 4037 IJ/dish,the IJ rate of 92%.It was no effect on nematode development to add 50 mg/L of the Amp or 0.4mM IPTG or 50 mg/Amp +0.4 mM of IPTG L in NGM medium.2.In this study,conserved sequence of other nematodes che-11 genes was analysed to designed degenerate primers.PCR amplified was performed to obtain homologous fragments of che-11 gene and then to construct the recombinant plasmid of LA440-che-11.The vector was transformed into E.coli strain HT115 to get RNAi expression strain targeted che-11 gene.3.S.carpocapsae was fed with RNAi expression strain of E.coli targeted che-11 gene.SYBR-Green real-time fluorescence quantitative PCR was employed to test che-11 expression level of nematode.It was found that che-11 gene expressed downregulated of 0.63 times.It suggested the feeding RNAi technology of entomopathogenic nematode worked.