Silencing Of V-atpase Subunit B In Mythimna Separata Though RNA Interference

RNA interference(RNAi)is a specific gene silencing mechanism mediated by double-stranded RNA(dsRNA)that can be used to study individual gene function.The V-ATPase is widely distributed in prokaryotes and eukaryotes including endosomes,lysosomes,Golgi-derived vesicles,secretory vesicles,eukaryotic cellular membranes,principal cell apical membranes,and for some cells,the plasma membrane.The main function of V-ATPases is hydrolysis ATP and proton transport,or controls the cytoplasmic pH in neutrophiles and macrophages.In insect,the V-ATPase provide the energy absorption of amino acids,which is the crucial substances in process of insect growth and development.There for,any interference of the insect V-ATPase B would dramatic effect the insect growth and development,and even result the death of the insect.In this study,the Oriental armyworm(Mythimna separata,Walker)V-ATPase subunit B cDNA fragment was cloned by RACE technology.The dsRNA of the V-ATPase B were synthesized in vitro and micro-injected armyworm(M.separata)larvae.Phenotype changes and the expression of V-ATPase were analyzed by qRT-PCR.The results showed that the relative expression level of V-ATPAse B in M.separata which injected by the dsRNA of the coding sequences of V-ATPAse B were suppressed and decrease insect survival rates.The results indicated that the RNAi effect was influenced by the dose and the time of injection.The dose for each larva should be as high as 0.45 ug or around,the best developmental period for injection was at early third instar.Transmission Electron Microscope(TEM)results showed that the projections of goblet cells were disorganized,with apical portions slightly inflated.The structure of the microvillus was disordered.It was clear that the nuclei were getting fewer and smaller.These results propose that V-ATPases affect microvilli synthesis in the midgut.In addition,this result further verify the results of the changes caused by the injection of dsRNA from internal parasite morpholog.These results also provides a theoretical basis for screening of candidate genes which had the potential to pest control and supports our previous suggestion that V-ATPase should be considered for a potential pesticide target.The V-ATPase subunit B was silenced successfully in this experiment and the establishment of RNAi technique can help reveal the gene function of M.separata V-ATPaseB and other Lepidoptera insects.