Preliminary Study On RNAi Targeting Of The P35 Of Autographa Californica Mutiple-capsid Nuclear Polyhedrosis Virus

RNA silencing or interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA.The RNAi encompasses related pathways found in a broad range of eukaryotic organisms, including RNA interference of animal, quelling of fungi, posttranscriptional gene silencing and cosuppression of genes in plants, etc. It plays important role in immunity against exogenous genetic factors (such as retrotransposons transgen and virus) and regulates gene expression of eukaryotic organisms.Silencing triggers are nucleic acids which are converted into 21-27-nt-long "small RNAs"in vivo. All RNA silencing pathways are triggered by "small RNAs"—a term that encompasses small interfering RNAs (siRNAs), repeat-associated small interfering RNAs (rasiRNAs), and micro RNAs (miRNAs). Small RNAs guide RNA silencing effector complexes, like the RNA-induced silencing complex (RISC) or the RNA-induced initiation of transcriptional gene silencing (RITS) complex. At last, effector complexes containing the small RNA silence target mRNA at transcriptional or posttranscriptional level.It has been reported that the p35 gene of Autographa Californica Mutiple-capsid Nuclear Polyhedrosis virus (AcMNPV) possesses anti-apoptosis function, the knocking down of the p35 induce AcMNPV infected-cell apoptosis.In this paper we cloned a fragment corresponding to a part of p35 gene into pLITMUS28i to generate a recombinant vecor pLITMUS-p35 and transcripted it into dsRNA in vitro. Upon transfection of dsRNA into sf9 cells infected by AcMNPV, no obvious cell apoptosis was observed. It is probably due to transfection efficiency of transfection reagents. Our transfection efficiency experiments showed that the tansfection efficiency is very low. The RNAi technique needs to be optimized.