Identification Of A Tn5-mediated Baculovirus Mutant And Orf66 Gene Function

Baculoviruse is believed to be important biopesticides,effiecient expression vectors in genetic engineering and potential gene therapy vectors. Although many efforts have been made to sequence their genomes,and characterize the functions of the important genes,the functions of most of their genes remain unknown.The construction of a library of AcMNPV genome (bacmid) with random insertional mutations using Tn5 transposon-mediated in vitro mutagenesis will play a meaningful roll in functional genomics research of the virus.A mutant incapable of producing progeny virus in Sf9 cells was isolated from the library and named as ipra165.88 primers from the whole AcMNPV genome and 4 primers from the thransposon were applied in different combinations in PCR assays.After 5 rounds PCR and 2 gene sequencings,the transposon was determined to be conversely inserted at the site of 85bp down stream of ORF66' s ATG.In order to exclude the possibility that Apra165 had other mutations that contribute to the defect in virus replicaiton,a recombinant bacmid,DB165,with targeted disruption in orf66 while carrying an egfp report gene was constructed.The transfection of DB165 in sf9 cells proved that the disruption in orf66 truly lead to the defect in progeny virus production and the expression of the report gene.Further co-transfections of DB165 with gene fragments indicated that the insertion of the transposon in the promotor area of DNA polymerase gene disabled the expression of the essential DNA polymerase thus lead to the replication incapability.These results suggest that orf66 might be a non-essencial gene for virus.AcMNPV ORF66 contains three conserved domains,namely Desmo_N,CorA and Smc.CorA-like Mg²⁺ transporter protein might be an RNA splicing protein.The transcriptional phases of orF66 in sf9 ceils was detected by RT-PCR proved that orf66 is a late gene,whose function remains to be further studied.In summary,a replication-defective mutant was selected from the AcMNPV mutant library.The whole genome PCR assay was applied for the determination of the transposon's insertion site.With the construction of a targeted disrupted recombinant bacmid,the mechanism of mutant' s replication incapability and the expression and function of orf66 was studied preliminarily.The protocol of this research can be further used in the investigation of other mutants from the library,thus will be conducive to the functionalgenomics study ofbaculoviruses.In addition,more researches are needed for the understanding of the function of otiS6.