Analysis Of Partial Genomic Sequence From EupsNPV And Construction Of The Recombinant HearSNPV Containing The Scorpion Toxin

Tea tussock moth,Euproctis pseudoconspersa Strand (Lepidoptera: Lymantriidae),is mainly distributed on Hubei, Fujian, Zhejiang, Guangxi, Guizhou and Anhui provinces of China. It can be also found in Japan and India. Tea tussock moth is one of the main pests attacking tea bushes. Euproctis pseudoconspersa nucleopolyhedrovirus (EupsNPV) is a pathogen specific to Tea tussock moth and as an important pest control agent it has been extensively used for many years. In this research, the genomic DNA of EupsNPV was extracted and a DNA library was constructed partial genomic DNA fragments were sequenced and analyzed. The phylogenetic relationship of EupsNPV with other known NPVs was also determined. The detailed results are as follows:An EcoRI-XhoI 5942bp DNA fragment from the Euproctis pseudoconspersa nucleopolyhedrovirus (EupsNPV) genome was cloned and its sequence was determined. Five Open Reading Frames (ORFs) encoding P24, Rr1, 38.7K, Lef1 and Ep-ld124 were identified in the fragment. Comparative and phylogenetic analysis of the 5 genes with their homologues from other baculoviruses revealed that EupsNPV was a nucleopolyhedrovirus distinct from other known baculoviruses. The result also showed that EupsNPV was one of the NPV II members, and was most closely related to Ectropis obliqua SNPV (EcobSNPV).The genomic region containing the polyhedrin gene was sequenced. The coding region of polyhedrin was 738 nucleotides in length and potential to encode a predicted protein of 28.9 kDa. The sequence upstream of the initiation codon, ATG, was rich in AT, the conserved ATAAG motif, a typical baculovirus very late promoter localized 52bp upstream of the initiation codon. Two putative polyadenylation signal AATAAA were found 25 nucleotides and 80 nucleotides separately downstream of the termination codon. The 14bp conserved sequence upstream of ATG initiation codon was CCAATAAGTATTTT, with C at the -55bp instead of A or T as in other NPV'ph gene.Insect-selective neurotoxins classified to protein toxins are considered most potent agents for developing recombinant baculoviral insecticides. The scorpion toxin gene with ph gene was introduced into the HearNPV Bac to Bac system to build the recombinant HearNPV.