The DNA Replication Origins And Characterization Of ORF63from Spodoptera Litura Nucleopolyhedrovirus Ⅱ

Spodoptera litura is an omnivorous agricultural and forestry pests of LepidopteraNoctuidae. The host plants of Spodoptera litura include300kinds of100families. As theSpodoptera litura feeding miscellaneous, strong breeding potentialg and strong resistance, soit is difficult to control. Chemical synthesis of various pesticides are serious pollution of theenvironment and pest resistance to chemical synthetic pesticides growing circumstances,biological control more and more is important and urgent. Insect baculovirus has been a goodbiological control weapon because of its host specificity, and the environmental friendship.SpltMNPVⅡ is a kind of strong reproduction rate and the highly virulent new virus variants,its whole genome sequence has been determined complete, including149Open ReadingFrames（ORFs）. However, the genomic DNA replication origins and functions of its geneswere unknown. In this study, we analyzed the genomic DNA replication origins and the newgene ORF63of SpltMNPVⅡby the means of the promoter activity analysis, cloning andexpression, preparation of polyclonal antibodies, transcription phase analysis and expressionphase analysis and laocation of gene products in infected host cells. The object of this study isto enhance our understanding of baculovirus molecular biology. The major findings are asfollows:1. SpltMNPVⅡgenomic DNA sequence was analyzed by bioinformatics analysis. In thegenome sequence of SpltMNPVⅡ, seven homologous region（shrs）were identified, including3-864-bp imperfect palindrome around a PvuI site in the center. The seven hrs also consist ofsome direct and inverted repeats and other motifs. The conservation of forty64-bp imperfectpalindrome is up to90%by sequence analysis.2. Constructing SpltNPVⅡ seven hrs functional plasmid, by transfection assays and areal-time PCR method the seven putative origins of DNA were identified to be bifunctional,serveing as oris of SpltNPVⅡ DNA replication and involving in the enhancement oftranscription. The replication and enhancer potential of the seven hrs varied from1.52×105copies/μg DNA（hr5） to more than4.88×106copies/μg DNA（hr4）. The hrs-mediatedenhancement of the ie1promoter varied from3-fold to more than13-fold in infected spli cells. hr1and hr7increased luciferase activity by11-and13-fold respectivel. Enhancement by theother four hrs varied between6-and9-fold.3. A luciferase activity assay and real-time PCR were carried out with a single64-bpimperfect palindrome. Real-time PCR results showed no detectable replication abovebackground. The luciferase activity was not significantly different to the control group. Thecis-elements of palindromes, repeat sequences, and characteristic motifs were significantlypositively correlated with both replication potential and enhancer efficiency. However,replication potential was most closely related to the number of repeat sequences, and enhancerefficiency to characteristic motifs. In addition, no significant Spearman’s correlation wasobserved between the replication potential and enhancer efficiency.4. SpltMNPVⅡORF63gene structure analysis hane been done, ORF63gene was1071bp, encoding356aa, predicted protein molecular weight of41.3kDa, both early promotermotif CAGT and late promoter motif TAAG were found. Promoter activity and transcriptionphase were analyzed, the results suggested that the ORF63may be a gene that both express inearly and late phase in the viral life cycle.5. Part of SpltMNPV Ⅱ ORF63gene sequences were expressed by prokaryoticexpression system,which products were purified and idenified by mass spectrometry.Theimmunization of guinea pigs produced a polyclonal antibody, antibody titers were determinedby ELISA, antibody titers of polyclonal antibodies which produced from the ORF63geneexpression product were high, more than in1:3200. Using polyclonal antibodies as firstantibody, products from ORF63gene expression in the host cell were analyzed. orf63geneexpressed in infected cells as early as4h after infection, as early as8h after infection in larva,the ORF63gene was expressed both early and late phase, and its promoter analysis andspecial libraries is consistent with the results.6. The cellular localization of GP63was determined in spli221cell infected after24h bythe immunocytochemistry and it suggested that GP63existed both in cytoplasm and cellnucleus.