Characterization Of Egt Gene Promoters From BmNPV And AcMNPV And Regulation Of Diapause Hormone On Activity Of Bombyx Mori Trehalase Gene Promoter

Baculoviruses are potential insecticides without pollution. However, a primary disadvantage of inability to kill target insects rapidly has strongly limited their use as insecticides. Baculoviruse ecdysteroid UDP-glucosyltransferase (egl) genes are non-essential for the replication of viruses. Host insects infected by egf-minus baculoviruses succumbed to the viral infection sooner than those infected by wild-type viruses. Thereby the efficiency of recombtnant baculoviruses as insecticides can be improved by deletion of egt genes. Meanwhile, selective marking genes or valuable protein genes can be cloned downstream the egt promoter to facilitate the selection of recombinants and processing of gene expression products.This article characterized the egt gene promoters from Bombyx mori nucleopolyhedrovrius (BmNPV) and Autographa californica nucleopolyhedrovrius (AcMNPV) and trehalase gene promoter of B. mori. And the regulatory effects of ecdysone and juvenile hormone (JH) on egt promoter activities were investigated as well as diapause hormone (DH) on trehalase gene promoter of B. mori.1. Characterization of egt gene promoters from BmNPV and AcMNPVThe egt gene promoter fragments of different lengths were generated from genomic DNAs of BmNPV strain ZJ-8 and AcMNPV, respectively. Reporter plasmids were constructed with luciferase (luc) gene as reporter driven by egt promoter. Transient expression showed that egt promoters from BmNPV and AcMNPV shared similar properties. Their transcriptional activities require the transactivation of viral factor(s) and observed first at 24 hours post infection (hpi) of viruses.2. Effect of BmNPV hr3 on egt promoterTo investigate the effect of BmNPV homologous region (hr) on egt promoter, BmNPV hr3 was cloned into reporter plasmids downstream luc gene. Transient expression showed that hr3 increased egt promoter activity by 1200- to 1600-fold in vitro and 130-fold in vivo. But egt promoter activities still required the transactivation of viral factor and transcription occurred at 18 hpi.3. The functional region of egt promoters from BmNPV and AcMNPVExpression assay of reporter genes driven by different lengths of egt promoter fragments was conducted. The results revealed that promoter fragment from egt translation initiation ATG to -159 bp upstream contains the basal promoter structure but its transcriptional activity is almost abolished; and the main m-acting elements for viral factors are located in the nucleotide sequence between -159 to -309 bp upstream the translation initiation site.4. Effects of foreign insect ecdysone and JH on egt promoter activityForeign insect ecdysone showed negative regulatory effect on egt promoter activity in vitro. Ecdysone of 0.5ug/ml showed little effect, but it of over 1.0 ug/ml significantly down regulated egt promoter activity. In contrast to in vitro, ecdysone increased egt promoter activity by 2.6- to 15-fold invivo when larvae given an ecdysone injection of 4-8 ug per 05. JHA showed positive regulatory effects on egt promoter activity. JHA of 0.5-2.0 ug/ml in vitro or 50-150 ug/ml by spreading onto larvae increased promoter activities by 0.76- to 1.46-fold and 0.76- to 4.4-fold, respectively.5. Characterization of Trehalase promoter from Bombyx mori and Bombyx mandarinaTrehalase promoters both from B. mori and B. mandarina revealed low but significant transcriptional activities in Bm5 and Sf21 cells. B. mori trehalase promoter activity was higher in homologous Bm5 cells than that in heterologous Sf21 cells.6. Regulatory effect of diapause hormone on trehalase Promoter from Bombyx moriIn cell media, DH concentration at the range from 13 to 33 nmol/L enhanced the promoter activity significantly. Of which 20-27 nmol/L increased the most, by about 1.3 times. When DH dose rose to 50 nmol/L, no significant effect was observed. It revealed that DH has a regulatory effect on B. mori trehalase promoter activity with a typical dose-dependent pattern in vitro. Moreover, there possibly exist DH receptors in the ovary-derived Bm5 cel...