Analysis On Promoter Of Ecdysone Receptor And Ultraspiracle Genes In Silkworm, Bombyx Mori

The active form of ecdysteroid is20-hydroxyecdysone (20E), considered to be theprincipal determinant of developmental timing in insects, is involved in the control ofdiverse biological processes, including morphogenetic, apoptotic, physiological,reproductive and behavioral responses. The ecdysone receptor (EcR) and ultraspiracle(USP) are the nuclear receptors found in insects, they are the molecular target of the steroidhormone, ecdysone. The reserch of mode of action and regulation mechanism of EcR andUSP with the ecdysone has theoretical significance and application value.Constracting a dual fluorescent plasmid of FHNLuc-A3RL, in which Fluc gene isdriven by BmEcR and BmUSP gene promoter, and Rluc is driven by A3promoter. Weprepared recombinant baculovious through the Bac-to-Bac system, and injected them intofifth instar silkworm, and analysis the luciferase activity. The results shown that thereexisted positive regulatory elements at position-637to-518bp and-278to-177bp forBmEcR-A and positiveregulatory elements at position-325to-198bp in BmEcR-B1promoter region with or without0.002g/L20E. In the BmUSP promoter, the region from-485to-374bp existed positive regulatory elements are located within this region.However, it was also shown that the region from-374to-281bp may contain thenegatively regulatory elements. The serial deletions of BmEcR-A, BmEcR-B1and BmUSPgene promoter shown that different levels of activity in various tissues, higher in fat bodyand lower in midgut and Malpighian tubules.To further study the core prompter of BmEcR-A, dual luciferase reporter gene assaysystem was performed in this study. we constructed different lengths deletions andmutations of pGL3-Basic plasmid. The internal control vector pRL-TK was co-transfectedinto BmN cell with recombinant plasmids. The results shown that: the promoter activity ofBmEcR-A gene were raised by ecdysone. In the BmEcR-A promoter, the region from-637to-612bp is important for the basal transcriptional activity of the BmEcR-A promoter. Wealso analysed of the region from-197to-177bp by excision analyses and it is essential for the promoter activity of BmEcR-A. Sequence analysis of the region from-197to-177bprevealed no predictedtranscriptional regulatory elements, butin the region from-637to-612bp contain HSF transcription factor binding sites.Further analysis thetranscription factor binding sites of-197to-177bp by use ofstreptavidin agarose resin technology and design of biotinylated probes, add silkworm fatbody tissue extracts were incubated, then eluted protein, purification. Protein bandsobserved by SDS-PAGE electrophoresis and identified by mass spectrometry. To findrelated proteins combination with BmEcR-A promoter important regulatory regions. Theresults shown that the sequence-197to-177bp may be combined with the silkwormtransketolase.To further study the core promoter of BmEcR-B1and BmUSP, dual luciferase reportergene assay system was performed in this study.The results shown that in the BmEcR-B1promoter, the region from-325to-295bp contain important regulatory elements werenecessary for transcriptionalregulation of BmEcR-B1gene promoter. Sequence analysis ofthepromoter revealed several transcriptional regulatory elements contains Dfd and HSFtranscription factor binding sites. In the BmUSPpromoter, deletion of the region from-485to-445bp and-307to-281bp upstream of BmUSP gene abolished and increased itspromoter activity, respectively. This region contains AP-1, Dfd transcription factor bindingsites.This study analyzed the luciferase activity of BmEcR and BmUSP in silkwormbyusing dual luciferase reporter gene assay system and obtaining some important regulatoryregions. So further analyzed these important regulatory regions of BmEcR and BmUSP inBmN cells, the core promoter region and associated protein factors were obtained. Thisstudy would provide an useful clue for investigating transcriptional regulation mechanismof BmEcR and BmUSP in the silkworm Bombyx mori.