To understand the transcriptional regulation of E74B by low concentrations of ecdysone, the promoter activity of Bombyx mori E74B was assessed in the B. mori wing disc using a transient reporter assay. We identified the transcription start sites of BmE74B and found that the core promoter region consists of initiator (Inr) and downstream promoter elements (DPE). The 3.6-kb upstream promoter region of BmE74B was responsive to 20-hydroxyecdysone (20E) in a dose-dependent manner, and the highest luciferase activity was observed in the presence of 0.2 μg/ml 20E. Moreover, the upstream BmE74B promoter activity was induced by 20E in a stage-specific and time-dependent manner, and the 3.6-kb promoter contained essential elements for the temporal regulation of BmE74B. Furthermore, we found a set of putative ecdysone response elements (EcREs). Five of these elements are highly conserved, capable of binding to the ecdysone receptor. Mutation of more than three putative EcREs, followed by introduction into the wing discs, abolished the activation of the BmE74B promoter by a low concentration of ecdysone. The results confirmed the role of ecdysone response elements in the transcription regulation of BmE74B and demonstrated that multiple putative EcREs were involved in the maximum response of BmE74B to low concentrations of ecdysone.
KEYWORDS: Bombyx mori, E74B, Ecdysone, Ecdysone response element, Wing disc