Cuticular protein genes showing peaks at different stages are probably regulated by different ecdysone responsive transcription factors during larval-pupal transformation

We aimed to explain the reason and function of the successive expression of ecdysone-responsive transcription factors (ERTFs) and related cuticular protein (CP) genes during transformation from larva to pupa. The regulation of the expression of CP genes by ERTFs was examined by in vitro wing disc culture and reporter assay using a gene gun transduction system. Two CP genes that showed expression peaks at different stages-BmorCPG12 at W3L and BmorCPH2 at P0 stage-were selected and examined. Reporter constructs conveying putative BHR3, beta FTZ-F1, BHR39, and E74A binding sites of BmorCPG12 and BmorCPH2 showed promoter activity when introduced into wing discs. In the present study, we showed the functioning of the putative BHR3 and E74A binding sites, together with putative beta FTZ-F1 binding sites, on the activation of CP genes, and different ERTF binding sites functioned in one CP gene. From these, we conclude that BHR3, beta FTZ-F1, and E74A that are successively expressed bring about the successive expression of CP genes, resulting in insect metamorphosis. In addition to this, reporter constructs conveying putative BHR39 binding sites of BmorCPG12 and BmorCPH2 showed negative regulation.