The present study was conducted to clarify the regulatory mechanism of cuticular protein genes of Bombyx mori expressed in wing discs in the prepupal stage. BHR3, BHR4, E74A, and ßFTZ-F1 were successively expressed in wing discs at the pre-pupal stage. BHR3 showed different ecdysone responsiveness from other ecdysone-responsive transcription factors (ERTFs) and was induced by ecdysone addition but showed decrease by ecdysone removal after treatment (ecdysone pulse). BHR4 and E74A were induced by the ecdysone addition and by the ecdysone pulse. ßFTZ-F1 was not induced by the ecdysone addition but was induced by the ecdysone pulse. Thus, ERTFs showed different hormone responsiveness, resulting in the different expression timing. We selected cuticular protein genes that showed the same expression stage with each transcription factor and examined their ecdysone responsiveness. The developmental expression and ecdysone responsiveness of BmorCPH5, BmorCPR34, BmorCPR23, and BmorCPR99 resembled those of BHR3, BHR4, E74A, and ßFTZ-F1, respectively. The results of the transient reporter assay strongly suggested the regulation of each cuticular protein promoter by ERTF. These ERTFs regulated different cuticular protein genes and determined their expression timing and probably the nature of the cuticle layer of insects.
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