Analysis of control system in the expression of insect cuticle protein genes

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BAC clone, including cuticle protein genes (CPG) of Bombyx mori, was sequenced. and BMWCP2, 3, 4, and 5 were found to locate in the same clone. We isolated a new CPG (BMWCP11), and clarified five group CPGs, having different developmental expression pattern and hormonal responsiveness, in wing discs of B. mori.About two kb upstreams of BMWCP2 and BMWCP4 were cloned into reporter plasmid, to examine the region controlling expression of CPGs. First, we identified Bombyx cell lines, having ecdysone responsiveness. Next, reporter assay system was constructed by using wing discs which have stage specificity. Sequences responding to ecdysone pulse were examined with the wing discs culture system. For the confirmation of DNA induction, GFP was used. Ecdysone pulse responsive sequences were identified by EMSA method, using mutagenesis. Luciferase assay was operated by using reporter plasmid which included several length upstream of BMWCP2. Plasmid induction into wing tissues were performed by using gene gun. By this method, we were able to screen the region controlling CPG expression in response to ecdysone. It was confirmed that expression of BMWCP2 was induced by FTZ-F1, which responded to ecdysone pulse, through deletion analysis of BMWCP2 upstream region, mutagenesis of FTZ-F1 binding site and EMSA analysis. We clarified the transcription factor and its binding site relating to induction of BMWCP2.