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

对家蚕BAC克隆进行了序列测定，包括角质层蛋白基因（CPG）。BMWCP 2、3、4、5定位于同一克隆。在B翅盘中分离到一个新的CPG（BMWCP 11），并阐明了五组具有不同发育表达模式和激素反应性的CPG。将BMWCP 2和BMWCP 4的上游约2kb的片段克隆到报告质粒中，以检测控制CPGs表达的区域。首先，我们鉴定了具有蜕皮激素反应性的p53细胞系。其次，利用具有阶段特异性的翅盘构建了报告基因检测系统。用翅盘培养系统检测蜕皮激素脉冲反应的序列。为了确认DNA诱导，使用GFP。通过EMSA方法，使用诱变鉴定蜕皮激素脉冲响应序列。荧光素酶测定通过使用包括BMWCP 2上游几个长度的报告质粒来操作。用基因枪将质粒导入翅组织。通过这种方法，我们能够筛选控制CPG表达的区域，以响应蜕皮激素。通过对BMWCP 2上游区的缺失分析、FTZ-F1结合位点的突变分析和EMSA分析，证实了BMWCP 2的表达是由FTZ-F1诱导的，FTZ-F1对蜕皮激素脉冲有反应。阐明了与BMWCP 2诱导相关的转录因子及其结合位点。

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.