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

昆虫角质层蛋白基因表达控制系统分析

基本信息

批准号：
16580036
负责人：
KAWASAKI Hideki
金额：
$ 2.05万
依托单位：
Utsunomiya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580036/
关键词：
Bombyx mori ecdysone cuticle protein gene metamorphosis promoter gene gun GFP FTZ-F1 クチクラ蛋白質クチクラタンパク質 BACクローン

项目摘要

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.

测定了家蚕BAC克隆的角质层蛋白基因（CPG）序列。BMWCP2、3、4和5位于同一克隆中。在家蚕翅盘中分离到了一个新的CPG (BMWCP11)，并明确了具有不同发育表达模式和激素反应性的5组CPG。将BMWCP2和BMWCP4上游约2kb克隆到报告质粒中，检测CPGs的表达控制区域。首先，我们鉴定出具有蜕皮激素反应性的家蚕细胞系。其次，采用具有阶段特异性的翅片构建报告基因检测体系。用翅片培养系统检测对蜕皮激素脉冲响应的序列。为确认DNA诱导，采用绿色荧光蛋白。采用诱变技术，用EMSA法鉴定蜕皮激素脉冲响应序列。荧光素酶测定采用BMWCP2上游数个长度的报告质粒进行。用基因枪将质粒诱导入翅膀组织。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BMWCP2上游区域的缺失分析、FTZ-F1结合位点的突变分析和EMSA分析，证实BMWCP2是由响应蜕酮脉冲的FTZ-F1诱导表达的。我们明确了与BMWCP2诱导相关的转录因子及其结合位点。