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.

对包含家蚕角质层蛋白基因 (CPG) 的 BAC 克隆进行了测序。发现BMWCP2、3、4和5位于同一克隆中。我们在家蚕翅盘中分离出一个新的CPG（BMWCP11），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BMWCP2和BMWCP4上游约2 kb克隆到报告质粒中，以检查该区域控制 CPG 的表达。首先，我们鉴定了具有蜕皮激素反应性的家蚕细胞系。接下来，利用具有阶段特异性的翼盘构建了报告基因检测系统。用翼盘培养系统检查对蜕皮激素脉冲作出反应的序列。为了确认 DNA 诱导，使用了 GFP。蜕皮激素脉冲响应序列通过 EMSA 方法使用诱变进行鉴定。荧光素酶测定通过使用包括BMWCP2上游的几个长度的报告质粒进行。使用基因枪将质粒诱导入翅组织中。通过这种方法，我们能够筛选响应蜕皮激素而控制 CPG 表达的区域。通过BMWCP2上游区域的缺失分析、FTZ-F1结合位点的诱变和EMSA分析，证实BMWCP2的表达是由响应蜕皮激素脉冲的FTZ-F1诱导的。我们阐明了与 BMWCP2 诱导相关的转录因子及其结合位点。