Ali Md Saheb, Rahman R F, Swapon Ahsanul Haque
Bangladesh Jute Research Institute, Manik Mia Ave., Dhaka 1207, Bangladesh
Bangladesh Jute Research Institute, Manik Mia Ave., Dhaka 1207, Bangladesh.
J Insect Sci. 2015 Apr 5;15(1). doi: 10.1093/jisesa/iev019. Print 2015.
Cuticular protein genes are good models to study the molecular mechanisms of signaling by ecdysteroids, which regulate molting and metamorphosis in insects. The present research demonstrates on hormonal regulation and analysis of the regulatory sequences and transcription factors important for Bombyx mori cuticular protein glycine-rich13 (CPG13) gene expression. Expression of CPG13 was strong at prepupal stage in wing tissues of B. mori. CPG13 expression was induced by the addition of 20E, which was inhibited by cycloheximide in the wing disc. The upstream region of the CPG13 gene was analyzed using a transient reporter assay with a gene gun system and identified two BR-Z2 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. It was found that the regions carrying the binding sites for the ecdysone-responsive transcription factor BR-Z2 were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z2 binding sites decreased the reporter activity suggesting that the BR-Z2 isoform can bind to the upstream region of the cuticle protein gene, CPG13 and activates its expression.
表皮蛋白基因是研究蜕皮甾类信号传导分子机制的良好模型，蜕皮甾类可调节昆虫的蜕皮和变态。本研究展示了对家蚕富含甘氨酸的表皮蛋白13（CPG13）基因表达的激素调控以及对其调控序列和转录因子的分析。CPG13在家蚕翅组织的预蛹期表达强烈。在翅盘中添加20E可诱导CPG13表达，而环己酰亚胺可抑制该表达。使用基因枪系统通过瞬时报告基因测定法分析了CPG13基因的上游区域，确定了两个BR-Z2结合位点是蜕皮激素脉冲激活荧光素酶报告基因转录的重要顺式作用元件。对这些位点进行定点诱变，然后导入翅盘，显著降低了报告基因活性。结果发现，携带蜕皮激素应答转录因子BR-Z2结合位点的区域负责翅盘中报告基因活性的激素增强作用。BR-Z2结合位点的突变降低了报告基因活性，这表明BR-Z2亚型可以结合到表皮蛋白基因CPG13的上游区域并激活其表达。
