用于细胞水平家蚕转录调控元件研究的简单基础启动子的构建

Construction of a simple basal promoter for the research of transcription regulatory elements in the silkworm (Bombyx mori) at the cell level

【目的】本研究致力于构建一种能够在家蚕Bombyx mori细胞水平稳定表达的简单基础启动子,从而更准确地反映单一转录调控元件对基因启动子活性的影响,为研究家蚕乃至其他昆虫的基因转录调控奠定基础。【方法】本研究在本课题组已报道的能在家蚕细胞中稳定表达且基本不含上游转录调控元件的BmVgP78M启动子的基础上,通过PCR技术在其上游添加一定长度的间隔序列和能够应答20-羟基蜕皮激素(20E)且增强启动子活性的BrC-Z2转录因子结合基序(BrC-Z2 element, BrC-Z2E);通过基因克隆技术构建细胞转染载体;通过细胞转染技术和双荧光素酶报告基因系统检测启动子活性的变化。【结果】通过在BmVgP78M启动子上游添加28 bp间隔序列,成功构建了一个简单基础启动子,命名为VgP78ML,并证明其为可用于研究目标转录调控元件的简单基础启动子。经实验验证表明,该简单基础启动子不仅可以在家蚕细胞中稳定表达,且其本身活性不受20E及转录因子BrC-Z2的影响;当该启动子上游连接BrC-Z2E时,可以显著地应答20E及BrC-Z2转录因子,从而调控报告基因的表达。【结论】VgP78ML能够作为简单基础启动子应用于细胞水平对家蚕基因转录调控进行研究。同时,其构建方法也为其他物种构建研究转录调控的简单基础启动子提供了参考。

【Aim】 This study aims to construct a simple basic promoter, which can be stably expressed in the silkworm (Bombyx mori) cells, so as to reflect the influence of single transcriptional regulatory element on gene promoter activity more accurately and lay a foundation for studying the transcriptional regulation of the silkworm and even other insects.【Methods】 Based on the BmVgP78M promoter previously reported in our research group, which can be stably expressed in silkworm cells and hardly contains upstream transcriptional regulatory elements, interval sequences of a certain length and BrCZ2 transcription factor binding motif (BrCZ2 element, BrCZ2E) that can respond to 20hydroxyecdysone (20E) and enhance promoter activity were added to the upstream of BmVgP78M by PCR technique. The cell transfection vectors were constructed by gene cloning technique, and the changes of promoter activity were detected by cell transfection technique and dual luciferase reporter gene system.【Results】 A simple basal promoter named VgP78ML was constructed successfully with a 28 bp interval sequence added to the upstream of the BmVgP78M promoter, and proved to be a simple basic promoter to study target transcriptional regulatory elements. The verification test results showed that this simple basal promoter not only could be stably expressed in silkworm cells, but also was not affected by 20E and transcription factor BrCZ2. When BrCZ2E was linked to the upstream of this promoter, it could significantly respond to 20E and BrCZ2 transcription factor, thereby regulating the expression of reporter gene.【Conclusion】 VgP78ML can be used as a simple basic promoter to study the transcriptional regulation of genes in silkworm at the cell level. At the same time, its construction method also provides a reference for constructing simple basic promoter to study the transcriptional regulation in other species.