The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.
本研究旨在筛选调控北极狐毛色基因CBD103 的启动子活性区域及转录因子结合位点，为揭示CBD103基因调控北极狐毛色形成的分子遗传机制提供依据。克隆获得了北极狐CBD103 基因5′侧翼区2 123 bp 的片段，并构建了4 个不同长度的启动子缺失片段表达载体，通过双荧光素酶检测系统对启动子活性进行检测。对启动子活性最高区域预测出的3 个特异性蛋白1 (Sp1) 转录因子结合位点分别进行点突变并构建3 个突变载体，利用双荧光素酶检测系统测定其活性。结果显示，在构建的4 个不同长度启动子缺失片段中1 656 (−1 604/+51) 区域活性最高，在此区域构建的3 个突变载体的启动子活性较野生型 (片段1 656) 均显著降低，说明−1 604/+51 区域为北极狐CBD103 基因的核心启动子区，−1 552/−1 564、−1 439/−1 454 和−329/−339 区域为正调控区域。文中成功获得了北极狐CBD103 基因的核心启动子区域和正调控区域，这为进一步研究该基因调控北极狐被毛颜色分子遗传机制奠定了基础。.
Keywords: Arctic fox; CBD103 gene; promoter activity; transcriptional regulatory element.