水貂TYRP1基因启动子活性和Sp1结合位点

Promoter activity and Sp1 binding sites of mink TYRP1 gene

以同源性较高的雪貂TYRP1基因序列（Gen Bank：NW_004569257．1）为模板，通过PCR扩增7个不同长度的启动子缺失片段，定向克隆至pGL3-Basic载体中，利用脂质体转染到A375和293T细胞，通过双荧光素酶检测系统检测活性，利用多个在线软件分别分析启动子区活性及转录因子结合位点，并构建突变体进行活性验证。结果显示：成功构建7个不同长度的启动子缺失片段重组质粒，其中6个片段具有明显的启动子活性。-367／＋70区域为水貂丁豫．P1基因核心启动子区域，-367／-144区域的缺失，启动子活性无明显变化，-144／-37区域的缺失使启动子活性明显下降，表明该区域可能存在正调控元件。通过生物信息学方法预测可能存在Spl转录因子结合位点，构建的Mut-Spl-2突变体活性明显低于野生型pGL3—215，差异极显著（P〈0．01）。结果表明：成功筛选了水貂TYRPl基因核心启动子区域（-367／＋70），确定Spl（-56／-45）结合位点为转录的正调控区域。

The ferret TYRP1 gene （GenBank. NW 004569257. 1） sequence with high homology as template, seven different length promoter deletion fragments were amplified by PCR and cloned into pGL3-Basic vector,transfection of A375 and 293T cells by liposome. The promoter activity and transcription factor binding sites were analyzed by dual luciferase assay system and multiple online software. The mutants were constructed to verify the activity. Construction of seven recombinant plasmids with different lengths of promoter deletion fragments,and in which the six fragments had obvious promoter activity. The --367/＋ 70 region was the TYRP1 gene core promoter region of mink. There was no significant change in promoter activity in the absence of --367/-144,the deletion of --144/-37 region significantly reduced promoter activity. The potential positive regulatory elements enhanced mink TYRP1 gene promoter activity. The Spl transcription factor binding sites were predicted by bioinformatics method, and the activity of Mut-Spl-2 mutant was significantly lower than that of wild type pGL3-215,and the difference was significant （P〈0.01）. This study successfully screened mink TYRP1 gene core promoter region （--367/＋70）,Sp1 （--56/- 45） binding sites for transcription regulation region.