水牛STAT5a和STAT5b基因启动子克隆及其活性分析

Cloning and Activity Analysis of Buffalo STAT5a and STAT5b Promoters

为了解转录激活与信号转导因子5(STAT5)在水牛乳腺发育及泌乳生理中的功能,对水牛STAT5a和STAT5b基因的5′调控区启动子序列进行了克隆,并比较其活性.根据GenBank已公布的牛STAT5a和STAT5b基因序列设计特异性引物,以广西本地水牛乳腺组织基因组DNA为模板,通过PCR法分别扩增不同长度STAT5a和STAT5b基因启动子片段并进行生物信息学分析.结果表明:克隆得到的水牛STAT5a基因启动子片段(P1和P2)大小是500bp,700bp,STAT5b基因启动子片段(P3,P4和P5)大小是500bp,800bp,1 500bp.在线分析结果显示,仅P2片段中存在高甲基化位点,且富含SP1,AP2等转录因子结合位点.将构建的不同长度启动子片段分别连入pGL3-Basic载体,分别转染水牛乳腺上皮细胞,检测荧光素酶表达水平.与未转染组相比,P1~P5质粒转染组的荧光素酶比值均显著提高(p<0.05);添加5mg/mL质量浓度催乳素(PRL)处理乳腺上皮细胞,P3质粒转染组的荧光素酶比值显著高于其他各组(p<0.05).以上结果表明,水牛STAT5a和STAT5b基因启动子活性均受到PRL调节,两者表达水平在水牛乳腺上皮细胞中不同,且STAT5b基因表达受PRL调控更为明显.

In order to understand the role of transcriptional activation and signal transduction factor 5（STAT5）protein in the development of the mammary gland and lactation in buffalo,we cloned and analyzed the promoter sequences of 5regulatory region of STAT5aand STAT5b genes and compared their activity.We designed specific primers according to the STAT5aand STAT5b gene sequences published in GenBank,used the mammary gland tissue genomic DNA of Guangxi local buffaloes as a template,amplified the promoter fragments of STAT5aand STAT5b genes by PCR,and made bioinformatics analysis of them.The size of the promoter fragments（P1and P2）of the buffalo STAT5agene was shown to be 500bp and 700bp,and the size of the promoter fragments（P3,P4and P5）of the STAT5b gene was 500bp,800bp and 1500bp,respectively.The results of on-line analysis showed that there were hypermethylation sites in P2fragment,which were also rich in transcription factor binding sites such as SP1and AP2.The promoter fragments were inserted into pGL3-Basic vector,and transfected into buffalo mammary epithelial cells respectively.Detection of the expression level of luciferase showed that the ratio of luciferase in P1~P5plasmid transfected group was significantly higher than that of in untransfected groupadding 5mg/mL PRL（prolactin）to mammary epithelial cells,the promoter activity was significantly higher in P3than the other groups（p〈0.05）.The above results showed that the promoter activity of the STAT5aand STAT5b genes was regulated by PRL,and such regulation was more obvious for STAT5b.