ssc-miR-148a启动子克隆及其特征分析  

Cloning and Analysis of ssc-miR-148a Promoter

作　　者：王萍[1] 郝文艳[1] 曹丽华[1] 沈开元[1] 代小丽[1] 李海艳[1] 梁贤威[2] 石德顺[1] 李湘萍[1] 

机构地区：[1]广西大学,亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [2]中国农业科学院广西水牛研究所,南宁530001

出　　处：《畜牧兽医学报》2016年第10期1969-1976,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广西壮族自治区研究生教育创新计划项目(YCBZ2014013);国家自然科学基金(31560632);广西自然科学基金项目(2014GXNSFAA118084);广西水牛遗传繁育重点实验室开放课题项目(SNKF-2014-03)

摘　　要：为研究猪miR-148a(ssc-miR-148a)的转录调控机制,对其启动子进行了克隆及分析。本试验首先设计特异性PCR扩增引物,分别得到ssc-miR-148a前体上游3个片段,并将其连接到荧光素酶报告载体pGL3-Basic上。通过生物信息学方法,在线分析ssc-miR-148a启动子大概区域、甲基化部位和转录因子结合部位。将重组报告质粒转染293T细胞,分析启动子活性。采用不同浓度碱性成纤维生长因子(bFGF)处理猪成纤维细胞和转染有重组报告质粒的猪成纤维细胞,检测ssc-miR-148a和DNA甲基化转移酶1(DNMT1)的表达,及其对启动子活性的影响。结果显示,克隆得到的ssc-miR-148a启动子区2 043bp片段具有启动子活性,该序列存在5个CpG岛、Sp1及AP2等转录因子结合位点。0、5和10ng·mL-1浓度bFGF处理猪成纤维细胞和转染重组报告质粒的猪成纤维细胞后,ssc-miR-148a表达均显著下降(P<0.05),DNMT1 mRNA显著增加(P<0.05)。启动子活性均显著下降(P<0.05),5和10ng·mL-1浓度间无显著差异(P>0.05)。结果表明,ssc-miR-148a启动子位于前体上游2 043bp片段内,启动子区域有转录因子SP1结合位点,其表达受bFGF的调控。In order to understand the transcriptional regulatory mechanism of ssc-miR-148 a,we have cloned and analyzed its promoter.Firstly,we designed specific PCR primers to amplify 3upstream fragments of ssc-miR-148 aprecursors,then inserted them into pGL3-Basic expression vector.Based on the amplified fragments,ssc-miR-148 apromoter region,methylation sites and transcription factor binding sites were predicted by bioinformatics.The plasmid was transfected into293 Tcells to analyze the promoter activity.Expression of ssc-miR-148 aand DNA methylation transferase 1（DNMT1）in treated porcine fibroblasts by Basic Fibroblast Growth Factor（bFGF）with different concentrations was detected.The results showed that the cloned 2 043 bp sequence had promoter activity,which had 5CpGs and transcriptional factor binding sites,such as Sp1,AP2.After treated porcine fibroblasts with 0,5and 10ng·mL-1 bFGF,the expression of sscmiR-148 awas decreased significantly（P〈0.05）,DNMT1 mRNA level increased significantly（P〈0.05）.The promoter activity significantly reduced（P〈0.05）,but no significant difference between 5and 10ng·mL-1 concentrations（P〉0.05）.The results indicate that the promoter region of ssc-miR-148 alocate at-2 043 bp,which has Sp1 transcription factor binding sites.The expression of ssc-miR-148 ais regulated by the bFGF.

关 键 词：ssc-miR-148a 启动子 克隆 BFGF DNMT1 

分 类 号：S828[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]