猪NLRC5基因启动子克隆及生物信息学分析  

Cloning and bioinformatics analysis of porcine NLRC5 gene promoter

作　　者：刘翔 尹灵丹 薛美[1] 李亮[1] 赵立媛 蒋成凡 王文哲 冯力[1] 刘平黄[2] LIU Xiang;YIN Lingdan;XUE Mei;LI Liang;ZHAO Liyuan;JIANG Chengfan;WANG Wenzhe;FENG Li;LIU Pinghuang(Harbin Veterinary Research Institute,Chinese Academy of Agriculture Sciences,Harbin 150069,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,哈尔滨150069 [2]中国农业大学动物医学院,北京100193

出　　处：《黑龙江畜牧兽医》2021年第13期1-5,143,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31802198)。

摘　　要：为了构建猪NLR家族含CARD结构域5(NOD-like receptor family caspase recruitment domain family domain-containing 5,NLRC5)基因启动子双荧光素酶报告质粒,确认其活性,进而探索其转录调控机制,试验采用PCR方法从猪睾丸细胞(swine testicular cell,ST细胞)中扩增出猪NLRC5基因启动子,将其与pGL3-Basic载体连接,测序正确后将猪NLRC5基因启动子双荧光素酶报告质粒转染至ST细胞中,通过双荧光素酶报告检测试剂盒检测荧光素酶的表达活性,并使用生物信息学软件预测该基因启动子区域的CpG岛、调控元件及转录因子结合位点。结果表明:试验成功构建猪NLRC5基因启动子双荧光素酶报告质粒,该质粒的相对荧光素酶活性极显著高于pGL3-Basic载体(P<0.01)。NLRC5基因启动子区域中存在2个CpG岛,TATA box、CCAAT box和GC box等多种调控元件,以及IRF1、IRF3、STAT1、STAT3、STAT4和NF-κB转录因子结合位点。说明猪NLRC5基因的表达可能受甲基化的影响,同时也可能受顺式作用元件以及干扰素和NF-κB相关转录因子的调控。In order to construct the dual luciferase reporter plasmid containing porcine NOD-like receptor family caspase recruitment domain family domain-containing 5(NLRC5) gene promoter, confirm its activity, and then preliminary investigate the transcriptional regulatory mechanisms. NLRC5 promoter gene was amplified from swine testicular cells(ST cells) by PCR method and inserted it into the pGL3-basic vector. After verification by sequencing, ST cells were transiently transfected with dual luciferase reporter plasmid of porcine NLRC5 gene promoter. The luciferase reporter gene assay kit was used to detect luciferase expression activity and bioinformatics software was utilized to predict CpG islands, promoter elements and transcription factor binding sites in promoter region of NLR15 gene. The results showed that the dual luciferase reporter plasmid of porcine NLRC5 gene promoter was successfully constructed. Dual-luciferase activity of porcine NLRC5 promoter was extremely significant higher than that of pGL3-Basic vector(P<0.01). There are several regulatory elements of two CpG islands, TATA box, CCAAT box, GC box, etc., and transcription factor binding sites of IRF1,IRF3,STAT1,STAT3,STAT4,NF-κB in promoter region of NLRC5 gene. It indicated that the expression of NLRC5 gene might be affected by methylation, and might also be regulated by cis-acting elements, interferon and NF-κB related transcription factors.

关 键 词：启动子质粒 双荧光素酶 NLR家族含CARD结构域5(NLRC5)基因 启动子调控元件 转录因子结合位点 生物信息学 

分 类 号：S828[农业科学—畜牧学] Q78[农业科学—畜牧兽医]