转录因子MZF1对大鼠RGC32基因启动活性的影响及可能的结合部位  

Construction of rat RGC32 promoter and identification of its binding sequence with MZF1

作　　者：王璐璐[1] 何风霞[1] 赵聃[1] 虞天一 张婧[1] 卢燕来[1] 邱文[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2015年第2期143-148,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(31470853;81273333;81471626);江苏省自然科学基金面上项目(BK20131386)资助

摘　　要：目的 :构建大鼠补体应答基因32(response gene to complement,RGC32)启动子(全长和截短)荧光素酶报告质粒,并观察人胚肾细胞(HEK293)过表达髓锌指基因1(myeloid zinc finger gene1,MZF1)对大鼠RGC32基因启动活性的影响。同时,筛选其可能的MZF1结合位点。方法:将RGC32基因启动子全长(-686^-1 nt)插入到荧光素酶报告基因载体p GL3-basic中,获得RGC32基因启动子全长荧光素酶报告质粒(p GL3-RGC32-FL)后,再将p GL3-RGC32-FL与本课题组前期构建的大鼠野生型MZF1表达质粒(p IRES2-EGFP-MZF1)共转染HEK293细胞,检测其荧光素酶活性,以确定MZF1对RGC32基因的启动作用。另用生物信息学软件预测RGC32基因启动子上转录因子MZF1潜在的结合位点,并据此构建3个RGC32基因启动子截短的荧光素酶报告质粒(即p GL3-RGC32-1、p GL3-RGC32-2和p GL3-RGC32-3)。将上述RGC32基因启动子全长和各截短的荧光素酶报告质粒与MZF1过表达质粒共转染HEK293细胞,再行荧光素酶活性测定,以筛选MZF1的结合位点。结果:菌液PCR及核酸测序证实,大鼠RGC32基因启动子(全长和各截短)的荧光素酶报告质粒均构建成功。将p GL3-RGC32-FL和p IRES2-EGFPMZF1共转染HEK293细胞发现,RGC32基因启动子活性显著增加。而将p GL3-RGC32-FL、p GL3-RGC32-1、p GL3-RGC32-2和p GL3-RGC32-3分别与p IRES2-EGFP-MZF1共转染HEK293细胞后显示,p GL3-RGC32-3的启动活性显著低于p GL3-RGC32-FL、p GL3-RGC32-1和p GL3-RGC32-2。提示MZF1可能结合在RGC32基因启动子的-286^-86 nt区域。结论 :成功构建了大鼠RGC32基因启动子全长及截短荧光素酶报告质粒,证实过表达MZF1可促进RGC32基因的启动,并初步筛查出转录因子MZF1在RGC32基因启动子上可能的结合区域。Objective：To construct luciferase reporter plasmids of full-length and truncated promotors of rat response gene to complement 32 （RGC32） gene and detect their activity in HEK293 cells in response to myeloid zinc finger gene 1 （MZFI） overexpression, screening the possible binding sites for MZF1. Methods：Rat RGC32 promoter （-686--1 nt） was amplified by PCR and cloned into the luciferase reporter plasmid （pGL3-basic）. The recombinant plasmid （pGL3-RGC32-FL） and rat MZFI expression plasmid （pIRES2-EGFP-MZF1） were co-transfected into HEK-293 cells and then the luciferase activity was detected to determine the role of MZF1 in RGC32 gene transcription. Meanwhile,the potential MZF1 binding sites within RGC32 promoter were predicted by bioinformatics software. Based on the predicted results,different luciferase reporter plasmid of truncated MZF1 gene promotor that named pGL3-RGC32-I ,pGL3-RGC32-2 and pGL3-RGC32-3 were constructed. The promoter laciferase reporter plasmids of pGL3- RGC32-FL or pGL3-RGC32-1,pGL3-RGC32-2,pGL3-RGC32-3 and the plasmid of pIRES2-EGFP-MZF1 were co-transfected into HEK293 cells. Then,the luciferase activity was detected to screen the MZF1 binding sites. Results：It was verified that different kinds of plasmids were all constructed correctly by PCR analysis and nucleotide sequencing. The plasmids of pGL3-RGC32-FL and pIRES2-EGFP-kIZF1 were also co-transfected into HEK293 cells,and then the luciferase activity was detected. The results showed that the transcriptional activity of RGC32 gene was increased markedly in response to MZF1 overexpression. In addition,the plasmids of pGL3-RGC32-FL or pGL3-RGC32-1, pGL3-RGC32-2 and pGL3-RGC32-3 and pIRES2-EGFP-MZF1 were co-transfected into HEK293 cells,and then the luciferase activity in different groups was determined. The result displayed that the activity of pGL3-RGC32-3 was much lower than that it] pGL3- RGC32-FL,pGL3-RGC32-1 and pGL3-RGC32-3,indicating that the region of rat RGC32 promoter （-286--86 nt） might contain MZF

关 键 词：补体应答基因32 髓锌指基因1 荧光素酶报告质粒 启动子活性 

分 类 号：R392.11[医药卫生—免疫学]