【摘要】 采用PCR方法扩增了ZNF313基因的启动子序列,构建了含人ZNF313基因启动子不同片断的荧光素酶报告基因表达体系.以pRLTK为内参照质粒,瞬时转染HEK293T细胞,48h后收集细胞,测定荧光素酶的相对表达活性.结果发现,在ZNF313基因的启动子区域构建了4种荧光素酶报告基因表达体系,即pGL3215(-215bp～+38bp)、pGL3160(-160bp～+38bp)、pGL3133(-133bp～+128bp)和pGL38(-8bp～+128bp).其中pGL3215表达载体的荧光素酶相对表达活性最高;pGL3160和pGL3133表达载体的荧光素酶相对表达活性几乎相同,且是pGL3215的75%;而pGL38的荧光素酶相对表达活性急剧下降,接近于零.这表明,-133bp～-8bp区域内含有人ZNF313基因转录所必需的启动子序列.生物信息学的分析表明,两个SP1、一个AP2和一个TAg是人ZNF313基因启动子所必需的.图4参19更多还原

【Abstract】 To analyze the promoter of human spermatogenesis-related novel gene ZNF313, the promoter sequence of ZNF313 gene was amplified by PCR and luciferase expression vectors containing different promoter fragments of ZNF313 were constructed. Using pRL-TK plasmid as control, the expression vectors were instantly transfected into HEK293T cell line and the relative activity of luciferase was measured 48 h later. The result indicated that four luciferase expression vectors, pGL3-215 (-215 bp～+38 bp), pGL3-160 (-160 bp～+38 bp), pGL3-133 (-133 bp～+128 bp) and pGL3-8 (-8 bp～+128 bp) were constructed. The relative activity of luciferase of pGL3-215 was the highest, the activities of pGL3-160 and pGL3-133 were much the same, and 75% of the highest activity and the activity of pGL3-8 significantly declined nearly to zero. These indicated that the region from -133 bp to -8 bp included the essential promoter sequence for human ZNF313 gene transcription. The analysis of bioinformatics showed that two SP1 elements, one AP-2 element and one T-Ag element were also necessary for the transcription of human ZNF313 gene. Fig 4, Ref 19更多还原