【摘要】 ZBED6是锌指蛋白家族的一员,在胎盘哺乳动物中极其保守,可通过对IGF2的调控参与骨骼肌生长。为进一步探究ZBED6基因自身的表达调控机制,本研究以民猪基因组DNA为模板,通过常规PCR扩增ZBED6基因启动子区系列截短片段,构建克隆质粒,通过双酶切和连接反应定向连入pGL3-basic载体,利用PK15细胞和双荧光素酶检测系统测定重组质粒的相对荧光素酶活性;利用在线软件预测启动子区的转录因子结合位点,使用重叠PCR定点缺失转录因子结合位点,构建突变载体并在PK15细胞中检测突变载体的相对荧光素酶活性。结果表明:ZBED6基因启动子区-2 053～-1 777 bp存在多个转录因子结合位点,尤其是-1 808～-1 777 bp,该片段缺失造成启动子活性下降(P<0.01);利用在线软件在该区间预测到3个转录因子HINFP、Adf-1和CREB3,经实验验证后发现这3个转录因子均可调控ZBED6基因的转录,其中Adf-1效果最为明显。据此推测,民猪ZBED6基因的转录调控机制较为复杂,其启动子区存在HINFP、Adf-1和CREB3等多个调控元件的结合位点。更多还原

【Abstract】 ZBED6, a member of the zinc finger protein family, is extremely conserved in placental mammals. It regulates the growth of skeletal muscle through IGF2. In order to further explore the mechanism of expression and regulation of ZBED6,the genomic DNA of Min pig was used as template to amplify the series truncated fragments of ZBED6 gene promoter by PCR. The cloning plasmid was constructed and linked into pGL3-basic vector by double enzyme digestion and linkage reaction. The relative luciferase activity of the recombinant plasmid was predicted by online software, and the relative luciferase activity of the mutant vector was detected in PK15 cells. The results showed that there were multiple transcription factor binding sites in the promoter region of ZBED6 gene-2 053～-1 777 bp, especially in the region-1 808 ～-1 777 bp. The deletion of this fragment would cause a significant decrease in promoter activity(P<0.01). Three transcription factors, HINFP,Adf-1 and CREB3, were predicted by on-line software. The experiment results showed that these three transcription factors could regulate the transcription of ZBED6 gene, and Adf-1 was the most effective. The conclusion is that the transcription regulation mechanism of ZBED6 gene is relatively complicated, and there are many binding sites including HINFP, Adf-1 and CREB3 in the promoter region.更多还原