鸡TATA结合蛋白的亚细胞定位及基因组织表达特性分析

Subcellular localization and tissue expression characteristics of the chicken TATA binding protein

为探究鸡TATA结合蛋白(TATA-binding protein,TBP)的亚细胞定位以及TBP基因在鸡胚不同发育阶段不同组织中的表达情况,首先扩增鸡TBP基因的蛋白质编码区(CDS),将其亚克隆至真核表达载体pCMV-Myc构建重组真核表达载体pCMV-Myc-TBP,转染人胚胎肾细胞(HEK-293T),然后利用蛋白质印迹法和间接免疫荧光法分别检测重组蛋白的表达和亚细胞定位;进一步通过RT-qPCR方法检测TBP基因在鸡胚胎期14 d(E14 d)、出壳后1日龄(1 d)、7日龄(7 d)和14日龄(14 d)4个时间点各组织中的表达情况。结果表明,成功构建了鸡TBP基因的重组真核表达载体pCMV-Myc-TBP,转染细胞后获得与预期大小一致的Myc-TBP重组蛋白条带。亚细胞定位分析结果显示,鸡TBP蛋白主要定位在细胞核。RT-qPCR检测结果显示,鸡TBP基因在E14 d到14 d的各组织中均有表达,但均以肺脏中的表达量高,其中在1 d鸡的肺脏中表达量最高。对鸡TBP基因组织表达变化趋势分析发现,在E14 d至14 d,TBP基因在眼球、脑、心脏、肝脏、肌胃、胸肌、腿肌中的表达整体呈下降趋势,没有明显的变化规律;而肺脏中的TBP基因表达水平先升高后降低再升高,整体呈"N"型变化趋势。本研究证实鸡TBP蛋白主要定位在细胞核,TBP基因在鸡胚发育不同阶段的各组织中都有表达,但以肺脏中的表达量高。

To explore the subcellular localization of the chicken TBP protein and its expression in different tissues at different embryonic growth stages of chickens,the CDS region of the chicken TBP gene was amplified and then sub-cloned into the eukaryotic expression vector pCMV-Myc to construct pCMV-Myc-TBP.HEK-293 T cells transfected with the plasmids were used to detect the expression and subcellular localization of the recombinant protein by Western blot and indirect immunofluorescence assays.In addition,the qRT-PCR method was performed to examine the expression of the TBP gene at 4 time points including the embryonic period 14 d(E14 d),and the time points of 1 day(1 d),7 day(7 d)and 14 day(14 d)after hatching.The results showed that the recombinant eukaryotic expression vector pCMV-Myc-TBP was successfully constructed,and the recombinant protein Myc-TBP was correctly expressed when it was transfected into cells.The results of the fluorescence localization analysis revealed that the chicken TBP protein was mainly localized in the nucleus.The RT-qPCR results showed that the TBP gene was expressed in each tissue from E14 d to 14 d,and had the highest expression in the lung,especially at 1 d.The expression tendency of the TBP gene in various tissues at different developmental stages indicated that the expression levels of the gene showed a downward trend in eyeball,brain,heart,liver,muscular stomach,chest and leg muscles of the chicken from E14 d to 14 d,with no obvious change patterns.However,the expression of the TBP gene in the lung was first increased,then decreased and increased again,showing the expression pattern of N.This study demonstrated that chicken TBP protein was mainly localized in the nucleus,and that the TBP gene was expressed in various tissues at different embryonic growth stages of chickens,having especially the highest expression in the lung.