水牛转录抑制因子CTCF基因克隆分析及不同组织中的表达研究

Cloning of Buffalo CTCF Gene and its Expression Pattern Investigation in Different Tissues

本研究克隆了水牛转录抑制因子CTCF基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,此外还对CTCF基因在水牛不同组织中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆获得了长2239bp水牛CTCF基因序列,其中编码区全长2184bp,编码727个氨基酸,理论蛋白质分子质量82.7ku,等电点为6.57。多重序列比较分析显示,水牛CTCF核苷酸序列与牛、猪、马、人和小鼠相应序列的相似性分别为99%、96%、96%、94%和92%,结合系统进化树分析结果推测,CTCF基因在不同物种及进化的过程中具有高度的保守性。对水牛CTCF蛋白的二级和三级结构分析结果发现,其存在连续11个锌指C2H2结构,预测其为重要的DNA结合蛋白。定量表达分析结果显示,CTCF在水牛肝脏组织中相对表达量最高,大脑、肌肉和肾脏次之,卵巢和皮肤表达量较低。

Buffalo CTCF gene was cloned in the present study,the CTCF sequence was systemically analysed by bioinformatics techniques,and the expression patterns of CTCF in different tissues were also assayed with Real-time fluorescence quantitative-PCR（QRT-PCR）.The results showed that,with RT-PCR a 2239 bp buffalo CTCF gene fragment was cloned and sequenced,including the whole CDS of 2184 bp（coded 727 amino acids）.The molecular weight and isoelectric point of buffalo CTCF protein were predicted as 82.7 ku and 6.57 respectively.The sequence multialigned results showed that buffalo CTCF gene shared 99%,96%,96%,94% and 92% of similar nucleotide sequence with Bos taurus,Equus caballus,Sus scrofa,Homo sapiens and Mus musclus,respectively.It was predicted that buffalo CTCF protein contained eleven ZnF_C2H2 domains,which meaned that it was a DNA binding protein.In addition,we also analyzed the tissue expression of CTCF gene through QRT-PCR.The results showed the mRNA of CTCF gene existed in all the six detected buffalo tissues with the most abundant expression in liver,followed by brain,muscle,spleen and ovary,the minimal expression in skin was observed.