水牛ALK5基因克隆分析与组织表达模式研究

Cloning analysis of buffalo ALK5 gene and the study of its tissue expression pattern

为了阐明水牛ALK5基因在水牛卵泡发生过程中的作用,了解其在水牛不同组织中的表达模式,试验采用了RT-PCR及QRT-PCR方法对ALK5基因进行了研究,参考已公布的Gen Bank中牛ALK5基因序列,设计特异性引物应用RT-PCR方法扩增克隆获得目的基因片段;应用生物信息学方法,系统分析和预测了水牛ALK5基因的遗传进化、蛋白质的理化性质、二级和三级结构,并应用QRT-PCR技术对ALK5基因在水牛组织的表达进行了差异分析。结果表明:水牛ALK5基因的编码区全长为1 512 bp,共编码503个氨基酸;水牛ALK5核苷酸序列与牛、绵羊、猪、人和小鼠相应序列的相似性分别为99%、97%、95%、93%和90%,结合系统进化树分析结果推测ALK5基因在不同物种以及进化的过程中具有高度保守性;该蛋白呈弱碱性,有信号肽,细胞亚定位于胞膜上,存在low complexity和GS结构,呈典型跨膜蛋白结构;水牛ALK5基因在肺脏、垂体、下丘脑组织中不表达,在水牛大脑、肝脏、骨骼、卵巢、肌肉和心脏等12个组织细胞中具有不同程度的表达,其中卵巢中表达量最高,心脏和肾脏次之,肌肉表达量最低。说明试验成功地克隆了沼泽型水牛ALK5基因,其具备典型的TGF-β受体家族结构,表达具有一定的组织特异性。

To elucidate the role of buffalo ALK5 genes in the process of buffalo folliculogenesis,and understand its expression patterns in different tissues of the buffalo,the methods of RT- PCR and QRT- PCR method were used to study the ALK5 gene. A pair of specific primers was designed referring to the sequence of bovine ALK5 gene published in Gen Bank. The ALK5 gene was amplified and cloned to obtain the target gene fragment by RT- PCR,the bioinformatics method was used to systematically analyze and predict the genetic evolution,physical and chemical properties of proteins,and the secondary and tertiary structures of the buffalo ALK5 gene. A differential analysis for the ALK5 gene expression in the tissues of the buffalo were conducted using QRT- PCR technique. The results showed that the the full- length of the coding region of the buffalo ALK5 gene was 1 512 bp,encoding for a protein of 503 amino acids. The nucleotide sequence of the buffalo ALK5 gene shared99%,97%,95%,93% and 90% similarities with that of Bos Taurus,Ovis aries,Sus scrofa,Homo sapiens and Mus musculus,respectively.It was speculated that the ALK5 gene was highly conserved in the different species and the process of evolution based on the results of phylogenetic analysis. The ALK5 protein was weakly alkaline,which had signal peptide,cellular sublocalization in the membrane,and with the presence of low complexity and GS（ GS motif） structure,showing a typical transmembrane protein structure. In addition,the expression pattern analysis results showed that the buffalo ALK5 gene was not expressed in the lung,pituitary and hypothalamus tissues,but there were different levels of expression in the 12 tissues of the buffalo including brain,liver,skeleton,ovary,muscle,heart and so on,thereinto,the highest expression was in the ovary,followed by heart and kidney,the lowest expression was in the muscle. The results indicate that the ALK5 gene of the swamp buffalo is successfully cloned,it has the typical structure of TGF- β receptor family,and has a certain tissue- specific expression.