绵羊EDAR基因的克隆、序列分析及其在毛囊发育过程中的表达

Cloning,Sequencing and Quantitative Expression of EDAR Gene in Sheep During Hair Follicle Development Period

旨在获得中国美利奴羊EDAR(ectodysplasin A receptor)基因的CDS区全序列并进行生物信息学分析,同时分析EDAR在绵羊毛囊发育过程中的表达特性,为深入研究该基因在绵羊毛囊生长发育过程中的作用及其表达调控提供基础资料。采用PCR扩增获得绵羊EDAR基因全长编码区,并克隆到PCRTM-BluntⅡ-TOPO@vector进行测序;利用生物信息学方法预测蛋白结构;应用qRT-PCR技术检测EDAR基因在绵羊毛囊发育过程中的表达特征。结果表明,绵羊EDAR基因的CDS序列长度为1 350bp(GenBank登录号:KX900497),编码449个氨基酸,该氨基酸序列与其他物种相比一致性较高;进化树分析表明,绵羊EDAR氨基酸序列与牛的进化关系较近,与斑马鱼较远;预测结果显示绵羊EDAR蛋白存在一个信号肽和一个跨膜结构域;qRT-PCR分析表明,EDAR基因在绵羊胎儿毛囊发育过程中均有表达,在毛囊发育第55天和第135天表达较高,第75天表达最低。获得绵羊EDAR基因完整的编码区序列和毛囊发育过程中的表达特性,生物信息学分析发现EDAR基因的编码区序列具有物种间的保守性,同时该基因在绵羊毛囊不同发育阶段的皮肤组织中表达,由此表明EDAR基因可能在绵羊毛囊的生长发育过程中发挥重要作用。

The aim of this study was to clone the coding sequence（CDS）of sheep EDAR（ectodysplasin A receptor）coding sequence（CDS）, to analyze its sequence characteristics, and to investigate its expression during hair follicle different development periods. It could provide the data for further investigation on the function and the expression regulation of EDAR Gene in the hair follicle growth and development period. PCR products were cloned into PCRTM BluntⅡ TOPO@ vector and sequenced. The protein structure of sheep EDAR was analyzed by bioinformatic methods. The expression patterns during hair follicle development period were detected by real time RT PCR. The results showed that the sheep EDAR CDS was 1 350 bp （GenBank accession KX900497 ） in length, which coded 449 amino acids and shared highly identity with other species. The phylogenetic tree analysis indicated that the sheep EDAR was closely related to cattle, but distantly related to the zebrafish. The sheep EDAR protein contained a signal peptide and a transmembrane helice; According to the analysis of real time PCR, mRNA expression of sheep EDAR was detected during hair follicle development period, and EDAR mRNA expression levels was higher in the 55th day and the 135 th day, and it was the lowest in the 75th day. Therefore, EDAR was cloned and its expression patterns during hair follicle period were investigated. The sequence characteristics of EDAR was conservative in different species. EDAR expression in the hair follicle development period indicated that EDAR may play an important role in the hair follicle growth and development period.