乌骨绵羊NR5A2基因编码区序列克隆及特征分析

Cloning and characterization of coding sequences of NR5A2 gene in blackbone sheep

为了研究NR5A2基因在乌骨绵羊中的序列特征,利用PCR和克隆测序技术获得乌骨绵羊NR5A2基因的外显子序列,用DNAMAN软件和DNAStar软件对序列进行比对和拼接、运用Clustal软件与其他物种相应序列进行同源比对分析,MEGA5.1软件用来构建哺乳动物NR5A2蛋白质系统进化树,用DNA池方法对其编码区序列的SNPs位点进行筛选。结果显示:乌骨绵羊NR5A2基因编码区全长1 488 bp,共编码495个氨基酸;乌骨绵羊的NR5A2基因编码区核苷酸序列与湖羊和牛的同源性分别为99.87%和85.51%;氨基酸序列的一致性分别为99.41%和89.66%。与湖羊相比,乌骨绵羊NR5A2基因编码区共发现3个单碱基突变(C1069A、C1419T和G1485A),其中C1069A位点突变引起了氨基酸的改变,从亮氨酸变为异亮氨酸。在乌骨绵羊的编码区现了一个单核苷酸突变位点G999C。

To analyze the sequence characterization of NR5A2 gene in blackbone sheep,exon sequences of NR5A2 were obtained using PCR and cloning and sequencing technology,assembled in turn using DNAStar and DNAMAN software and then blasted with its corresponding sequences of other species with Clustal software.The full length of coding region of NR5A2 was 1 488 bp in blackbone sheep encoding 495 amino acids.The NR5A2 coding region shared 99.87% and85.51% similarities in nucleotide sequence with Hu sheep and cattle,respectively,and 99.41% and 89.66% similarities in amino acids sequence,respectively.The phylogenetic tree constructed based on NR5A2 protein using MEGA5.1software revealed blackbone sheep and Hu sheep were clustered closely together,and then formed a cluster with cattle.Blackbone sheep,Hu sheep and cattle exhibited a closer genetic relation to livestock,such as swine,horse,and dog than primate.Blackbone sheep was distinct from poulty and fishery.Compared with Hu sheep,three single nucleotide polymorphism（C1069 A,C1419T and G1485A） in coding region of NR5A2 of blackbone sheep were detected,among which,C1069 A mutation caused the changes of amino acid,from leucine to isoleucine.A SNP site,G999 C,was found in the coding region of blackbone sheep using DNA pool.