水牛Sohlh2基因的克隆分析及其表达

Cloning and expression pattern of buffalo Sohlh2 gene

为阐明广西沼泽型水牛Sohlh2基因在水牛不同组织中的表达规律,根据Gen Bank已公布的牛Sohlh2基因序列,设计其编码区(CDS)扩增引物,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,系统分析了水牛Sohlh2的遗传进化以及蛋白质的理化性质、二级和三级结构;并应用RT-QPCR技术对Sohlh2基因在水牛组织(器官)的表达进行了分析。结果表明:水牛Sohlh2基因编码区全长1 281 bp,共编码426个氨基酸。多重序列比较分析结果显示水牛Sohlh2核苷酸序列与牛、绵羊、山羊、猪、马、人和小鼠相应序列的相似性分别为98%、96%、96%、85%、86%、81%和72%,系统进化树显示Sohlh2基因在不同物种以及进化的过程中具有高度保守性。Sohlh2蛋白质呈弱碱性,无信号肽,在细胞中亚定位于细胞核中,存在b HLH结构。水牛Sohlh2基因在水牛垂体、大脑、肝脏、骨骼、卵巢和睾丸等11个组织(器官)细胞中具有不同程度的表达,其中睾丸细胞中表达量最高,垂体和生殖脊细胞中表达量次之,卵丘细胞中表达量低,心脏、肺脏、肌肉细胞和颗粒细胞中不表达。

To identify the expression pattern of Sohlh2 gene in different tissues of Guangxi swamp buffalo, a pair of special primers for CDS of Sohlh2 gene was designed according to the released sequence of bovine Sohlh2 in GenBank. The Sohlh2 gene was amplified by RT-PCR, and its genetic evolution, protein physical and chemical properties, the secondary and tertiary structure were systemically analyzed by bio-informatics techniques. A 1 281 bp whole length coding region enco-ding 426 amino acids of buffalo Sohlh2 gene was cloned and sequenced. The multiple sequence alignment revealed that buffalo Sohlh2 gene shared 98%, 96%, 96%, 85%, 86%, 81% and 72% similarities in nucleotide sequence with Bos Taurus, Ovis aries, Capra hircus, Sus scrofa, Equus caballus, Homo sapiens and Mice, respectively. Phylogenetic tree analysis showed that Sohlh2 gene was highly conserved in different species during their evolution. Sohlh2 protein was weakly alkaline, signal peptide free, and was localized in nuclear. The secondary structure of protein Sohlh2 contains 20 alpha helixes, 4 beta sheets, 23 beta turns and 18 random coils. RT-qPCR results showed that the expression levels of buffalo Sohlh2 varied in 11 of 15 tested samples, with the＆nbsp;most abundant expression in testis, followed by hypophysis and germinal ridge, and the minimal expression in cumulus cells. There was no expression in lung, heart, muscle and granular cells.