山羊ST13基因的克隆及表达特性分析

Cloning and expression characteristic analysis of goat ST13 gene

摘要:

本研究旨在克隆获取山羊ST13基因完整的蛋白质编码区(sequence coding for aminoacids in protein,CDS)序列，并对其进行表达特性分析，同时探究该基因在不同组织和诱导分化不同阶段的山羊皮下前体脂肪细胞中的表达规律。利用逆转录PCR (reverse transcription PCR,RT-PCR)技术克隆ST13基因，利用在线工具和软件对该基因序列进行生物信息学分析，利用实时荧光定量RT-PCR (quantitative RT-PCR,qRT-PCR)技术检测其在山羊各个组织及皮下前体脂肪细胞不同分化阶段中的表达规律。克隆获得山羊ST13基因序列长度为1 380 bp，其CDS区长度为1 101 bp，共编码366个氨基酸；蛋白预测结果显示，ST13蛋白存在26个磷酸化位点，部分序列有强烈的亲水性和不稳定性，并且属于非跨膜和非分泌蛋白；亚细胞定位预测显示，ST13大部分分布在细胞核中，占69.6%；进化树显示，山羊ST13与绵羊的亲缘关系最近；组织表达谱显示，ST13基因在山羊心、肝、脾、肺、肾等13种组织中均有表达，尤其在臂三头肌和皮下脂肪中的表达量最高(P<0.01)，在心、肝、脾、肺、肾、大肠、小肠和胰腺中的表达量无显著性差异(P>0.05)；时序表达谱显示，该基因在诱导分化后的脂肪细胞中表达上调，在诱导分化第108小时表达最高，显著高于其他诱导分化时间段(P<0.01)；研究表明，该基因在山羊组织中广泛表达，对山羊皮下脂肪细胞分化过程有重要调控作用。

Abstract:

In this study, we cloned the complete sequence coding for aminoacids in protein (CDS) of goat ST13 gene, analyzed the bioinformation of it, and explored the expression pattern in different goat tissues and goat subcutaneous preadipocytes at different differentiation stages. To be specific, ST13 gene was cloned by reverse transcription PCR (RT-PCR), and the bioinformation was analyzed by online tools or software. The expression in various goat tissues and subcutaneous preadipocytes at different differentiation stages was detected by quantitative reverse transcription PCR (qRT-PCR). The results showed that the cloned goat ST13 gene was 1 380 bp, with CDS of 1 101 bp, encoding 366 amino acids. Protein prediction results showed that ST13 had 26 phosphorylation sites and that some sequences were highly hydrophilic and unstable. Moreover, ST13 was a non-transmembrane and non-secretory protein. Subcellular localization demonstrated that ST13 was mostly distributed in the nucleus (69.6%). Phylogeny analysis suggested that goat ST13 had the highest identity to sheep ST13. Tissue expression pattern showed that ST13 gene expressed in all of the collected 13 tissues of goat, including heart, liver, spleen, lung and kidney, especially in triceps brachii and subcutaneous fat (P<0.01) and that the expression among heart, liver, spleen, lung, kidney, large intestine, small intestine and pancreas was insignificantly different (P>0.05). In addition, according to the temporal expression pattern in adipocytes, the expression of ST13 was up-regulated in differentiated adipocytes, and the expression was the highest at the 108th hour of induction, significantly higher than that at other time points (P<0.01). In conclusion, this gene expresses in various tissues of goat and regulates the differentiation of goat subcutaneous adipocytes.