摘要： 旨在克隆山羊载脂蛋白E（Apolipoprotein E，APOE）基因序列并进行生物信息学分析，明确APOE基因在山羊各组织及分化前后脂肪细胞中的表达模式，利用RT-PCR及3'RACE方法克隆山羊APOE基因序列，利用实时荧光定量PCR（Real-time quantitative PCR，qPCR）方法检测该基因在山羊心、肝、脾、肺、肾、肌肉、脂肪等13个组织中的表达水平以及在皮下前体脂肪细胞成脂分化过程中的表达变化情况。结果表明，RT-PCR方法获得山羊APOE基因序列970 bp，其中ORF 951 bp，5'UTR 7 bp，3'UTR 12 bp（GenBank登录号：MN049956）；3'RACE法获得3'UTR 152 bp（登录号：MN049957）；Targetscan和Mirbase预测得知miR-22-3p可能靶标山羊APOE基因；蛋白预测显示山羊APOE编码316个氨基酸，是一个具有信号肽、无跨膜结构域的不稳定酸性蛋白；亚细胞定位结果发现APOE在细胞外、细胞质、液泡、细胞核以及内质网中均发挥生物学作用；进化树显示该基因在各物种的同源性较高，与绵羊、藏羚羊和牛的亲缘关系最近；基因组织表达谱显示山羊APOE在皮下脂肪中的表达量最高，极显著高于其他组织（P<0.01）；时序表达结果显示随着皮下前体脂肪细胞分化的进行，APOE基因表达呈上升趋势且在诱导分化60 h时表达量最高。结果为最终进一步揭示APOE基因在脂肪细胞分化、脂肪沉积及脂质代谢中的作用提供了基础理论数据。

关键词: 山羊, APOE, 克隆, 组织表达, 时序表达

Abstract: The aim of this study was to clone goat apolipoprotein E (APOE) gene sequence, analyze its biological characteristics and elucidate the expression patterns of APOE in goat different tissues and differentiated stages of preadipocytes. The goat APOE gene sequence was cloned by RT-PCR and 3'RACE technique. The biological characteristics of goat APOE were analyzed by a variety of online tools. In addition, the expression levels of goat APOE in different tissues and differentiated stages of intramuscular adipocytes were investigated by qPCR technique. The obtained length of goat APOE sequence was 970 bp, including 951 bp ORF, 7 bp 5'UTR and 12 bp 3'UTR (GenBank accession number:MN049956); 152 bp 3'UTR cloned by 3' RACE technique(Accession number:MN049957). Targetscan and mirbase predicted that miR-22-3p may target the goat APOE. The goat APOE encoded 316 amino acids and formed an unstable acidic protein with SignalIP 4.1 peptide without transmembrane domain. Subcellular localization results showed that APOE playing biological role in extracellular, cytoplasm, vacuole, nucleus and endoplasmic reticulum. The phylogenetic tree showed that the homology of APOE was close to Ovis aries, Pantholops hodgsonii and Bos mutus. Tissue expression profile revealed that goat APOE gene had highest expression level in subcutaneous fat, which was significantly higher than other analyzed tissues (P<0.01). Meanwhile, time-series expression profile in subcutaneous preadipocytes indicated that the highest expression level of APOE reached at 60 h. This study provides basic theoretical data for further revealing the role of APOE gene in adipocyte differentiation, fat deposition and lipid metabolism.

Key words: Goat, APOE, Cloning, Tissue expression, Temporal expression

中图分类号: 

• Q78

• S826