摘要： 为获得山羊PHKG1基因序列，明确其生物学特征，阐明其在山羊各组织及诱导分化不同阶段皮下和肌内脂肪细胞中的表达特性，以简州大耳羊为试验动物，屠宰后采集心脏、肝脏、脾脏、肾脏、肺脏、背最长肌、皮下脂肪等组织样品，提取组织中总RNA，利用RT-PCR方法克隆山羊PHKG1基因序列，利用各种在线工具分析其生物学特性，利用实时荧光定量PCR （Real-time quantitative PCR，qPCR）技术检测其在各个组织及不同分化阶段的前体脂肪细胞中的表达水平。结果显示，克隆得到的山羊PHKG1基因序列1 233 bp，其中CDS区1 164 bp，共编码387个氨基酸，形成无信号肽的不稳定亲水酸性非跨膜蛋白，亚细胞定位显示其主要存在细胞质中；山羊PHKG1氨基酸序列与绵羊、牛、猪、马、人的相似性均在90%以上，说明该基因在不同物种中具有较高保守性；构建进化树显示，山羊和绵羊在同一分支，符合物种进化规律；组织表达谱显示，PHKG1基因在山羊心脏、肝脏、脾脏、肾脏、肺脏、背最长肌、皮下脂肪中广泛表达，且在背最长肌中表达水平最高（P <0.01），时序表达谱显示，PHKG1基因在成脂诱导分化60 h的肌内脂肪细胞和96 h的皮下脂肪细胞中的表达水平分别显著（P <0.05）和极显著（P <0.01）高于前体脂肪细胞中的表达水平。结果可为进一步研究PHKG1在山羊脂肪细胞分化过程中的作用奠定基础。

关键词: 山羊, PHKG1, 基因克隆, 组织表达, 时序表达

Abstract: The aim of this experiment was to obtain the sequence of goat PHKG1,to clarify its biological characteristics,and to elucidate the tissue expression level and cell expression profiles of subcutaneous and intramuscular adipocytes. Jianzhou Big-eared goats were selected as experimental materials,the heart,liver,spleen,kidney,lung,longissimus muscle,subcutaneous fat were harvested after slaughtering,and the total RNA was extracted from these tissues. The sequence of PHKG1 gene was cloned by Real-time PCR(RT-PCR),the biological characteristics were analyzed by online tools,and the tissue and cell temporal expression of PHKG1 was detected by Real-time quantitative PCR (qPCR). The results showed that the cloned PHKG1 gene sequence was 1 233 bp in length,of which the CDS region was 1 164 bp. The PHKG1 gene encoded 387 amino acids,forming an unstable hydrophilic acidic nontransmembrane protein with no signal peptide,and subcellular localization indicated that it was mainly present in the cytoplasm. The amino acid sequence similarity between goats and sheep,cattle,pigs,horses and humans was above 90%,indicating that PHKG1 was highly conserved in different species. Constructing phylogenetic tree showed that goats and sheep were in the same branch,in line with the evolutionary laws of species. The PHKG1 gene was widely expressed in goat-tissues and was the highest in the longissimus muscle(P <0.01)and the expression levels in intramuscular adipocytes at 60 h and subcutaneous adipocytes at 96 h of adipogenic differentiation were significantly(P<0.05)and extremely significantly(P<0.01)higher than those in the preadipocytes. This study laid the foundation for further study of the role of PHKG1 in the differentiation of goat adipocytes.

Key words: Goat, PHKG1, Gene cloning, Tissues expression, Temporal expression

中图分类号: 

• Q78

• S826