甘肃鼢鼠甘油三酯脂酶基因(ATGL)CDS克隆及组织表达分析

CDS Cloning and Expression Analysis of Adipose Triglyceride Lipase Gene(ATGL)in Gansu Zokor(Myospalax cansus)

以甘肃鼢鼠(Myospalax cansus)脂肪组织作为研究对象,采用PCR技术克隆鼢鼠ATGL基因,运用生物信息学方法分析序列特征、系统进化关系以及预测ATGL的理化性质与分子结构,利用qRT-PCR技术检测鼢鼠ATGL基因mRNA表达水平。结果表明,鼢鼠ATGL基因CDS序列全长1 461bp(GenBank登录号:KY030728),编码486个氨基酸。序列比对结果显示,鼢鼠ATGL基因序列与小鼹形鼠(Nannospalax galili)、中国仓鼠(Cricetullus griseus)、小鼠(Mus musculus)、大鼠(Rattus norvegicus)、人(Homo sapiens)、猪(Sus scrofa)、牛(Bos taurus)等物种相比均具有较高的相似性(81%以上)。多物种ATGL氨基酸序列的系统进化分析表明,鼢鼠ATGL与中国仓鼠、小鼠和大鼠的亲缘关系较近。ATGL分子结构预测发现,其N端含有1个Patatin结构域和具有酯酶活性的GASAG结构。组织表达分析结果显示,ATGL基因在被检测的8个组织中均有表达,表达量由高到低依次为棕色脂肪组织、皮下脂肪组织、肝脏、内脏脂肪组织、心脏、脾、肌肉和肾,且棕脂中ATGL的表达量显著高于其他组织(P<0.05)。研究获得了鼢鼠ATGL基因CDS全长及组织表达规律,表明棕脂对于鼢鼠野外生存具有重要意义,为进一步阐明鼢鼠适应地下生活的能量代谢方式提供理论依据。

Abstract：Gansu zokor （Myospalax cansus） is a typical subterranean rodent which lives in burrows with hypoxia and high carbon dioxide, and excavation habits lead to some energy metabolism differences between Gansu zokor and subaerial rodents. Adipose triglyceride lipase （ATGL） is the key enzyme that catalyzes the initial step of triglyceride hydrolysis and plays an important role in lipid catabolism and energy homeostasis. The aim of this study was to clone the coding sequence （CDS） of ATGL gene of Gansu zokor and analyze its expression profile. Adipose tissue of Gansu zokorwas used as experimental material in this stud- y. Primers were designed with reference to mouse （Mus rnusculus） and mole rat （Nannospalax galili） ATGL gene sequences,and ATGL gene was cloned by PCR. The sequence characteristics, phylogenetic relationship, physico-chemical properties and molecular structure of ATGL were analyzed using bioinformatits softwares,and the expression level of ATGL mRNA in various tissues was detected by qRT-PCR. The results showed that a 1 461 bp of ATGL gene CDS （GenBank accession number： KY030728） was cloned, which encoded a protein of 486 amino acids. Sequence alignment showed nucleotide sequence and amino acid sequence of Gansu zokor ATGL exhibiting high similarity to that of mole rat, hamster （Cricetulu sgriseus ） , mouse, rat （Rattus norvegicus ）, human （ Homo sapiens ）, pig （ Susscro f a domestica ）, bovine （Bos taurus）and other species （more than 800//00）. Phylogenetic analysis based on the amino acid sequence alignment of a multi species revealed that Gansu zokor ATGL with close genetic relationship with hamster, mouse and rat. ATGL molecular structure analysis illustrated that the protein N-terminal contains one single Patatin domain and a GASAG structure with esterase activity. The results of expression analysis indicated that the ATGL gene mRNA existed in all of the 8 detected tissues. Brown adipose tissue significantly displayed the highest abundance （P〈0.05） of ATGL mRNA, followed by subcutaneous adipose tissue,liver, visceral adipose tissue, heart, spleen, muscle and kidney. The results of the current study indicated the necessity of brown adipose tissue for the Gansu zokor survival in the wild,which provided the basic data for further enriching the function of ATGL gene and clarifying its energy metabolism mechanism of adapt to underground life in Gansu zokor.