西农萨能奶山羊甘油三酯水解酶基因(ATGL)的cDNA克隆与组织表达分析  

Cloning and Tissue Expression Analysis of Adipose Triglyceride Lipase Gene (ATGL) of Xinong Saanen Dairy Goats

作　　者：滕炎玲[1] 罗军[1] 李君[1] 赵旺生[1] 王桢[1] 王维[1] 胡仕良[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2010年第6期1134-1142,共9页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No2088AA10Z135);公益性行业(农业)专项科研经费项目(No3-45);转基因生物新品种培育项目(No2009ZX08009-162B)共同资助

摘　　要：甘油三酯水解酶(adipos etriglyceride lipase,ATGL)是水解甘油三酯的关键酶。本研究采用RT-PCR和RACE技术,分离并克隆了西农萨能奶山羊(Capra hircus)ATGL基因的cDNA序列,其全长2074bp(GenBank收录号为:GQ918145),包括5'UTR141bp,CDS1461bp,和3'UTR472bp,该基因编码486个氨基酸(GenBank收录号为:ADD25174)。经氨基酸序列分析发现,山羊与GenBank中牛(Bos taurus)、大鼠(Rattus norvegicus)、小鼠(Mus musculus)、猪(Sus scrofa)和人(Homo sapiens)的ATGL的相似性较高,均在85%以上。经蛋白质结构分析表明,其蛋白质的分子量为53243.4D,等电点为7.4,具有GXSXG(氨基乙酸-X-丝氨酸-X-氨基乙酸)脂肪酶活性特征结构和Patatin结构域及α/β水解酶折叠域,在N端存在跨膜螺旋结构,并且整个序列中不含信号肽。此外,本研究还通过实时荧光定量PCR(RT-qPCR)技术对ATGL基因进行了组织表达分析。结果表明,在所检测的12个组织或器官中均有ATGLmRNA存在,其中皮下脂肪组织中表达最丰富,并且远远高于其它组织或器官,肺、乳腺次之,心脏相对最少。ATGL基因在奶山羊的乳腺组织中具有较高的表达水平,为该基因的功能研究提供了实验依据。Adipose triglyceride lipase （ATGL） is a key enzyme in lipid lipolysis. In this experiment, the whole cDNA length ATGL gene with 2 074 bp （GenBank accession No. GQ918145） was isolated and cloned by RT-PCR and RACE （rapid amplification cDNA ends） from Xinong Saanen dairy goats （Capra hircus）. ATGL gene contains 141 bp 5＇UTR, 472 bp 3＇UTR, and 1 461 bp coding sequences （CDS） which encode a protein of 486 amino acid residues （GenBank accession No. ADD25174）. AA sequence alignment showed that the AA of goat ATGL had high score similarity, more than 85%, when compared with bovine （Bos taurus）, rat （Rattus norvegicus）, mouse （Mus musculus）, pig （Sus scrofa）, and human （Homo sapiens） in GenBank. The protein structure had a Patatin domain, α/β hydrolase folds and GXSXG （Gly-X-Ser-X-Gly） structure. A transmembrane helix was speculated in the N-terminal and no signal peptide found in the entire sequence. The protein molecular weight was 53 243.4 D and the isoelectric point was 7.4. Furthermore, the ATGL gene expressions in various organs and tissues were analyzed by real-time fluorescence quantitative-PCR. The results showed that the ATGL mRNA existed in all of the 12 detected organs and tissues. The most abundant expression was detected in subcutaneous adipose tissue, which was significantly higher than that of the other tissues, and the minimal expression was observed in heart tissue. The higher expression level of ATGL gene in the mammary gland of dairy goats provided the experimental evidence for its function analysis.

关 键 词：西农萨能奶山羊 甘油三酯水解酶 基因克隆 组织表达 

分 类 号：S827[农业科学—畜牧学]