中国农业科学 ›› 2014, Vol. 47 ›› Issue (12): 2435-2445.doi: 10.3864/j.issn.0578-1752.2014.12.016

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

兰州大尾羊PPARG全长cDNA克隆及生物信息学分析

 金方园1, 徐红伟2, 达小强1, 柏家林1, 冯玉兰1, 臧荣鑫1, 杨具田1   

  1. 1、西北民族大学生命科学与工程学院,兰州 730030;
    2、西北民族大学实验中心,兰州 730030
  • 收稿日期:2013-11-06 出版日期:2014-06-15 发布日期:2014-03-24
  • 通讯作者: 杨具田,E-mail:jutianyang988@163.com;臧荣鑫,E-mail:rongxinzang2000@163.com
  • 作者简介:金方园,E-mail:373995197@qq.com
  • 基金资助:

    国家自然科学基金(31160440,31260533,31360529)、甘肃省科技支撑计划(1011NKCA051)、兰州市科技计划(2011-1-113)、西北民族大学研究生科研创新项目(ycx13175)

Cloning and Bioinformatics Analysis of PPARG Gene from Lanzhou Fat-Tailed Sheep

 JIN  Fang-Yuan-1, XU  Hong-Wei-2, DA  Xiao-Qiang-1, BAI  Jia-Lin-1, FENG  Yu-Lan-1, ZANG  Rong-Xin-1, YANG  Ju-Tian-1   

  1. 1、College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou 730030;
    2、Science Experimental Center, Northwest University for Nationalities, Lanzhou 730030
  • Received:2013-11-06 Online:2014-06-15 Published:2014-03-24

摘要: 【目的】克隆兰州大尾羊过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor gamma, PPARG)基因,分析其序列及编码蛋白的生物学特性,阐明PPARG基因在绵羊中的生物学作用,为生产应用提供理论依据。【方法】根据绵羊PPARG基因CDS序列设计特异性引物,利用RACE和RT-PCR技术克隆获得兰州大尾羊PPARG基因序列,结合生物信息学方法分析其生物学特性。【结果】克隆获得兰州大尾羊PPARG cDNA 序列全长1 774 bp(GenBank序列号:KF727439),其CDS区片段长1 428 bp,编码475个氨基酸,编码区两侧翼分别具有5’-UTR 131 bp(1-131 nt)和3’-UTR 214 bp(1 560-1 774 nt)。预测兰州大尾羊PPARG蛋白分子量为54.40 kD,理论等电点为4.94。预测PPARG编码蛋白疏水性最大值为3.600,最小值为-2.744,为非跨膜的疏水性蛋白。亚细胞定位主要在细胞质(65.2%)和细胞核(21.7%)中,少量作用于细胞支架(4.3%)和过氧化物酶体(4.3%),无信号肽,不属于分泌蛋白。预测其氨基酸序列有26个磷酸化位点,无糖基化位点,含有1个ZnF_C4结构域、1个HOLI结构域和1个LCR结构域,二级结构以随机卷曲为主。同源性分析显示兰州大尾羊PPARG核苷酸序列与山羊、野牛、水牛、绵羊、虎鲸、野猪和人类核苷酸序列间的同源性分别为99% 、98% 、97% 、99%、 94%、92%和90%,兰州大尾羊PPARG氨基酸序列与山羊、野牛、水牛、绵羊、虎鲸、野猪和人类核苷酸序列间的同源性分别为100% 、99.8% 、100% 、100%、 98.7%、98.5%和97.5%。系统发育树表明,兰州大尾羊与山羊、绵羊和水牛的进化水平更为接近,与鱼类、人类和鼠类较远。其基因第486位发生碱基转换(C←→T),第828位发生碱基转换(C←→A),但其所编码氨基酸不变。【结论】兰州大尾羊与其他物种PPARG在结构上相似性较高,说明该基因具有高度的保守性。推测PPARG蛋白大部分在游离核糖体上合成,其功能与过氧化物酶体有关,该预测结果符合其过氧化物酶体增殖物激活受体的身份。PPARG氨基酸序列有26个可以成为蛋白质激酶磷酸化的位点,某些位点被磷酸化后,可导致一系列肥胖相关基因的功能失调和表达变化, 如胰岛素增敏激素,脂联素( adiponectin)的表达减少。其序列包含的ZnF_C4锌指结构域具有与脂质结合的功能,HOLI即LBD配体结构域在激素信号转导过程中发挥重要作用,其中PPARG锌指结构域中的磷酸化位点Ser112,被MAPK磷酸化后,可以抑制PPARG同配体的结合,从而降低PPARG蛋白的活性,两个结构域均与成脂分化过程相关联。文章为进一步研究PPARG在成脂分化过程中的作用提供了参考。

关键词: 兰州大尾羊 , PPARG , cDNA末端快速扩增 , 生物信息学

Abstract: 【Objective】The objective of this study is to reveal the biological function of peroxisome proliferator-activated receptor gamma (PPARG) gene in sheep and provide theoretical data for application by cloning PPARG and analyzing its sequence as well as genetic features.【Method】The specific primers on the CDS template of PPARG gene were designed and then the PPARG gene sequence of Lanzhou fat-tail sheep was cloned using RACE and RT-PCR technology. After that, its genetic characteristics were analyzed by bioinformatics methods. 【Results】 The 1 774 bp full-length cDNA of PPARG of Lanzhou fat-tailed sheep (Genbank No.: KF727439) was obtained, in which the length of CDS is 1 428 bp. It encodes 475 amino acids in total. The coding regions on both sides of the wing are respectively provided with 5 '-UTR 131 bp (1-131 nt) and 3' -UTR 214 bp (1560-1774 nt). It was predicted that the molecular weight of PPARG protein of Lanzhou fat-tail sheep is 54.40 kD and its theoretical isoelectric point is 4.94. In addition, its maximum hydrophobic value is 3.600 and minimum hydrophobic value is -2.744. It is a non-transmembrane hydrophobin with no signal peptide, which locates mostly in cytoplasm (65.2%), seldom in nucleus (21.7%), scaffold (4.3%) and peroxisome (4.3%) by the result of subcellular level prediction and does not belong to the secretory protein. Its amino acid sequence contains 26 phosphorylation sites, 0 glycosylation site, 1 ZnF_C4 domain, 1 HOLI domain and 1 LCR domain. Also, its secondary structure is mainly randomly curly. Nucleotide sequence analysis revealed that the gene nucleotide sequence of Lanzhou fat-tail sheep is similar with those of the capra (99%), bos (98%), bubalus (97%), ovis (99%), orcinus (94%), sus (92%) and homo (90%). The similarity of amino acid sequence of PPARG between Lanzhou fat-tailed sheep and capra, bos, bubalus, ovis, orcinus, sus and homo are 100%, 99.8%, 100%, 100%, 98.7%, 98.5% and 97.5%. The phylogenetic tree indicates that Lanzhou fat-tail sheep is close to capra, ovis and bubalus, but far from fish, human and mouse. Base transitions occur in the 486th and 828th sites, which are (C vs. T) and (C vs. A), respectively, but the corresponding amino acids do not change.【Conclusion】The fact that the structure of PPARG of Lanzhou fat-tail sheep highly similar with other species indicates that the gene is highly conservative. It was predicated that most of the PPARG protein synthesises at the ribosome and its function is related to peroxisome. The results are in good agreement with its status of peroxisome proliferator activated receptor. The amino acids sequence of PPARG has 26 protein kinase phosphorylation sites. After phosphorylation some sites will lead to a series of dysfunction and expression of obesity-related genes, such as decreased expression of insulin sensitizing hormone and adiponectin. In PPARG , HOLI (also named LBD ligand) structural domain plays an important role in the process of hormone signal transduction, while ZnF_C4 zinc finger structural domain has the function of combining with lipid, which means that the Ser112 in PPARG zinc finger domain, after phosphorylation by MAPK, can inhibit the PPARG binding with the ligand, thereby reducing the activity of the PPARG protein. Both domains are associated with the differentiation process of lipid. The paper has laid a biological foundation for further study on the function of PPARG in lipid differentiation.

Key words: Lanzhou fat-tailed sheep , PPARG gene , race , bioinformatics analysis