绵羊TMEM182基因克隆、生物信息学及组织表达谱分析

Cloning of Sheep TMEM182 Gene and Its Bioinformatics and Tissue Expression Profile Analysis

【目的】本试验旨在克隆绵羊TMEM182基因编码区(CDS)全长序列,并通过在线软件分析TMEM182基因结构和表达蛋白序列,以探究其在绵羊各部位组织中表达水平的变化。【方法】从绵羊尾部脂肪组织中克隆获得TMEM182基因,利用在线生物学信息软件对TMEM182蛋白的理化性质、亲疏水性、跨膜区、糖基化位点、信号肽、二级结构、三级结构和蛋白互作等进行预测分析。同时,采用实时荧光定量PCR方法检测TMEM182基因在绵羊13个组织中的相对表达量,这些组织包括大脑、小脑、心、肾、脾、肝、肺、皮肤、卵巢、尾部脂肪、皮下脂肪、肾周脂肪和肌肉。【结果】绵羊TMEM182基因CDS区全长序列为690bp,编码229个氨基酸,TMEM182基因序列在不同物种间具有较高的保守性。生物信息学预测分析显示,TMEM182属于稳定的疏水性蛋白,具有跨膜结构,无信号肽,具有3个N-糖基化位点,其蛋白二级结构主要由无规则卷曲(38.43%)和2α螺旋(30.57%)组成,而延伸链(24.89%)和β转角(6.11%)的占比相对较少,三级结构预测结果与二级结构预测结果相符。实时荧光定量PCR结果显示,TMEM182基因在肌肉、心和脂肪中表达量显著高于肝、肾、大脑、小脑、皮肤等其它部位组织(P<0.05)。【结论】成功克隆了绵羊TMEM182基因的CDS区片段,分析了该基因在绵羊肌肉组织和脂肪组织中特异性表达。这些研究结果为进一步探究TMEM182基因在绵羊肌肉生长发育和脂肪沉积过程中的分子作用机制提供了理论依据。

【Objective】The objective of this study was to clone the full-length sequence of the coding sequence(CDS)region of TMEM182 gene in sheep,analyze its structure and the sequence of expression protein using online software,so as to the changes in its expression level in various parts of sheep tissues.【Methods】TMEM182 gene was cloned from sheep tail adipose tissue.The physical and chemical properties,hydrophilicity,transmembrane region,glycoylation site,signal peptide,secondary structure,tertiary structure and protein interaction of TMEM182 protein were predicted by online biological information software.Real-time fluorescence quantitative PCR was used to detect the relative expression levels of TMEM182 gene in 13 kinds of sheep tissue samples,including brain,cerebellum,heart,kidney,spleen,liver,lung,skin,ovary,tail fat,subcutaneous fat,perirenal fat and muscle.【Results】The full length of CDS region of sheep TMEM182 gene was 690 bp,encoding 229 amino acids,and the sequence was highly conserved among different species.Bioinformatics prediction results showed that TMEM182 was a stable hydrophobic protein with transmembrane structure and 3 N-glycoylation sites,no signal peptide.The secondary structure of TMEM182 protein was mainly composed of random coil(38.43%)and 2α-helix(30.57%),while the proportion of extension chain(24.89%)andβ-turn(6.11%)was relatively small.Meanwhile,the prediction results of tertiary structure were consistent with those of secondary structure.Real-time fluorescence quantitative PCR results showed that the expression levels of TMEM182 gene in muscle,heart and fat were significantly higher than those in liver,kidney,brain,cerebellum,skin and other tissues(P<0.05).【Conclusion】The CDS region fragment of sheep TMEM182 gene was successfully cloned,and the gene was specifically expressed in sheep muscle and adipose tissues.A theoretical basis was provided for further exploring the molecular mechanism of TMEM182 gene in the process of muscle growth and fat deposition in sheep.