动物组织中GLUD1基因表达与GDH蛋白结构分析

目的]研究动物不同组织中GLUD1基因的表达情况,比较不同物种中GDH蛋白的结构差异,明晰GLUD1基因的表达模式与GDH蛋白的功能。方法]分别采集大鼠、绵羊、梅花鹿的肝脏、肾脏、皮肤、脂肪和肌肉5种新鲜组织样品。以β-actin为内参基因,采用荧光定量PCR法（qRT-PCR）分析3种动物不同组织中GLUD1基因的表达情况;利用无重复双因素分析方法表征GLUD1基因在不同组织中的表达量差异。比较人、小鼠、大鼠、山羊、绵羊、牛和猪7个物种GDH蛋白氨基酸序列,模拟这7个物种的GDH蛋白三维空间结构。结果]GLUD1基因在大鼠、绵羊和梅花鹿的5个组织中均有表达,在肝脏中的表达量均高于其他组织;该基因在大鼠和梅花鹿肝脏组织中的表达量极显著（P<0.01）高于绵羊肝脏组织中的表达量,在大鼠肾脏组织中的表达量极显著（P<0.01）高于梅花鹿肾脏组织中的表达量。人GDH蛋白的氨基酸序列与猪、牛、山羊和绵羊的序列相似度较高,而大鼠与小鼠GDH蛋白的氨基酸序列相似度达到99.10%。大鼠、人、猪的GDH蛋白氨基酸序列中分别存在2个（第8位丙氨酸→缬氨酸、第40位丙氨酸→缬氨酸）、1个（第23位丙氨酸→丝氨酸）、2个（第33位丙氨酸→苏氨酸、第39位丙氨酸→苏氨酸）物种特异性突变位点。小鼠、大鼠、人的GDH蛋白预测为三聚体结构,山羊、绵羊、牛和猪的GDH蛋白预测为同源六聚体结构。结论]GLUD1基因在大鼠、绵羊、梅花鹿不同组织中的表达量存在差异,在肝脏中都有较高水平的表达。不同物种GDH蛋白的氨基酸序列相似度较高,但依旧存在一定的序列差异性,并且存在数个对应物种特异性的氨基酸突变位点。

Expression of GLUD1 Gene in Animal Tissues and Structural Analysis of GDH Protein

Objective]To characterize the expressions of GLUD1 gene in different tissues of animals, to compare the structural differences of GDH protein in different species, and to clarify the expression mode of GLUD1 gene and biological function of GDH protein. Method] Five tissue samples, including liver, kidney, skin, fat and muscle, were collected respectively from rat, sheep and sika deer for detecting the relative expression of GLUD1 gene by qRT-PCR using β-actin as reference gene. The differential expression of GLUD1 gene in various tissues was characterized by non-repetitive two-factor analysis. The amino acid sequences of GDH proteins from 7 species including human, mouse, rat, goat, sheep, cattle and pig were compared, and the three-dimensional structure of GDH proteins from different species was simulated. Result] The expression of GLUD1 gene was observed in the five tested tissues of rat, sheep and sika deer, and higher expression level was found in liver for all of the three kinds of animals. The expression level of GLUD1 gene in liver of rat and sika deer was extremely significantly （P<0.01） higher than that of sheep, and the expression level of GLUD1 gene in kidney of rat was extremely significantly （P<0.01） higher than that of sika deer. The amino acid sequence of human GDH protein had high similarities to that of pig, cattle, goat and sheep, while the amino acid sequence similarity of GDH protein between rat and mouse reached up to 99.10 %. Rat, human and pig had 2 （position 8 and 40 from Ala to Val）, 1 （position 23 from Ala to Ser）, and 2 （position 33 and 39 from Ala to Thr） species-specific mutations in amino acid sequence of GDH protein, respectively. The GDH proteins of mouse, rat and human were predicted as trimer structure, and those of goat, sheep, cattle and pig were predicted as homologous hexamer structure. Conclusion] The GLUD1 gene has differential expression in various tissues of rat, sheep and sika deer, and exhibits a higher level of expression in liver. While the amino acid sequence similarity of GDH proteins among different species is high, there are still sequence differences, especially the corresponding species-specific amino acid mutation sites.