【摘要】 采用RT-PCR和RACE方法克隆建鲤(Cyprinus carpio Var.Jian)葡萄糖激酶(GK)基因全长cDNA序列,结果表明:建鲤GK基因cDNA全长2041bp,含1个1431bp的开放阅读框(ORF),编码476个氨基酸,GK蛋白分子量53.6kD,等电点5.13,建鲤GK氨基酸序列保守基序包括:1个己糖激酶标签序列Leu156-Phe181、2个N-连接糖基化位点Asn176和Asn214、1个细胞黏附序列Arg202-Asp204和1个糖胺聚糖黏附位点Ser455-Gly458,其氨基酸序列与翘嘴红鲌、人和鼠的GK氨基酸序列相似百分比分别为96.2%、79.8%和79.1%;以β-actin基因为内标,采用半定量RT-PCR方法对GK基因在肠系膜脂肪组织、肝脏、心脏、肌肉和大脑等组织的表达进行研究,结果表明GK基因仅在肝脏和大脑中表达,在肝脏中的表达水平高于大脑。更多还原

【Abstract】 A full-length cDNA encoding glucokinase (GK) was cloned from liver cells of Jian carp (Cyprinus carpio var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) methods. The analysis on GK gene cDNA sequence indicated that this gene contained a 1431 bp open reading frame (ORF) and encoded a 476-aa polypeptide. The GK protein encoded had a calculated molecular weight of 53.6 kD and a PI of 5.13. Some conserved functional sites were found, including one conserved hexokinases signature Leu156-Phe181, two N-linked glycosylation sites Asn176 and Asn214, one cell attachment sequence Arg202-Asp204 and one glycosaminoglycan attachment site Ser455-Gly458. The amino acid sequence had a high similarity to those of other species like Topmouth culter, human and rat, with the percent identity values of 96.2%, 79.8% and 79.1%, respectively. The tissue distribution of GK mRNA in mesenteric adipose tissue, liver, heart, muscle and brain of Jian carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as the internal control. The results showed that this gene only could be expressed in liver and brain and the expression level in liver was higher than that in brain.更多还原