关键词: 建鲤, 葡萄糖激酶基因, 快速扩增cDNA 末端, 全长cDNA

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 semiquantitative 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.

Key words: Cyprinus carpio var. Jian, glucokinase gene, RACE, full-length cDNA