cDNA sequence and tissue expression analysis of glucokinase from liver of grass carp (Ctenopharyngodon idella)

Abstract

A full-length cDNA coding glucokinase (GK) was cloned from the liver of grass carp (Ctenopharyngodon idella) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The cDNA obtained was 2066 bp exclusive of poly (A) residues with a 1431 bp open reading frame encoding 476 amino acids. The GK protein has a calculated molecular weight of 53.7 kDa and isoelectric point of 5.11. Some conserved functional sites were found including one conserved hexokinase signature sequence Leu¹⁵⁶-Phe¹⁸¹; two N-linked glycosylation sites Asn¹⁷⁶ and Asn²¹⁴; one cell attachment sequence Arg²⁰²-Asp²⁰⁴; one glycosaminoglycan attachment site Ser⁴⁵⁵-Gly⁴⁵⁸. The amino acid sequence has a high similarity to GK of other species, the percent identity compared with topmouth culter, common carp, human and rat are 98.1, 96.8, 80.3 and 79.8%, respectively. Tissue distribution of GK mRNA in brain, mesenteric adipose tissue, spleen, white muscle and liver of grass carp was analyzed by SYBR real-time fluorescence quantitative RT-PCR method using β-actin as an internal control for cDNA normalization. The result shows that the expression level of GK mRNA in liver was significantly higher than in mesenteric adipose tissue, spleen and brain (p<0.05). Relative expression profile of GK mRNA in liver normalized with β-actin level was 31, 454 and 649-fold compared with the levels in mesenteric adipose tissue, spleen and brain, respectively. Meanwhile, GK mRNA was not detected in white muscle.

Key words: Ctenopharyngodon idella, glucokinase, full-length cDNA, tissue distribution, real-time PCR.