彭泽鲫葡萄糖激酶基因全长cDNA克隆及表达分析  

Cloning and Expression Analysis of a Full-length cDNA Encoding Glucokinase from Pengze Crucian Carp (Carassius auratus var. Pengze)

作　　者：程汉良[1] 姬南京[1] 彭永兴[1] 申欣[1] 吴陈晨 许建和[1] 董志国[1] 

机构地区：[1]淮海工学院,江苏省海洋生物技术重点实验室,连云港222005

出　　处：《动物营养学报》2011年第7期1167-1175,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：江苏省自然科学基金(BK2008191);浙江省近岸水域生物资源开发与保护重点实验室开放基金(J2010010);江苏省海洋生物技术重点实验室开放基金(2009HS15);江苏省高校自然科学研究重大项目(10KJA240002)

摘　　要：本试验采用RT-PCR和cDNA末端快速扩增(RACE)方法克隆获得了彭泽鲫(Caras-sius auratusvar.Pengze)葡萄糖激酶(GK)基因全长cDNA序列。结果表明,该cDNA全长2 050bp,含1个1 431bp的开放阅读框(ORF),编码476个氨基酸,GK蛋白计算分子量为53.78ku,等电点为5.14。彭泽鲫GK氨基酸序列的ATP结合位点、葡萄糖结合位点、己糖激酶标签序列、N-连接糖基化位点、细胞黏附序列和糖胺聚糖黏附位点等主要功能位点与其他脊椎动物相比基本保守。彭泽鲫GK氨基酸序列与建鲤、人和鼠GK氨基酸序列相似百分比分别为98.1%、79.8%和79.1%。以β-actin为内标,采用半定量RT-PCR方法对GK基因在彭泽鲫大脑、白肌、脾脏、肠系膜脂肪和肝胰脏各组织中的表达进行了研究。结果表明,GK基因主要在肝胰脏中表达;在脾脏、肠系膜脂肪和大脑中也检测到微量表达,但表达量显著低于肝胰脏(P<0.05);白肌中没有检测到GK基因表达。A full-length cDNA coding glucokinase（GK） was cloned from Pengze crucian carp（Carassius auratus var.Pengze） by RT-PCR and rapid amplification of cDNA ends（RACE） methods.The cDNA sequence obtained is of 2 050 bp length with a 1 431 bp open reading frame（ORF） encoding 476 amino acids.The GK protein has a calculated molecular weight of 53.78 ku and isolectric point of 5.14.The main domains of GK,such as ATP-binding domain,glucose-binding amino acids,hexokinases signature,N-linked glycosylation sites,cell attachment sequence and glycosaminoglycan attachment site for the Pengze crucian carp are basically conservative compared with other vertebrates.The amino acid sequence has a high similarity to GK of other species,and the percent identity compared with common carp,human and rat are 98.1%,79.8% and 79.1%,respectively.Tissue distribution of GK mRNA in brain,white muscle,spleen,mesenteric adipose tissue and liver of Pengze crucian carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control.The result showed that the expression level of GK mRNA in liver was significantly higher than that in spleen,mesenteric adipose tissue and brain（P0.05）.GK mRNA did not be detected in white muscle.

关 键 词：彭泽鲫 葡萄糖激酶 全长CDNA 组织表达 

分 类 号：Q785[生物学—分子生物学] Q786