鲢IGFBP-1基因全长cDNA的克隆及表达分析  

Molecular cloning and expression analysis of the full length cDNA of IGFBP-1 gene in Hypophthalmichthys molitrix

作　　者：丁为群[1] 梁宏伟[2] 邹桂伟[2] 王晓阳[2] 曹磊[2] 刘迪秋[1] 李忠[2] 

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500 [2]中国水产科学研究院长江水产研究所,湖北武汉430223

出　　处：《西北农林科技大学学报（自然科学版）》2013年第5期1-8,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31101894);公益性行业(农业)科研专项(200903045)

摘　　要：【目的】克隆鲢胰岛素样生长因子结合蛋白1(IGFBP-1)基因,分析其编码蛋白的结构与功能,并分析低氧胁迫下该基因在鲢肝脏中的表达情况。【方法】采用cDNA末端快速扩增技术(Rapid amplification of cDNAends,RACE)扩增鲢IGFBP-1基因的全长cDNA,通过半定量RT-PCR法对鲢肌肉、心脏、脑、肾脏、肝脏、腮和脾等组织器官IGFBP-1mRNA的表达水平进行检测,利用Real-time PCR法检测急性低氧胁迫0,2,4,6,8,10和12h后鲢肝脏组织中IGFBP-1表达量的变化。【结果】鲢IGFBP-1全长cDNA为1 081bp,具有73bp的5′非编码区(Un-translated Regions,UTR)、789bp的开放读码框(Open reading frame,ORF),以及219bp的3′UTR,编码含262个氨基酸的蛋白质。RT-PCR结果表明,IGFBP-1在肌肉、心脏、脑、肾脏、肝脏、腮和脾等7种组织中均有表达,其中在肝脏中的表达量最高,心脏和肌肉次之,在脑中的表达量最低;Real-time PCR结果表明,在低氧胁迫6h时,IGFBP-1cDNA在肝脏中的表达量显著增高,随后略有降低但仍显著高于未进行低氧胁迫处理组。【结论】克隆得到鲢IG-FBP-1全长cDNA,该基因在肝脏组织中大量表达;随着低氧胁迫时间的延长,鲢肝脏中IGFBP-1mRNA表达量升高,达最大值后继续低氧胁迫其表达量略有降低。【Objective】 The study aimed to clone the cDNA of IGFBP-1 and analyze the encoded amino acids and the expression level under hypoxia in liver.【Method】 The method of Rapid amplication of cDNA ends（RACE）was used to obtain the complete IGFBP-1 cDNA sequence.Semi-quantitative RT-PCR was performed using the tissues of muscle,heart,brain,kidney,liver,gill and spleen of silver carp.The expression patterns of IGFBP-1 under the condition of acute hypoxia for 0,2,4,6,8,10 and 12 h was analyzed by real-time PCR.【Result】 The full-length cDNA of IGFBP-1 was 1 081 bp and the open reading frame was 789 nucleotides flanked by a 73 nucleotides of 5’-UTR and a 219 nucleotides of 3’-UTR.The ORF encoded a predicted polypeptide of 262 amino acids.RT-PCR showed that IGFBP-1 mRNA was expressed in muscle,heart,brain,kidney,liver,gill and spleen and showed high expression in liver,heart and muscle and low expression in brain.Real-time PCR revealed that in liver,the expression level increased significantly at 6 hours under hypoxia,and decreased a little after then but kept at a high level compared to the control group.【Conclusion】 A full-length cDNA of IGFBP-1 was cloned.IGFBP-1 mRNA was abundantly expressed in liver and the expression level increased after acute hypoxia.The peak was at 6 h after hypoxia and it slightly decreased after the peak.

关 键 词：白鲢 低氧 IGFBP-1 RACE REAL-TIME PCR 

分 类 号：S965.113.6[农业科学—水产养殖] Q786[农业科学—水产科学]