| 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 cDNA ends，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-1 mRNA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12 h后鲢肝脏组织中IGFBP-1表达量的变化。【结果】鲢IGFBP-1全长cDNA为1 081 bp，具有73 bp的5′非编码区(Untranslated Regions，UTR)、789 bp的开放读码框(Open reading frame，ORF)，以及219 bp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6 h时，IGFBP-1 cDNA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IGFBP-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-1 mRNA表达量升高，达最大值后继续低氧胁迫其表达量略有降低。 |
| 关键词: 白鲢 低氧 IGFBP-1 RACE Real-time PCR |
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| 基金项目:国家自然科学基金项目(31101894）;公益性行业(农业)科研专项(200903045) |
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| Molecular cloning and expression analysis of the full length cDNA of IGFBP1 gene in Hypophthalmichthys molitrix |
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DING Weiqun, LIANG Hongwei, ZOU Guiwei,et al
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Kunming University of Science and Technology，et al
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| Abstract: |
| 【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. |
| Key words: Hypophthalmichthys molitrix hypoxic IGFBP-1 RACE Real-time PCR |
