gip克隆、序列分析及其基因表达的调节

Cloning and sequence analysis of common carp (Cyprinus carpio) gip and the regulation of its mRNA expression

  • 摘要: 为研究GIP (葡萄糖依赖性肠促胰岛素,glucose-dependent insulinotropic polypeptide)在鲤体内的生理功能,实验利用RT-PCR (reverse transcription-polymerase chain reaction)技术从鲤前肠克隆得到gip,对其进行生物信息学分析,并对其mRNA的表达进行检测。结果显示,鲤gip的开放阅读框 (open reading frame,ORF)为324 bp,编码107个氨基酸。经过结构预测,鲤Gip前体蛋白包括信号肽、N端结构域、Gip成熟肽和C端结构域。氨基酸序列比对及系统进化树分析结果显示,鲤Gip蛋白与斑马鱼Gip蛋白的亲缘关系最近。Real-time PCR结果显示,gip在鲤各个组织中均有表达,但在前肠和垂体组织中表达量最高。葡萄糖灌喂实验结果表明,鲤的脑和前肠中gip的表达量在灌喂葡萄糖1和2 h后显著增加。在饥饿再投喂实验中,鲤前肠中gip的表达量在饥饿后显著降低,而再投喂后表达量显著升高。在饲养实验中,高糖和高脂均能显著增加鲤前肠中gip基因的表达量。本研究克隆了鲤gip,并且不同的营养状态可以调节其表达水平。研究结果初步阐明了Gip的生理功能,为探究Gip调节鱼类糖脂代谢的机制提供理论依据。

     

    Abstract: As an important incretin, GIP (glucose-dependent insulinotropic polypeptide) is involved in regulation of glucose uptake and lipid deposition. To investigate the role of Gip in Cyprinus carpio, the gip was cloned from the foregut by RT-PCR (reverse transcription-polymerase chain reaction). The sequence was analyzed by bioinformatics and the mRNA expression was detected by Real-time PCR. The result showed that the ORF (Open reading frame) of C. carpio gip is 324 bp, which encodes 107 amino acids. Based on amino acids sequence, the structure of Gip precursor protein contains four domains: signal peptide, N-terminal domain, mature Gip peptide and C-terminal domain. The result of sequence alignment and phylogenetic tree showed that the identity of C. carpio Gip protein was the closest to that of D. rerio. The Real-time PCR result showed that the gip was widely expressed in multiple tissues, and had higher expression in the intestine and pituitary of C. carpio. In OGTT experiment, the gip expression was significantly increased in C. carpio foregut and brain after glucose treatment. In the fasting and refeeding experiment, the gip expression in the foregut was markedly decreased after fasting for 7 days, and was significantly increased after refed. In the feeding experiment, the gip expression level was increased in the high glucose and high lipid groups. In this study, C. carpio gip was cloned, and the expression level was regulated by nutrient status. The results provide the data base for illustrating Gip biological functions and theoretical foundation for investigating the regulation of glucose and lipid metabolism in fish.

     

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