团头鲂(Megahbrama amblycephala) Caspase 9 基因全长cDNA的克隆及在氨氮胁迫下的表达分析

Molecular Cloning and Expression Analysis of Caspase 9 Gene from Blout Snout Bream (Megahbrama amblycephala) Under Ammonia-N Stress

中文关键词: 团头鲂  细胞凋亡因子  基因克隆  氨氮胁迫  组织表达

英文关键词: Megahbrama amblycephala  Caspase 9  Gene cloning  Ammonia-N stress  Tissue expression

中文摘要:

      为了解团头鲂(Megahbrama amblycephala) Caspase 9基因序列特征及其在氨氮胁迫过程中的作用，应用末端快速扩增(Rapid amplification of cDNA ends, RACE)技术克隆得到团头鲂Caspase 9基因全长1613 bp的cDNA序列，包括185 bp的5¢末端非翻译区(Untranslated regions, UTR)、96 bp的3¢UTR、1332 bp的开放阅读框(Open Reading Frame, ORF)。氨基酸多序列比对显示，平均同源性为77.74%，表明团头鲂Caspase 9基因具有较高的保守性；系统进化分析显示，该基因氨基酸序列与其他鱼类Caspase 9聚为一支，并与锦鲤(Cyprinus carpio) Caspase 9亲缘关系较近；荧光定量PCR结果显示，Caspase 9基因在团头鲂各组织中均有表达，在脑中表达量最高，在肌肉中表达量最低，同时，在胁迫和恢复过程中，该基因在肝和脑中的表达规律相似，均在胁迫期间出现表达量上调，整体呈类似波浪形表达谱。研究结果表明，氨氮胁迫下，Caspase 9基因参与团头鲂的免疫防御，并与细胞凋亡分子过程有关。本研究结果可为进一步了解团头鲂应答氨氮胁迫分子机制提供理论依据。

英文摘要:

      Caspase 9 is a key protease of the intrinsic apoptotic pathway. In this study, the cDNA encoding caspase 9 was cloned with rapid amplification of cDNA ends (RACE) approach from blout snout bream Megahbrama amblycephala. The full-length cDNA of caspase 9 was composed of 1613 bp, including a 185-bp 5′ untranslated region, a 96-bp 3′ untranslated region, and an open reading frame of 1332 bp. The amino acid similarity comparison and the phylogenetic tree analysis indicated that the caspase 9 gene was highly conserved among different fish species and had a close relationship with Cyprinus carpio. Quantitative real-time PCR analysis showed that the caspase 9 was expressed in most tissues of M. amblycephala, and the highest expression was in the brain and the lowest expression was in the muscles. In response to the ammonia-N stress, the relative expression level of caspase 9 in both the liver and the brain displayed an “up-down-up” pattern and reached the highest level in 72 h ammonia-N stress. After a 72 h recovery, the level of expression decreased. We speculated that the caspase 9 gene was involved in the molecular mechanism of ammonia-N stress. Our study provided insight into the molecular responsive mechanisms of M. amblycephala exposed to environmental stresses