Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China.
Wuxi Fishery College Nanjing Agricultural University, Wuxi 214081, PR China.
Fish Shellfish Immunol. 2014 Jun;38(2):340-7. doi: 10.1016/j.fsi.2014.03.036. Epub 2014 Apr 13.
The full-length mitochondrial manganese superoxide dismutase cDNA of blunt snout bream Megalobrama amblycephala (denoted as MamMnSOD) was identified in liver using homology cloning and rapid amplification of cDNA ends. The full-length cDNA of MamMnSOD consisted of 986 bp, with an open reading frame encoding 224 amino acids, a 58-bp 5' untranslated region and a 256-bp 3' untranslated region. The deduced amino acid sequences of MamMnSOD showed high sequence homology to mitochondrial MnSODs from crustaceans. Several motifs, including three mitochondrial MnSOD signatures, amino acid residues responsible for coordinating the manganese, and the putative active center, were almost completely conserved in the deduced amino acid sequences of MamMnSOD. The mRNA expression of MamMnSOD in the tissues of heart, liver, spleen, kidney, muscle, intestine, and gill was examined by quantitative real-time PCR; the highest expression was in the liver. Transcription of MamMnSOD was kinetically modulated in response to nitrite stress in liver and gill tissues. The purified recombinant MamMnSOD showed potent antioxidant activity. Polyclonal antibodies generated from the recombinant product of MamMnSOD were used to specifically identify the native protein in liver of M. amblycephala. Collectively, the findings of this study strongly suggested that MamMnSOD combats oxidative stress and cellular damage induced by nitrite, by detoxifying harmful reactive oxygen species in M. amblycephala.
卵形鲳鲹（Megalobrama amblycephala）全长线粒体锰超氧化物歧化酶 cDNA 的鉴定（简称 MamMnSOD）采用同源克隆和 cDNA 末端快速扩增法从肝脏中获得。MamMnSOD 的全长 cDNA 由 986bp 组成，包含一个开放阅读框，编码 224 个氨基酸，一个 58bp 的 5'非翻译区和一个 256bp 的 3'非翻译区。MamMnSOD 的推导氨基酸序列与甲壳动物的线粒体 MnSOD 具有高度的序列同源性。几个基序，包括三个线粒体 MnSOD 特征、负责协调锰的氨基酸残基和假定的活性中心，在 MamMnSOD 的推导氨基酸序列中几乎完全保守。通过定量实时 PCR 检测了 MamMnSOD 在心脏、肝脏、脾脏、肾脏、肌肉、肠和鳃组织中的 mRNA 表达；肝脏中的表达最高。MamMnSOD 的转录在肝脏和鳃组织中对亚硝酸盐应激的动力学调节。纯化的重组 MamMnSOD 表现出强大的抗氧化活性。从 MamMnSOD 的重组产物中产生的多克隆抗体被用于特异性识别 M. amblycephala 肝脏中的天然蛋白。总之，本研究的结果强烈表明，MamMnSOD 通过解毒卵形鲳鲹中的有害活性氧物质来对抗亚硝酸盐引起的氧化应激和细胞损伤。
