Cheng Winton, Tung Ying-Hsiu, Liu Chun-Hung, Chen Jiann-Chu
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC.
Fish Shellfish Immunol. 2006 Apr;20(4):438-49. doi: 10.1016/j.fsi.2005.05.016. Epub 2005 Sep 8.
A cytosolic manganese superoxide dismutase (cytMn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cytMn-SOD cDNA clone consists of 1339 bp with an open reading frame of 858 bp encoding a protein of 286 amino acids. The calculated molecular mass of the mature proteins (286 amino acids) is 31 kDa with an estimated pI of 5.52. Two putative N-glycosylation sites, NXT and NXS were observed in the cytMn-SOD. Four conserved amino acids responsible for binding manganese were observed (H110, H158, D243 and H247). Sequence comparison showed that the cytMn-SOD deduced amino acid sequence of M. rosenbergii has an overall similarity of 77% and 54% to that of blue crab Callinectes sapidus and tiger shrimp Penaeus monodon, respectively. Quantitative real-time RT-PCR analysis showed that cytMn-SOD transcript in hepatopancreas of M. rosenbergii decreased 3h after Lactococcus garvieae injection, but no significant change in cytMn-SOD transcript was observed in the haemocytes 3-24 h after L. garvieae injection.
利用简并引物通过逆转录聚合酶链反应（RT-PCR）从罗氏沼虾的肝胰腺中克隆出一种胞质锰超氧化物歧化酶（cytMn-SOD）cDNA。通过cDNA末端快速扩增（RACE）方法分离出3'和5'区域。核苷酸序列分析表明，cytMn-SOD cDNA克隆由1339 bp组成，开放阅读框为858 bp，编码一个286个氨基酸的蛋白质。成熟蛋白（286个氨基酸）的计算分子量为31 kDa，估计pI为5.52。在cytMn-SOD中观察到两个推定的N-糖基化位点，NXT和NXS。观察到四个负责结合锰的保守氨基酸（H110、H158、D243和H247）。序列比较表明，罗氏沼虾推导的cytMn-SOD氨基酸序列与青蟹Callinectes sapidus和斑节对虾Penaeus monodon的序列总体相似性分别为77%和54%。实时定量RT-PCR分析表明，在注射加氏乳球菌3小时后，罗氏沼虾肝胰腺中的cytMn-SOD转录本减少，但在注射加氏乳球菌3至24小时后，血细胞中未观察到cytMn-SOD转录本有显著变化。
