Molecular cloning and characterisation of cytosolic manganese superoxide dismutase (cytMn-SOD) from the giant freshwater prawn Macrobrachium rosenbergii

Abstract

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 (H 110, H 158, 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 3 h after Lactococcus garvieae injection, but no significant change in cytMn-SOD transcript was observed in the haemocytes 3-24 h after L. garvieae injection. (C) 2005 Elsevier Ltd. All rights reserved.