Molecular cloning and characterisation of copper/zinc superoxide dismutase (Cu,Zn-SOD) from the giant freshwater prawn Macrobrachium rosenbergii

Abstract

A copper/zinc superoxide dismutase (Cu,Zn-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 the rapid amplification of cDNA ends method. Analysis of nucleotide sequence revealed that the Cu,Zn-SOD cDNA clone consists of 845 bp with an open reading frame of 603 bp encoding a protein of 201 amino acids with a 22 amino acid signal peptide. The calculated molecular mass of the mature proteins (179 amino acids) is 21 kDa with an estimated pI of 4.75. Two putative N-glycosylation sites, NXT and NXS, were observed in the Cu,Zn-SOD. Four conserved amino acids responsible for binding copper (H86, H89, H106 and H163) and four conserved amino acids responsible for binding zinc (H106, H114, H123 and D126) were observed. Sequence comparison showed that the Cu,Zn-SOD deduced amino acid sequence of M. rosenbergii has similarity of 60% and 64% to that of freshwater crayfish Pacifastacus leniusculus ecCu,Zn-SOD and blue crab Callinectes sapidus ecCu,Zn-SOD, respectively. Quantitative real-time RT-PCR analysis showed that Cu,Zn-SOD transcripts in haemocytes of M. rosenbergii increased 3h and 6h after injection of Lactococcus garvieae, whereas Cu,Zn-SOD transcripts decreased in the hepatopancreas 3h after L. garvieae injection.