Identification of antioxidant enzyme genes of the Indian edible oyster, Crassostrea madrasensis (Preston) through polymerase chain reaction

Abstract

When an organism is exposed to stress by way of environmental fluctuations or pathogenic attack, reactive oxygen species (ROS), which cause severe oxidative damage to the cells and hamper the cellular as well as membrane functions, are produced. In order to counter these effects, the cells activate the production of antioxidant enzymes which play pivotal role in removing ROS and maintaining the homeostasis within the cells. Crassostrea madrasensis is a promising bivalve species, living in intertidal region amidst a variety of stressors. In the present study, the RNA was isolated from gills and cDNA synthesised by Reverse Transcription PCR (RT- PCR) and amplification of these c DNA were carried out using a combination of different primers designed for super oxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPX).. The PCR generated amplicons of 464 bp, 171 bp and 147 bp of Cu/Zn-SOD, CAT and GPX respectively were purified and sequenced. Similarity search in NCBI - BLAST confirmed these sequences as the respective antioxidant enzyme genes. The amino acid profile of Cu/Zn-SOD deduced from the sequences representing original reading frame (ORF) on InterProScan analysis was found to contain the characteristic Cu and Zn binding domains. PCR with the SOD specific primer pair resulted in the amplification of ORF of SOD with both genomic DNA and C-DNA as templates. This indicates the intronless nature of SOD gene, an adaptation to initiate fast expression at times of stress, as observed in certain other stress related genes such as the inducible form of heat shock protein 70 (Hsp 70). This is the first report on the molecular detection and identification of antioxidant enzyme genes of Indian edible oyster.