Cloning And Function Of Two SOD Genes Of Procambarus Clarkii

The red swamp crayfish, Procambarus clarkii, is one of the important economic aquatic crustacean species in the southeast of China and has been artificially cultured in recent years. Generally, P. clarkii has great resistance to diseases in the natural environment. However, with the development of intensive aquaculture, various diseases have emerged and started to threaten the sustainability of the aquaculture populations of P. clarkii, severely affecting its production. Spiroplasma eriocheiris is a novel pathogen of aquatic crustaceans, which has been found in many commercially exploited crustaceans including Eriocheir sinensis and P. clarkii, causing serious diseases and catastrophic economic losses in aquaculture. Aeromonas hydrophila is also a common pathogen in aqauculture.These pathogens have the potential to result in significant economic losses in many crayfish producing farms in China. So an investigation into the mechanisms of immune defense against diseases of P. clarkii might be beneficial to the management of crayfish farming.The superoxide dismutases (SODs) are essential antioxidant enzymes that occur in virtually all oxygen-respiring organisms.In this study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, P.clarkii. This paper studied the immune defense mechanisms of SOD after S. eriocheiris and A. hydrophila stimulations, respectively.1. The characterization and biological activity analysis of superoxide dismutases (SODs) from P. clarkiiIn this study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene of the freshwater crayfish, P. clarkii were cloned and charactereized. The open reading frame (ORF) of ecCuZnSOD is612bp encoding a204amino acid (aa) protein with a24aa signal peptide, whereas the ORF of mtMnSOD is654bp and encodes a218aa protein with a20aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P.clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. 2. SOD mRNA expression pattern and determination of SOD activities under pathogen stimulationAfter S. eriocheiris challenge, the mRNAs of two SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNAs expressions of two SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila.