Molecular Cloning And Expression Characteristics Of Superoxidase And Lysozyme From Cristaria Plicata

Cristaria plicata is one of the most important "Freshwater pearl mussel" in our country. The understanding its immune system is essential for aqualculture. The present study is to clone superoxidase (CpSOD) and lysozyme (CpLYS) from Cristaria plicata, and to study on the expression characteristics of them.The CpSOD cDNAs full sequence and CpLYS partial cDNAs have been cloned using homologous cloning and RACE- PCR. The full cDNA of CpSOD is 891bp, which contains an open reading frame of 468 bp, 5' untranslated region 83 bp, 3' untranslated region 340 bp(including a polyA tail of 24 bp). The open reading frame encodes a protein of 155 amino acid residues, the predicted molecular weight is 15.8 kDa, isoelectric point is 5.80. The CpSOD contains four amino acid residues required for binding copper and four amino acid residues required for binding zinc, which are highly conserved in other Cu/Zn-SODs. There were no signal peptide found showed that the CpSOD are icCu/Zn-SOD. Blast analysis showed the CpSOD has highest similarity with Cu/Zn-SODs of other bivalves, and Neighbor-joining phylogentic tree of CpSOD and icCuZnSODs amino acid sequences from different species showed that CpSOD were clustered with Chlamys farreri, Crassostrea gigas and Mytilus edulis. The partial cDNAs of CpLYS is 530bp, which contains an 3' untranslated region 259 bp(including a polyA tail of 29 bp), encodes a protein of 89 amino acid residues. Blast analysis showed the CpLYS has highest similarity with i-type lysozymes of other bivalves, so the CpLYS was suspected to be an i-type lysozymes.Expression of CpSOD and CpLYS in hemocyte after injected by Aeromonas hydrophila 3h,6h,12h,24h,48h were determined by RT-PCR. The results showed that after injected by Aeromonas hydrophila, the expression of CpSOD were increased at first 12h, and decreased thereafter; hile the expression of CpLYS were increased from the first to 48h, which proved the CpSOD and CpLYS were important immune system in Cristaria plicata.Expression of CpSOD and CpLYS in different tissues of Cristaria plicata were determined by RT-PCR and the results showed the expression of both CpSOD and CpLYS were detected in mantel, conductor muscle, hepatopancreas, sexual gland and hemocyte.