Construction Of Head-kindney CDNA Subtructive Library Of Channa Argus And Clone And Analisis Of SOD Gene

Channa argus, feed on other fish, is one of Chinese special fish, which is considered as trash fish. With the deep awareness, this fish has gradually aroused people’s interest due to its outstanding features, such as fresh meat, medicinal properies and strong resistance. The problems brought by the increased scale of farming are urgent to be resolved. The enhanced virulence of pathogenic microorganisms and high morbidity caused great losses. SOD, one of nonspecial immune factors, plays an important role in anti-infection and lessening the damage.In this research, the head-kidney cDNA subtructive library was constructed at24h after the challenge with Aeromonas veronii to Channa argus. And then,84strains contained defferential gene fragments were obtained through cloning, PCR verification and reverse Northern Hybridization. The sequencing results showed that there were6groups besides30unknowed gene fragements:(1)cell structure gene,(2)metabolic-related gene,(3)protein expression-related gene,(4)immune gene,(5)signal transduction gene,(6)unclassified gene, besides8unknown genes and22no homology gene. Of the immune gene, Cu/Zn SOD gene, histone gene, IgM gene,, MHC molecule gene and MPO gene are important.The primers GSP1and GSP2were designed according to the suspected SOD gene fragment to amplify the5’cDNA and3’cDNA. After cloning and transformed, the full length was spliced based on the sequences of the5’cDNA and3’cDNA. The gene and its coding product were analyzed by the bioinformatic softwares. The gene with810bp shares the88%homologies with Rachycentron canadum and about70～80%homologies with other fishes. The phylogenetic tree showed the gene had a near relationship with it of fish. The ORF was465bp,5’-UTR was47bp and3’-UTR was298bp. The gene encoded154amimo acids, with16.1ku. The encode product without the signal peptides and transmernbrane domains may belong to cytCu/Zn SOD. Through prediction analysis, the Or and Zn2+bonding sites were located between the47th～121th and64th～84th amino acid. The second structure was predicted in Protean workplace of DNAstar. By Gamier-Robson, the encoded product had8a-helixs,10β-sheets,7β-turns and10coils; while byChou-Aasman, the encoded product had4a-helixs,7β-sheets,10β-turns and10coils.The head-kidney SSH library was constructed, and antibacterial related genes were obtained to provide basic data to deeply study these genes. SOD gene was amplified by the RACE and systematicly analyzed by molecular biology software to lay the foundation to study the functions of SOD in defensive and protective menchanism.