Cloning and expression analysis of cathepsin L gene in Trachinotus ovatus

ObjectiveThe aim of the study was to definite genetic evolution rule and tissue expression level of ca-thepsin L gene in Trachinotus ovatus(TroCatL),provide theoretical basis for researching biological function and disease resistant mechanism of CatL.MethodThe full length cDNA sequence of TroCatL were cloned by using RT-PCR and RACE technology,and the sequence features were analyzed by using bioinformatics methods. Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of TroCatL gene in healthy tissues and its correlation with Vibrio alginolyticus infection.ResultThe full length of TroCatL cDNA was 1492 bp(GenBank accession number:MH036350),including a 1011 bp open reading frame(ORF),a 120 bp 5'untranslated region(5'-UTR)and a 361 bp 3' untranslated region(3'-UTR). The TroCatL gene encoding 336 amino acid residues with theoretical isoelectric point(pI) of 5.7 and predicted molecular weight of 37.93 kD. It contained conservative domain structure(ERFNIN,GNFD and GCXGG motifs),and a cysteine protease active site formed by 139Cys,279His and 303Asn. The amino acid sequences of Tro- CatL shared 83.9%-95.2% homology with CatL amino acids of other fish species,especially sharing the closest relative re-lationship with Seriola dumerili. TroCatL mRNA were expressed in all detected tissues from healthy T. ovatus while the highest level was in liver and the lowest was in brain. TroCatL mRNA were significantly up-regulated in liver,spleen and blood after being infected by V. alginolyticus . TroCatL mRNA in liver and spleen reached the peak value 24 h post infec-tion,while that in blood reached the peak value 12 h post infection.ConclusionTroCatL protein domains and catalytic active sites are evolutionarily conserved in T. ovatus,and play an important role in against of bacteria or virus infection by participating in host immune response.