Molecular Cloning,expression And Functional Studies Of Chitinase From Roughskin Sculpin Trachidermus Fasciatus

Roughskin sculpin?Trachidermus fasciatus?,a catadromous fish with high ecological and economic value,is listed Order II in the National Key Protected Wildlife List in China.Aquaculture has become a crucial way to recover its population.The challenge of the aquaculture seems to be the disease such as mycotic infection.Chitinase is a kind of enzyme that hydrolysate chitin.It has many physiological functions,especially its important role in antifungal function.In order to achieve deeper understanding of chitinase of Roughskin sculpin,the whole cDNA length of a chitinase gene of Roughskin sculpin was cloned by RACE technique,and it was named Tf-Chl.The prokaryotic expression vector was constructed to obtain the recombinant protein,and its activity and function were detected.The full length of cDNA sequence of Tf-Chl was 1833 bp,and the open reading frame?ORF?was 1362 bp,encoding 453 amino acids.Tf-Chl contained a signal peptide and two domains,which were the N terminal Glyco₁8 domain and the C terminal CBM₁4 domain.The phylogenetic tree showed that all the fish chitinase was clustered into a large branch,and the chitinase of the other vertebrates was clustered into a large branch.Tf-Chl was closely related to the yellow croaker?Larimichthys crocea?.Vitro antibacterial experiments showed that Tf-Chl recombinant protein could inhibit the growth of fungi such as A.niger.The carbohydrate binding domain of Tf-Chl could bind to chitin and other carbohydrates,and had agglutination effect on yeast.Therefore,we speculated that Tf-Chl had a certain effect on the invasion of fungi in Roughskin sculpin.