Objective Chitinases are the important hydrolytic enzymes, which can degrade the shells of shrimp and crab containing chitin in fish food by hydrolyzing β-1, 4- glycosidic bonds. In this study, we tried to under the expression profile of chitinases in different tissues of tilapia and the characteristics of chitinase extracted from the stomach of tilapia.
Method Three chitinases, named tChit1a, tChi3 and tChit, were cloned from the stomach and intestines of GIFT tilapia. ORFs of these chitinases were analyzed by a series of bioinformatics software and their tissue distribution were detected by Real-time PCR. Next, the chitinases from stomach of tilapia were purified by affinity chromatography and the chitinase activity was detected by the 4-MU method.
Result The genes tChit1a, tChi3 and tChit encoded 453, 453 and 473 amino acids, respectively. Homology alignment results showed that the homology between the deduced amino acid sequences of tChit1a and tChi3 was 83.66%, while tChit showed lower homology to tChit1a and tChi3, with 49.89% and 50.11%, respectively. The phylogenetic analysis indicated that tChit1a, tChi3 and tChit were classified into fish chitinases-3(FCase-3)rather than acid fish chitinase-1/2(AFCase-1/2). The result of tissue distribution showed that tChit1a, tChi3 and tChit mainly expressed in the gastrointestinal tract of tilapia, with the highest mRNA expression in the midgut, foregut and midgut hind respectively. The purified chitinases from stomach of tilapia showed that there were two obvious bands with the size of about 40 ku, but no high homologous bands(Chitinase-1 of tilapia)were detected by polyclonal antibody of the Chitinase-1 of Epinephelus obliquus. The activities of hydrolyzed 4MU-(GlcNAc)2 and 4MU-(GlcNAc)3 were 1.73 and 4.89 U/g respectively at the optimum pH 5.
Conclusion The low expression and activity of chitinases extracted from stomach of tilapia suggest that tilapia may have little interest in ingesting chitinous substances.
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