Expression And Functional Analysis Of Djtry And Enzyme Activity Detection From Planarian Dugesia Japonica

Freshwater planarians (class Turbellaria;) are free-living members of the phylum Platyhelminthes (the flatworms), which can undergo dramatic changes in body size and regenerate their entire body plan from small pieces after cutting. This remarkable morphological plasticity has made them an excellent model in evolution, embryonic development and regeneration. In this study, Djtry was obtained from the cDNA library of Dugesia japonica, with an entire encoding sequence, and then the corresponding characterization, expression patterns and functional analysis of Djtry was reported in this study.Trypsin-like serine protease (Tryp_SPc) family is ubiquitous in animals and play diverse roles including dietary protein digestion, hemolymph coagulation, antimicrobial peptide and melanin synthesis, and the activation of a rapidly immune pathway in response to pathogen detection, which has a conserved catalytic triad:His (H), Asp (D), Ser (S).1218-271clone obtained from cDNA library comprised of706bp long with a polyadenylation sequence and a poly A tail, contains an opening reading frame (ORF) of609bp which encodes a peptide of203amino acids with a calculated molecular weight of about22.6kDa. Interestingly, in its catalytic triad, the primary His (H) was substituted by Lys (K), which indicated that it might be a Try homologue. Phylogenetic analysis suggests that Djtry is an ancient type of trypsin-like serine proteases.Results of Northern blot illustrated that the presence of a single bond of around700bp transcript in D. japonica. Whole-mount in situ hybridization revealed that Djtry is found to display a tissue specific expression pattern, with a predominant expression detected in whole gut region of intact and regenerating planarians, while the tissue-and stage-specific expression patterns during the embryonic development imply the roles of Djtry involved in gut formation.Moreover,quantitative real-time PCR was carried out to analyze the function of this protease in vivo after planarians were stimulated to a bacterial challenge and food. The results showed that Djtry increased after a bacterial challenge and was basically stable for food. Therefore, the trypsin-like serine protease might be involved in the innate defense reactions against bacterial infection.To investigate whether this substitution influenced the activity of trypsin, the recombinant vector pET-28a-Djtry was constructed and transferred into E.coli BL21. Induced by IPTG for expression, denaturation, renaturation, purification and Western blotting analysis, we obtained the soluble homogeneous protein with biological activities. Compared to bovine trypsin, taking BAEE as special substrate of trypsin, the enzyme activity of Djtry was detected. The results indicated that Djtry was expression as inclusion bodies and its molecular weight was approximately26kDa. Identification by Western Blotting and renaturation for inclusion bodies, this type of mutational typsin Djtry was keeping trypsin activities but a little weak relatively.