Molecular characterization of two kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum

Abstract

This study reports two kazal-type serine protease inhibitors (KPI) identified in a cDNA library from the surf clam Mesodesma donacium, and characterized through Rapid Amplification of cDNA Ends (RACE). The KPIs, denoted as MdSPI-1 and MdSPI-2, presented full sequences of 1139 bp and 781 bp respectively. MdSPI-1 had a 5′untranslated region (UTR) of 175 bp, a 3′UTR of 283 bp and an open reading frame (ORF) of 681 pb that encodes for 227 amino acids. MdSPI-2 showed a 5′UTR of 70 bp, a 3′UTR of 279 bp and an ORF of 432 bp that encodes for 144 amino acids. Both sequences presented two kazal-type tandem domains. Phylogenetic analysis of MdSPI-1 and MdSPI-2 shows a main clade composed by other bivalve species and closely related crustaceans. Real time PCR analysis showed that MdSPI-1 is mainly up-regulated in mantle, foot, gills and muscle tissues, while MdSPI-2 is expressed principally in foot tissue. Moreover, to evaluate the immune response of MdSPI-1 and MdSPI-2, infections with Vibrio anguillarum were performed. Herein, MdSPI-1 and MdSPI-2 transcription expression were significantly up-regulated at 2 and 8 h post-challenge. Our results suggest that MdSPI-1 and MdSPI-2 are important humoral factors of innate immunity in M. donacium.