I84 protease inhibitor family show significant expansion and functional differentiation in Crassostrea gigas

Protease inhibitors are extremely diverse proteins or peptides that inhibit the proteolytic activities of proteases. Protease inhibitors could inhibit the invasion of pathogens by inactivating pathogen proteases. The I84 protease inhibitor family is a new family in MEROPS database, and the role of its members in host defense has been studied to some extent. To investigate the distribution and function of I84 protease inhibitor family in Crassostrea gigas, in this study, 23 potential I84 family genes were identified in C. gigas. Based on phylogenetic analysis, 5 homeotic genes, Cgsi3, Cgsi5, Cgsi6, Cgsi16 and Cgsi19, were selected for gene expression and functional assessment. They were confirmed by cDNA cloning to represent expressible real genes and expressed in digestive gland, gills, adductor muscle, gonad, mantle, and hemocytes, with the highest level of expression detected in the digestive gland (P<0.05). In the larval oysters, none of the 5 selected genes was detected in zygote, and they showed different expression patterns in the remaining developmental stages. The expression level of CgSi3 increased significantly in the pediveliger stage, and then decreased. The expression level of CgSi6 increased significantly from the umbo larvae onwards. In oysters challenged with pathogen associated molecular patterns (PAMPs), gene expression pattern also differed with genes. In oysters injected with LPS and PGN, the expression level of CgSi6 changed significantly, while in those injected with Poly (I:C) and GLU the expression level of CgSi3 showed significant changes. This study revealed the gene expansion and functional differentiation of the I84 family protease inhibitors in C. gigas, and provides a basis for the comprehensive understanding of the biological functions of the I84 family protease inhibitors and the related mechanisms.