Molecular cloning and expression analysis of two type II crustin genes in the oriental river prawn, Macrobrachium nipponense.

Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.