FUNCTIONAL CHARACTERIZATION OF RELISH PROTEIN FROM BLACK TIGER SHRIMP Penaeus monodon

Abstract

In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. A PmRelish gene which encoded a protein of 1,195 amino acids was cloned from the cDNA of 48-h YHV-infected shrimp. RT-PCR study of the PmRelish gene revealed that it was constitutively expressed in all tissues tested and mostly up-regulated upon YHV infection. Real-time RT-PCR of PmRelish gene after bacterial and viral challenges showed that the expression of PmRelish gene was significantly increased. Under PmRelish silencing, the shrimp were more susceptible to infection by YHV with the 50% survival rate reduced from about 72 h to 42 h. The expression profiles of ALFPm3, crustinPm1, penaeidin3 and penaeidin5 determined using YHV and V. harveyi 639, as an inducer of the Imd pathway indicate that the expression of penaeidin3 and penaeidin5 is under the PmRelish regulation. Immunolocalization of PmRelish protein in shrimp hemocytes showed that the PmRelish was detected in the cytoplasm of all the hemocytes from both uninfected and YHV-infected shrimp. The activated PmRelish was detected in the YHV-infected hemocytes by Western blot analysis. The RNA interference (RNAi) and suppression subtractive hybridization (SSH) was employ to identify the immune genes up-regulated in response to YHV infection and under the PmRelish regulation. Sequencing of 252 and 99 cDNA clones from the respective forward and reverse libraries were done and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors, chaperones, clotting protein, and other defense and homeostasis proteins.