Cloning And Functional Studies Of The Key Gene (IM) Of IMD Signaling Pathogens Of Macrobrachium

Immune deficiency homologues (IMD) signaling pathway plays an important role in innate immunity of invertebrates. IMD and Relish are 2 key components in the IMD signaling pathway. In this study, a relish (MrRelish) and a IMD gene (MrlMD) were identified from the giant freshwater prawns, Macrobrachium rosenbergii. Their function in the innate immunity was further studied. The full length of MrRelish was 5072 bp, containing a 3510 bp open reading frame (ORF), encoding a protein of 1169 amino acids. MrIMD cDNA was 744 bp in length and its ORF was 551bp, encoding 184 amino acids polypeptide. The constructed phylogenetic tree showed that MrRelish together with Relish proteins from crustacean belong to one group. At the mRNA level, MrRelish was widely distributed in hemocytes, heart, hepatopancreas, gill, stomach and intestines. In compare to the expression level of MrRelish in other tissues, MrRelish has relatively higher expression level in hemocytes and intestine. MrIMD transcripts were also widely distributed in the heart, hepatopancreas, gill, stomach and intestine. The expression level of MrIMD in gills is the highest. In gills, MrRelish was upregulated by 6 h Vibrio anguillarum challenge. In gills, the expression level of MrIMD was gradually upregulated from 24 to 48 h white spot syndrome virus (WSSV) challenge. In the hepatopancreas, MrIMD went up to the highest expression level at 24 h viral challenge. In the hepatopancreas, MrIMD showed the highest expression level after 6 h Aeromonas hydrophila challenge. In Drosophila S2 cells, the expression of Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), Cecropin (CecA), and Penaeidin (Pen4)) could be induced by overexpression of MrRelish or MrIMD. In the gills, knockdown of MrRelish using RNA interference methods showed that the expression of antibacterial peptide (AMP) genes such as crustin (Cru)2, Cru5, Cru8 and lysozyme (lyso)1 and Lysol were inhibited. However, anti-lipopolysaccharide factor (ALF)1 and ALF3 expression was not obviously changed. In conclusion, a preliminary study on the two key components (relish and IMD) of IMD immune signaling pathways suggested that IMD signaling pathway was involved in anti-bacteria and antiviral immune response in the giant freshwater prawns.