Molecular characterization characterization of Rab5 and Rab6, and their involvement in innate immunity in red swamp crayfish Procambarus clarkii

Rab5 and Rab6 are members of the Rab GTPases protein family, and they play important roles in regulating trafficking organelles, especially in phagosome formation. In this study, full-length cDNA sequences of Rab5 and Rab6 were cloned from crayfish Procambarus clarkii, which were designated as PcRab5 and PcRab6, respectively. The full-length cDNA of PcRab5 is 1367 bp and contains an open reading frame (ORF) of 639 bp encoding 212 amino acids with an estimated molecular weight of 23.0 kDa. The full-length cDNA of PcRab6 is 1097 bp, and ORF is 636 bp encoding 211 amino acids with 23.7 kDa. The deduced amino acid sequence of PcRab5 displayed high identity to that of Penaeus vannamei (97.17%), and PcRab6 shared high similarity to that of Macrobrachium rosenbergii (99.05%). According to the results of multiple sequence alignment and phylogenetic analysis, the guanine-base binding motif (G), phosphate/magnesium-binding motif (PM) and Rab family motifs (RabF) of PcRab5 and PcRab6 are high conserved among different species, however, the N- and C-terminal regions were hypervariable. Besides, the mRNA transcripts of PcRab5 and PcRab6 were widely detected in various tissues of P. clarkii, with the highest expression levels in hemocytes and gill. The gene expression levels of PcRab5 and PcRab6 were significantly induced in hepatopancreas, gill, and intestines of P. clarkii after WSSV and Aeromonas hydrophila infection, respectively. RNA interference experiment was designed to further investigate the roles of PcRab5 and PcRab6 during pathogen infection. The results showed that the mortality rate of crayfish was influenced by silenced PcRab5 and PcRab6 after challenged by WSSV or A. hydrophila. These results suggested that PcRab5 and PcRab6 were involved in bacterial and viral infection in the immune response of P. clarkii