Molecular cloning, characterization and RNA interference assay of two toll-like receptors in giant freshwater prawn, Macrobrachium rosenbergii

Abstract

Toll-like receptors are a family of conserved membrane receptors which are involved in the regulation of a series of innate immune responses in invertebrates and vertebrates. Two different types of Toll-like receptors from giant freshwater prawn (Macrobrachium rosenbergii), which were designated as MrToll1 and MrToll2, were cloned in the present study. The full-length cDNA of MrToll1 is 4234 bp in length, which includes a 3015-bp open reading frame (ORF) encoding a deduced protein of 1004 amino acids. The sequence of MrToll2 cDNA is 4383 bp in length containing a 2739-bp ORF which encodes 912 amino acid residues. The results from the multiple alignments of amino acid sequences and phylogenetic analysis indicated the two MrTolls identified in the present study should be classified as crustacean type-1 and type-2 Toll-like receptor, respectively. MrToll1 and MrToll2 were constitutively expressed in all the examined tissues, including gill, stomach, intestine, eyestalk, muscle, heart, ganglion, brain, hemocytes and hepatopancreas. RNA interference assay demonstrated that knockdown of MrToll1 with specific-siRNA significantly suppressed the expression of myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor (ALF), crustin and prophenoloxidase (proPO) and enhanced the expression level of MrToll2 in M. rosenbergii. Additionally, an obvious down-regulation of MyD88 and crustin expression was observed in MrToll2-silenced prawns. These findings suggest that MrToll1 and MrToll2 might play essential roles in the innate immunity of M. rosenbergii through regulating the expression of immune-related genes.