Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii.

Myeloid differentiation factor 88 (MyD88) is an adapter protein that links toll-like receptor and interleukin 1 receptor-mediated signal transduction. In this study, we identified 20 MyD88 genes from eight mollusk genomes and found that MyD88 was expanded in bivalves. This expansion tends to be tandem duplication. Phylogenetic analysis suggested that the tandem duplication of MyD88 was formed before bivalve differentiation. All of the identified MyD88 contained both of death domain (DD) and toll/interleukin-1 receptor (TIR) domain, and 13 mollusks MyD88 have low complexity regions (LCRs), which were not found in the MyD88 from humans and zebrafish. The genomic structure showed that most of the mollusk MyD88 (14 of 19) contained five conserved introns, four of which were found in humans and zebrafish. Furthermore, the cDNA full length of PfmMyD88-2 (one of the two identified MyD88 in Pincatada fucata martensii) was obtained with 1591 bp, including 260 bp of 5'UTR, 257 bp of 3'UTR, and 1077 bp of open reading frame encoding 358 amino acids. Quantitative real-time PCR analysis demonstrated that PfmMyD88-2 mRNA was widely expressed in all detected tissues. The highest expression level was in the gills and followed by hepatopancreas and feet. After lipopolysaccharide stimulation, PfmMyD88-2 expression level increased and reached the highest level at 12 h and then gradually declined to the normal level. Over-expression of PfmMyD88-2 in HEK293T increased the luciferase activity of the pNF-κB-Luc reporter. We also identified that PfmmiR-4047 could regulate the expression of PfmMyD88-2. These results help us elucidate the mechanism underlying mollusk immune response.