Identification Of Toll-like Receptors In Hyriopsis Cumingii And Their Potential Roles In Innate Immunity

Hyriopsis cumingii is one of important aquaculture economic mussel in China.While disease caused by bacteria led to the outbreak of large-scale death of mussels,this events was reported at recent years.The economic losses directly influences the sustainable development of mussel aquaculture industry in china.Toll-like receptors play an important role in the recognition and clearance of pathogenic microorganisms.Therefore,The studies of TLR signaling pathway can help us to explore the the antibacterial and innate immune mechanism of mussels,and may provid theoretical basis for the healthy development of the industry.The EST sequences of TLR were obtained and primers were designed by searching the transcriptome database in our laboratory.Complete cDNA of HcToll8,9 and 10 genes were obtained using RACE PCR technique.The results showed that the full length sequence of the HcToll8,9,10 were 2538,3692 and 2612 base pairs,respectively,which they contained the maximum open reading frame of 2139,2157 and 2355 base pairs,and predicted encoding a polypeptide of 712,719 and 784 amino acids,respectively.Bioinformatics analysis showed that HcToll8,9 and 10 predicted proteins had an signal peptides,conserved domains LRRs and TIR,and a helix transmembrane domains.The multiple sequence alignment showed that HcToll8 and 9 had the highest homology with scallop M.yessoensis and A.irradians,with 31.38%and 32.65%homology,respectively.HcToll10 had the highest homology with C.gigas and the identity was 27.42%.Analysis of NJ phylogenetic trees showed that they were clustered in a branch with TLRs in bivalves.The transcripts of HcToll8,9 and 10 were expressed in hemocytes,gonad,heart,mantle,foot,kidney,gills,intestine,hepatopancreas and adductor muscle of healthy mussel.The expression levels of HcToll8 and 9 were the most in hepatopancreas and gill,and a lower levels in hemocytes.HcToll10 was mainly expressed in the hepatopancreas,followed by hemocytes and heart,and was the lowest in gills.After LPS,PGN and polyI:C stimulation,the transcription levels of HcToll8,9 and 10 were significantly up-regulated(p<0.05)at different time points in hepatopancreas and gill of mussels,and returned to normal level at 72 h.The time point of upregulation in gill was earlier than that in hepatopancreas,Recombinant HcToll9,10 proteins had the ability to bind gram-positive and gram-negative bacteria.as well as LPS,PGN and PolyI:C.The binding activity of recombinant HcToll9 was higher than that of HcToll10.The results of subcellular localization showed that the green fluorescence of HcToll9 and HcToll10 fusion proteins were mainly distributed in the cytoplasm and near the cell membrane,which was consistent with the fact that they were all transmembrane proteins.Dual-luciferase reporter gene assay showed that both HcToll9 and 10 could significantly activate NF-?B reporter gene,indicating that HcToll9 and 10 were involved in the antimicrobial immune response of H.cumingii.