Molecular Cloning And Expression Pattern Analysis Of Tolllike Receptors In Sea Perch (Lateolabrax Japonicas) After Bactriea Stimulation

As an important commercial aquatic animal in South China,the breeding scale of L.japonicas is becoming bigger.But the outbreak of pathogens infection caused enormous economic losses and restricted the development of L.japonicas aquaculture industry.The innate immunity is the first protective barrier to defense the pathogenic bacteria infection,and the activation of innate immunity in the fish is carried out by the pattern recognition receptor(PRR)which could recognize the pathogen-associated molecular pattern(PAMP).Toll-like receptor(TLR)is an important PRR belonging to the Ⅰ type transmembrane receptor and involved in the defense against the bacteria invasion.The function of TLR is based on its particular structure consisted of extracellular leucine-rich repeat(LRR)domains,transmembrane domain and intracellular Toll/interleukin-1 receptor(TIR).The different domain executes different function,the LRR domains are involved in the recognition of components and then TIR could carry the stimulation signals to the adapter proteins in order to activate the signaling pathway killing the microorganism.Then we cloned three TLR genes from L.japonicas including LjTLR1,LjTLR3 and LjTLR5 for the studies of sea perch TLRs against the different bacteria.According to the selected results of transcriptome,we got the EST sequences for the three TLR genes.We cloned the full-length cDNA sequence of three TLR genes by means of SMART RACE for the bioinformatics analysis.The tissues expression patterns of the TLR genes were carried out by RT-PCR.Then the bacteria infection experiments were challenged with the V.harveyi and S.agalactiae for the expression level changes analysis of the three TLR genes post the infection.In order to further verify the functions of the TLR genes,in situ hybridization experiments were carried out on spleen and head-kidney.The eukaryotic recombinant plasmids of the three TLR genes were constructed for transfection into the HEK-293 T cells and the further cellular localization of TLRs in the human cells.The HEK-293 T cells were dealt with the inactivated bacteria in order to confirm the protect functions of the three TLR genes.And the detailed results listed as follow:(1)The full-length of the LjTLR1,LjTLR3 and LjTLR5 cDNA sequence is 2755 bp,3265bp and 2676 bp respectively,and the predicted molecular weight is 93.55 kDa,103.85 kDa and 101.15 kDa respectively,and the theoretical isoelectric point is 6.56,8.73 and 6.10 respectively.The domains prediction results of the three TLR genes indicated that the three TLRs included the typical TLR protein domains contain the numerous LRR,LRRCT domain,transmembrane domain and the TIR domain.The results of multiple sequence alignment and phylogenetic tree suggested that the three L.japonicas TLRs showed great homology to the fish TLRs.(2)The tissues expression results showed extensive expression in all detected tissues and higher expression level in the immunity tissues of the both three TLR genes.LjTLR1 showed higher expression level in the head-kidney,intestines and liver,the lowest expression was detected in the muscle;LjTLR3 showed higher expression level in the spleen,head-kidney and liver,the lowest expression was detected in the heart;LjTLR5 showed higher expression level in the head-kidney,muscle and intestines,the lowest expression was detected in the liver.(3)The expression levels of the LjTLR1,LjTLR3 and LjTLR5 showed remarkable up-regulation after the V.harveyi and S.agalactiae infection in the immunity tissues and little difference in the muscle,meanwhile,the V.harveyi showed great stimulation than challenged with the S.agalactiae.In the head-kidney challenged with V.harveyi,the three TLR genes showed significant up-regulation,the up-regulation started at 6 h and the peak level reached at 48 h,48 h and 24 h of LjTLR1,LjTLR3 and LjTLR5 respectively;As well in the spleen,the similar expression patterns were found,the upregulation started at 6 h and the peak level reached at 48 h,48 h and 24 h respectively;The three TLR genes showed significant up-regulation in the liver,the up-regulation started at 6 h and reached the peak level at 72 h,24 h and 24 h respectively.After the S.agalactiae infection,the three TLR genes showed significant up-regulation in the head-kidney,the up-regulation started at 6 h and the peak level reached at 72 h,96 h and 24 h of LjTLR1,LjTLR3 and LjTLR5 respectively;In the spleen,three TLR genes showed significant up-regulation and the highest level was detected at 48 h,48 h and 24 h respectively;The three TLR genes showed significant up-regulation in the liver,the peak level reached at 24 h,6 h and 6 h respectively.Finally,the expression levels of the three TLR genes in the muscle showed up-regulation later.(4)In situ hybridization analysis showed that only a small number of positive signals were detected in the spleen and the head kidney after treated with PBS,and the positive signals of LjTLR3 and LjTLR5 showed significantly increase after different bacteria stimulation than those in the PBS control group.The positive signals of LjTLR3 and LjTLR5 challenged with V.harveyi were significantly stronger than S.agalactia in the same tissues.The results further demonstrated that the expression characteristics of LjTLR3 and LjTLR5 after bacterial stimulation.LjTLR1 did not show a significant positive signal.(5)We successfully constructed the eukaryotic recombinant plasmid and the overexpression of LjTLR1,LjTLR3 and LjTLR5 in the HEK-293 T cells.Three TLR recombinant proteins were screened closed to the cell membrane,indicating that all three belong to the typical TLR protein.