Expression Analysis Of Immune-related Genes, LBP, TLR4, MyD88, And AP-1 After LPS Induction And The Interaction Between TLR4 And MyD88 In Hyriopsis Schlegelii

In this study,the molecular structure characteristics,tissue expression patterns and interaction of immune-related genes with LPS-induced MyD88-dependent pathways were investigated for the first time in freshwater pearl mussel,(Hyriopsis schlegelii).The primers were designed based on partial cDNA sequences in the gonad gonad transcriptome and the full-length cDNAs of four genes,LBP,TLR4,MyD88,and AP-1 of H.schlegelii(the following are referred to as HsLBP,HsTLR4,HsMyD88,HsAP-1,respectively)were obtained by RACE-PCR.The results showed that the full-length cDNAs of the four genes were 1885 bp,3519 bp,1863 bp,and 1641 bp,respectively,and contained the maximum ORFs of 1506 bp,2694 bp,1428 bp,and 888 bp,respectively.The 3'UTRs of HsLBP,HsTLR4,and HsAP-1 contain the "AATAAA" tailing signal.Bioinformatics analysis showed that there are signal peptides in the Nterminus of HsLBP and HsTLR4,belonging to secretory proteins and transmembrane proteins;the HsLBP contains two domains,BPI1 and BPI2;HsTLR4 contains two conserved domains,LRRs and TIR,and a helix spanning transmembrane domains;the HsMyD88 contains two domains,which are DD and TIR;the HsAP-1 contains Jun-like transcription factor and BRLZ domains.Their NJ phylogenetic tree analysis showed that they had different degrees of conservation: HsLBP had the highest homology with HcLBP/BPI1 of Hyriopsis cumingii,and the similarity and identity were as high as 98%;HsTLR4 has the highest homology with MyTLR3 of Mizuhopecten yessoensis and the similarity and identity were 45% and 62%,respectively.HsTLR4-TIR has the highest similarity and identity with human TLR4,respectively,44% and 25%;HsMyD88 has the highest homology with HcMyD88-2 of Hyriopsis cumingii,and the similarity and identity are as high as 99%;HsAP-1 had the highest homology with RpAP-1 of Ruditapes philippinarum,and the similarity and identity were 69% and 60%,respectively.In H.schlegelii,HsLBP,HsTLR4,HsMyD88,and HsAP-1 were universality expressed in tissues.The mRNA of HsLBP was mainly expressed in gill,hemocytes and hepatopancreas,and detected lower levels in foot and adductor muscle;The mRNAs of HsTLR4 and HsMyD88 were mainly expressed in the gill and hepatopancreas,and detected lower levels in hemocytes and mantle;The mRNA of HsAP-1was mainly expressed in the hepatopancreas and detected lower levels in the mantle.After LPS induction,HsLBP mRNA was down-regulated at 6h,but it began to increase significantly at 12h(p<0.05).The mRNA expression patterns of HsTLR4 and HsMyD88 were similar,and they all showed significant up-regulation at 6 h(p<0.05)and the most obvious manifestation was in gill;The mRNA expression level of HsAP-1 began to increase significantly at 6h(p<0.05),the most obvious in the hemocytes,and which was highly significant up-regulation(p<0.01).In this study,we further explored the relationship between HsTLR4 and Hs MyD88.Firstly,we explored the interaction between HsTLR4 and HsMyD88 according to the principle of yeast two-hybrid.Secondly,we used bimolecular fluorescence interaction experiments in human hepatoma cells SMMC-7721 to further prove that there is a direct interaction between HsTLR4 and HsMyD88.And further use bimolecular fluorescence interaction experiments to explore the relationship between their domains.The results showed that the TIR domain of HsTLR4 can interacted with the Death domain or TIR domain of HsMyD88,but the intensity of the green fluorescence emitted by the TIRDeath interaction is weaker than the TIR-TIR.At the same time,we also demonstrated through GST-pulldown and western blot experiments that there is indeed an interaction between HsTLR4 and HsMyD88,and that TIR-Death can also interact with each other.The TIR-Death interaction may be due to the small electrostatic interaction.In this study,the full-length cDNAs of the innate immune-related genes,HsLBP,HsTLR4,HsMyD88 and HsAP-1 were obtained by RACE-PCR from hemocytes and gill of H.schlegelii.Bioinformatics and phylogenetic tree analysis showed that HsLBP,HsTLR4,HsMyD88 and HsAP-1 are conserved.After LPS induction,the mRNAs of HsLBP,HsTLR4,HsMyD88 and HsAP-1 was up-regulated.At the same time,it was explored that the direct interaction between HsTLR4 and HsMyD88 was discovered through yeast two-hybrid,bimolecular fluorescence interaction and GST-pulldown,and it was based on the interaction between TIR-TIR domains.HsLBP,HsTLR4,HsMyD88 and HsAP-1 were initially identified in H.schlegelii,and it revealed for the first time that HsTLR4 interacted directly with Hs MyD88 in freshwater mussel.Therefore,there are HsMyD88-dependent signaling pathways that respond to LPS in H.schlegelii.