Molecular Cloning And Expression Analysis Of TLR20 And TLR21 Genes In Japanese Eel,Anguilla Japonica

TLR is a kind of pattern recognition receptors in innate immunity.They can recognize a variety of pathogenic and activate immune responses by TLR signaling pathways.In the present study,the cDNA sequence AjTLR20,AjTLR21 which belong to the TLR11 family members,and the downstream molecules of TLR signaling pathways: AjMyD88,AjIRAK4,AjTIRAP were cloned in Japanese eel,and did some research.The putative protein of AjTLR20 contains 929 amino acids,which belongs to Type I transmembrane protein.It has an extracellular region with 6 LRRs(Leucine rich repeats,LRRs),a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor(TIR)domain with three conserved box.The putative protein of AjTLR21 contains 978 amino acids and possesses the characteristic domains and motifs mentioned above except the 14 LRRs in ectodomain.The results of quantitative real-time PCR showed that both AjTLR20 and AjTLR21 were widely expressed in blood,skin,liver,head kidney,spleen,heart,middle kidney,swim bladder,intestine,gill,stomach and sexual gland,with the highest expression in blood,and the least was sexual gland.Besides,the expression level of AjTLR20 in the same tissue/organ(except gill)is higher than that of AjTLR21.We studied the expression changes of AjTLR20 and AjTLR21 in different tissues/organs when challenged by LPS,Poly I:C or Edwardsiella tarda after 8 h,16 h,24 h or 72 h.To the AjTLR20,its expression was significantly up-regulated in blood at 8 h after challenged by Poly I:C and Edwardsiella tarda(P < 0.001).It was significantly up-regulated in skin at 8 h when challenged by LPS(P < 0.05).AjTLR20 in blood and skin was significantly up-regulated at 16 h when challenged by LPS.While to the AjTLR21,its expression was significantly up-regulated in spleen and kidney at 8 h,in blood,gill,intestine and spleen at 16 h after challenged by Poly I:C(P < 0.05).The result also show that the E.tarda can also up-regulate AjTLR21 in blood,intestine and middle kindey.We also studied the expression of AjTLR20 and AjTLR21 in eel PBMCs when added the LPS,Poly I:C or Edwardsiella tarda after 4 h,6 h,8 h or 10 h.Expression of AjTLR20 in eel PBMCs at 8 h added Poly I:C was extremely significantly raised(P < 0.001).The expression level of AjTLR20 is up-regulated when the PBMCs was stimulated by E.tarda after 6 h and 8 h.AjTLR21 is significantly up-regualted at 4 h(P < 0.05)when challenged by Poly I:C;AjTLR21 can also response to E.tarda in PBMCs.The result indicats that the AjTLR20 and AjTLR21 can response to Poly I:C,E.tarda or LPS mainly in blood.They can not only participate in antiviral immunoreactions,but also participate in anti-bacterial reactions,illustrating the diversity function of AjTLR20 and AjTLR21.We cloned 1875 bp fragment of the AjTLR21.The fragment contains several transcription factor binding sites such as C/EBP-?,AP-1,NF-?B,IRF and Sp-1.Luciferase reporter assay exhibited that the segment from-332 to-635 had key sites of transcription of AjTLR21.The segment,from-1179 bp to +117 bp,seemed to contain positive regulatory elements for AjTLR21 expression in response to Poly I:C treatment.We also cloned the cDNA sequence of three downstream molecules of TLR signaling pathways: AjMyD88,AjIRAK4 and AjTIRAP.They code 463 aa,282 aa and 423 aa respectively.AjMyD88 has the characteristic TIR domain and a death domain,AjIRAK4 has the characteristic death domain and a Ser/Thr kinase domain,AjTIRAP has the characteristic TIR domain.AjIRAK4 and AjTIRAP were widely expressed in examined tissues/organs such as blood,liver,middle kidney,with the highest expression in blood.