Gene Cloning And Function Study Of IRAK1and Tollip In Common Carp(Cyprinus Carpio L.)

IRAK1(IL-1receptor-associated kinase) and Tollip (Toll-interacting protein) is twoimportant molecules that participate in the TLR/IL-1R innate immune signaling pathway inmammal. IRAK1is a crucial kinase in the signaling pathway, while Tollip functions as negtiveregulation in the signal transduction. Although the mechanisms of the signaling pathway havebeen explained clearly in mammals, how the molecules function is not clarified in fish. In thepresent study, we first cloned IRAK1and Tollip gene in common carp and made a preliminarystudy on their function.In our study, we obtained the full length cDNA sequences of IRAK1and Tollip separately byusing RT-PCR and RACE. The compelet IRAK1cDNA was2800bp in length containing anopen reading frame (ORF) of2109bp encoding a peptide of702amino acids. Conserveddomain analysis with SMART server showed that IRAK1contained one DD (death domain),one Serine/Threonine protein kinases catalytic domain. While the full length of Tollip cDNAwas1284bp in length containing an open reading frame (ORF) of831bp encoding a peptide of276amino acids. Conserved domain analysis with SMART server showed that Tollip containedone TBD (Tom1-binding domain), one C2(conserved2) domain, one CUE (coupling ofubiquitin to endoplasmic reticulum degradation) domain.Then the deduced animo acid sequences of common carp IRAK1and Tollip were comparedwith IRAK1and Tollip proteins from the other species.Two phylogenetic trees were generatedusing the neighbor-joining method. We found that the deduced amino acid sequence of commoncarp IRAK1was highly similar to grass carp (84.5%). The phylogenetic analysis indicated thatthe common carp IRAK1fell into one clade with other species IRAK1, and the common carpIRAK1was found to show the closest relationship with the grass carp IRAK1. Whereas thecommon carp Tollip was most similar to grass carp (93.1%), zebrafish (93.8%). While in thephylogenetic analysis of Tollip, the common carp Tollip was clustered with the other bony fishTollip. The common carp Tollip was most closely related to the grass carp Tollip and zebra fish Tollip.In order to analyze the IRAK1and Tollip mRNA expression profile, the relative expressionlevels of IRAK1and Tollip mRNA was detected in eleven different tissues of healthy commoncarp by qRT-PCR. The results showed that the IRAK1and Tollip gene expressed in all tissuestested. The mRNA of IRAK1highly expressed in gills. We can see that the Tollip gene waspredominantly detected in brain.To investigate the antibacterial function of common carp IRAK1and Tollip, we analyzed thechange of IRAK1and Tollip expression level in common carp after i.p. injection with V.anguillarum. Expression levels of IRAK1and Tollip mRNA in common carp increased in allthe tested tissues. After stimulating with V. anguillarum, the upregulated expression level ofcommon carp IRAK1in liver was most significant among the immune-related tissues. Theupregulated espression level of IRAK1in common carp head kidney was less apparent.Whereas the upregulated level of common carp Tollip in spleen was most significant among theimmune-related tissues. The mRNA level of Tollip showed no significant change in head kidneypost-injection. These results showed that the common carp IRAK1and Tollip may play crutialroles in defensing the infection of V. anguillarum.To further investigate the antivirus function of common carp IRAK1and Tollip, we analyzedthe change of IRAK1and Tollip expression level in common carp after i.p. injection withpolyI:C. Expression level of IRAK1and Tollip mRNA in common carp increased in all thetested tissues. After stimulating with polyI:C, the upregulated expression level of common carpIRAK1in liver was most significant among the immune-related tissues. The upregulatedespression level of IRAK1in common carp head kidney was less apparent. Whereas theupregulated level of common carp Tollip in spleen was most significant among theimmune-related tissues. While the upregulated espression level of Tollip in common carp headkidney was less significant. These results showed that the common carp IRAK1and Tollip mayplay crutial roles in defensing the infection of polyI:C.Our results demonstrate the existence of Tollip and IRAK-1in common carp and providenovel insights into the role of the TLR signaling pathway in common carp.