Cloning, Characterization And Expression Analysis Of Common Carp TRAF6

The key position of teleost fishes in the evolution of innate immunity andadaptive immunity, so the study of teleost fishes become important reference forevolution of higher organisms immune pathways. Innate immune parameters inteleost fish are more active than in mammals. TRAF6is a key player at thecross-roads of development and immunity. TRAF6is involved in variousphysiological processes, including TLR/interleukin-1receptor innate immunity,adaptive immunity, osteoclastogenesis, developing epidermal appendixes, mammarygland and lymph node organogenesis, and the developing central nervous system. Anessential protein transducing the signals emanating from various PRRs and cytokinereceptors, including the TNF superfamily, TGFβ, IL-1/Toll-like and NOD-likereceptors, is the TRAF6. TRAF6is important in response to pro-inflammatorymediators such as IL-1and LPS. TRAF6is conserved from Drosophila to humans,like other TRAFs, TRAF6has a conserved carboxyl terminal TRAF-C domain, aTRAF-N domain and, with the exception of TRAF1, an amino terminal Ring finger.Recently, Study of Multiple myeloma, TRAF6may be considered as a potentialtherapeutic target for the treatment of MM.In this study, the cDNA sequence was labeled with DIG which was obtainedfrom EST sequencing used as a probe, then TRAF6was screened from common carpperipheral blood leukocyte cDNA library that mitogen stimulated by nucleic acidhybridization, then we obtained the full-length cDNA sequence of carp TRAF6. Wedesigned primes in the non-coding region and TRAF6cDNA obtained then used thespleen genomic DNA as plate to perform PCR, we got the full-length DNA sequenceof carp TRAF6. The TRAF6specific primers and β-actin internal reference primers were designed, we stimulated carp peripheral blood leukocyte using mitogens andabstract total RNA at different times after stimulation as plate, then we performedRT-PCR detection. Common carp treated with Aeromonas Hydrophila and extractRNA in seven different organs and detect its expression change in different timepoints by using RT-PCR.We obtained the full-length cDNA sequence of carp TRAF6, The sequence is2192bp in length containing an ORF (open reading frame)1632bp and predictivecoding543amino acids. There are instability (ATTTA) at127bp and64bp in thePoly(A) tail upstream, respectively. And there is a typical polyadenylation signal(AATAAA) in the3’-untranslated. We got the full-length DNA sequence of carpTRAF6, The sequence is5237bp. By the NCBI/GenBank BLAST, we found that thesequence showed the identity with known carp TRAF6genome (HM535646.1)97%.The genome contains six exons and five introns, and the splice sites between exonand intron are conserved supervision of the GT-AG. We performed RT-PCRdetection, the results show that with mitogens treated, the carp TRAF6levels inperipheral blood leukocytes changes over time, this is similar to TLRs. Commoncarp treated with Aeromonas Hydrophila by using RT-PCR. The result demonstratedthat TRAF6is expression in all tissues examined, and showed maximum expressionin the gill tissue, followed by liver and spleen. Induction with AeromonasHydrophila, the TRAF6in all tissues examined levels are upregulation. And TRAF6mRNA increased at12h post infection in maximum, and return to basal level at24hpost infection. The result indicated that TRAF6involved in the early immune incommon carp.