Cloning, Characterization And Expression Of A Myeloid Differentiation Factor88Gene In Tilapia, Oreochromis Niloticus And Oreochromis Aureus

Myeloid differentiation factor88(MyD88) is a universal adaptor protein able to activate nuclear factor-kappa B (NF-κB) through interactions with interleukin-1receptor (IL-1R) and the Toll-like receptors (TLRs), with the exception of TLR3. It plays a crucial role in the innate immune response. So far, no report about MyD88and MyD88-related factor in Tliapia (Oreochromisspp), one of the most promising and worldwide important species for freshwater fish culture in China, is available. In this paper, we cloned and analyzed MyD88gene in Oreochromis aureus and Oreochromis niloticus using RACE (rapid amplification of cDNA ends) method, and detectd the expression of MyD88in O. aureus and O. niloticus by real-time quantitative PCR.The full-length MyD88cDNA of O. aureus is1611bp, including a5’-terminal untranslated region (UTR) of155bp, a3’-UTR of589bp. And the full-length MyD88cDNA of O. niloticus is1572bp, including a5’-UTR of118bp, a3’-UTR of587bp. They both contain an open reading frame (ORF) of867bp encoding a polypeptide of288amino acids. The identity of the two nucleotide sequences is up to99%. To determine the structural domains in MyD88, the amino acid sequences were analyzed using the SMART program. The deduced amino acid sequences of MyD88both contain the typical N-terminal death domain (DD), intermediate domain and C-terminal TLR and IL-1R-related (TIR) domain. The TIR domain, with three conserved boxes—box1(FDAFICYCQ), box2(LCVFDRDVLPGSC) and box3(FWTRL), was found to comprise the carboxyl terminal half of the protein and exhibited sequence conservation more significantly than did the death domain. Homology analysis revealed that the MyD88sequence shares highly identity with the known MyD88members form other species. The predicted amino acid sequence of O. aureus MyD88was85.8%identical to Chinese perch (Siniperca chuatsi),69%-82%to other fishes,63%-64%to mammals, and31.8%to Zhikong scallop(Chlamys farreri),21.9%to Fruit flies (Drosophila melanogaster) with the lowest sequence similarity. The phylogenetic tree based on15species MyD88proteins showed that O. aureus MyD88grouped in the same clade as other piscine MyD88, with high bootstrap values, but it has distant relationship with invertebrates. The amino acid sequence shared the closest relationship with Chinese perch and large yellow croaker (Larimichthys crocea), which are all pertain to Perciformes.To establish Tilapia MyD88real-time PCR assays, we used β-actin as an endogenous gene and designed a pair of primers based on the sequence of the MyD88gene. The liver cDNA sample of O. niloticus was amplified with a series of5-fold dilution to make standard curve. The results show that the amplification efficiency (E values) of MyD88and β-actin were100%and96.7%, the correlation coefficients (R2values) were0.998and0.995, the melting curve appeared a single peak, and the difference value of the shope is no more than0.1, so the efficiencies of the target and reference genes are similar, and the△△CT calculation for the relative quantification of target can be used. A SYBR Green I real-time fluorescence quantitative PCR method was established and4tissues (liver、spleen、blood and muscle) of O. niloticus were preliminary detected using the2-△△Ct method in order to verify the established real-time PCR platform. The relative real-time PCR results show that the MyD88gene was expressed highly in immune tissues such as liver, spleen and blood.Using the established real-time fluorescence quantitative PCR platform, we studied MyD88expression in12tissues (gill, heart, kidney, liver, spleen, muscle, ovary, testis, brain, blood, skin and intestine) of healthy O. aureus and O. niloticus. The results revealed MyD88transcripts were expressed in all tested tissues, and the distribution in O. aureus and O. niloticus is consistent:strongly expressed in ovary, moderately expressed in intestine, spleen, liver, kidney, gill and blood, and weakly expressed in muscle, testis.The results imply that MyD88has an important role in the innate immune system in Tilapia as other piscine and mammals. Further research is needed to understand the dynamic role of MyD88with respect to other immuno-proteins (signaling receptor or costimulatory proteins) in the Tilapia innate immune system.