Abstract:

To analyze accurately the role of myeloid differentiation factor 88 (MyD88) during immune response of Nile Tilapia (Oreochromis niloticus), the specific primers were designed and synthesized in conserved region based on MyD88 EST sequence (GenBank Accession number:GR679416.1) and β-actin gene (GenBank Accession number:AB037865.1). The liver cDNA sample of Nile tilapia was amplified with a series of 5-fold dilution to make standard curve. Specificity of PCR primers was assessed by melting curve analysis of PCR products. With β-actin as an endogenous gene, a method of SYBR Green I real-time quantitative PCR was established to examine MyD88 gene expression in 4 tissues (liver, spleen, blood and muscle) of Nile tilapia by the 2?ΔΔCt method. The detection range of CT value of MyD88 and β-actin was 24–35 and 19–30. The amplification efficiency (Evalue) was 100% and 96.7%, and the correlation coefficient (R2 value) was 0.998 and 0.995. The melting curve appeared a single peak, with Tm value of 78.5℃ and 77.5℃ respectively. The results showed that MyD88 gene was expressed highly in immune tissues such as liver, spleen and blood.