Cloning, expression analysis and promoter structure of TBK1 (TANK-binding kinase 1) in Atlantic cod (Gadus morhua L.)

TBK1, also termed NAK or T2K, is a ubiquitous member of the IκB kinase (IKK) family that is required for innate and adaptive immune responses. We have identified and characterized the full-length TBK1 cDNA in Atlantic cod. The cod TBK1 gene consists of 2190 bp open reading frame encoding a polypeptide of 729 amino acids. According to a BLAST search, the cloned TBK1 gene has a high degree of sequence similarity (80.7-92%) to the various members of the TBK1 family, indicating that it is conserved during evolution. RT-PCR showed that the largest quantity of TBK1 transcripts was found in spleen, followed by the liver, gill, head kidney, gut, pyloric caeca, while the expression of TBK1 mRNA in muscle and skin was low. Both PMA, poly I:C and β-glucan promoted expression of TBK1 transcripts in vivo. Furthermore, we determined an 875 bp sequence upstream of the transcriptional start site (TSS) and found a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory regions of this gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. After the expression experiments, the results showed that the basal promoters and positive transcriptional regulator activities of cod TBK1 gene were dependent by sequences located from -875 to -425 bp and from -245 to +28 bp upstream of TSS. This study provides further insights into the transcriptional regulation of cod TBK1.