Cloning and analysis of nuclear factor E2-related factor 2 (Nrf2) and its function in the regulation of respiratory burst in grass carp (Ctenopharyngodon idella)

Nuclear factor E2-related factor 2 (Nrf2) plays a key role in the regulation of respiratory burst which can produce amount of reactive oxygen species (ROS). In the present report, we have cloned and analyzed the Nrf 2gene from grass carp (Ctenopharyngodon idella), generated its polyclonal antibody, and characterized its role in regulation of ROS. The obtained cDNA of Nrf2 gene was 1994 bp, containing an open reading frame with 1782 bp encoding 593 amino acids. The amino acid sequences of Nrf2 from grass carp were highly identical with those from common carp (Cyprinus carpio) with 87% similarity. Nrf2 from grass carp contained six typical Neh (Nrf2-Epoxy chloropropane (ECH) homology) domains, indicating it was highly conserved in evolution. The qRT-PCR and Western blot analysis showed that Nrf2 was expressed in all eight detected tissues. The total antioxidant capacity of CIKs were up-regulated but ROS generated from CIK cells were down-regulated and the mRNAs of Nrf2 and its downstream gene (GST and HO-1) were all up-regulated when CIK was exposed to the ROS inducer, tert-Butylhydroquinone (tBHQ). Similarly, the expression of Nrf2 protein was also increased and it was translocated in the nucleus of CIK exposed to tBHQ. In conclusion, evolutionary conserved Nrf2 was ubiquitously expressed in the tissues of grass carp and it is involved in down-regulating the generation of ROS in CIK cells via up-regulating the expression of itself and its downstream antioxidant genes, so as to regulate respiratory burst.