IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF P65 SUBUNIT OF NF-KB FROM REDLIP MULLET Liza haematocheila

Nuclear factor (NF)-kappaB is a family of structurally related transcription factors which plays a central role in innate immune response by controlling gene network expression.  All the members in the NF-κB protein family share a highly conserved DNA binding or dimerization domain called a Rel homology domain (RHD). The family can be subdivided into two subfamilies as "Rel" proteins and "NF-κB" proteins. RelA/p65, RelB, C-Rel are the members of "Rel" subfamily and p105 and p100 are the members of NF-κB subfamily.

The current experiment was designed to characterize homologs sequence for p65 subunit of NF-κB from red lip mullet. A nucleotide sequence for p65 was identified from mullet transcriptomic database. Bioinformatics tools and software were used in sequence characterization. Five redlip mullets were dissected and 12 different tissues including liver and spleen were collected. Groups of healthy red lip mullets were challenged with LPS, Poly I:C and Lactococcus garvieae. The spleen of fish (n=5) was sampled and pooled in 0, 6, 24, 48, 78 hours after the IP injection. mRNA extraction and cDNA synthesis were performed for tissue pools from both the studies respectively. qPCR was used to investigate the transcriptional modulation of Lhp65 gene in tissues of healthy fish and challenged fish.  

The Lhp65 gene is consisted of 1899bp and predicted polypeptide sequence carries 632 AA. The predicted molecular weight of the Lhp65 subunit is 69.36kDa. In the phylogenetic reconstruction, Lhp65 cladded with its fish counterparts. Multiple sequence analysis indicated a significant similarity in the domain structure of Lhp65with other vertebrate sequences.

The intestine shows the highest expression of Lhp65 among other tissues. The digestive tract is one of the primary entering routes of pathogens. That may cause high expression in the intestine. Lhp65 is highly stimulated in spleen tissue by all three PAMP's.  Interestingly, continuous upregulation is observed in poly I:C and L. garvieae challenges. The population density increase of live L. garvieae in challenged fish may induce the transcription of p65 subunit. The viral mimic poly I:C may induce the transcription, in order to inhibit the viral replication by activating NF-κB target genes. However, the extensive investigation is required to designate the function of p65 in the fish immune mechanism.