IMMUNE RESPONSES OF TWO THIOREDOXIN-LIKE GENES IN BLACK ROCKFISH Sebastes schlegelii

Thioredoxin-like 1 (TXN-like 1) and Mitochondrial Thioredoxin-like (MtTXN-like) are two different isoforms of thioredoxin, which are involved in the regulation of multiple biological processes. One of the main regulatory functions of both TXNs is to maintain the intracellular environment in a highly reduced state via their antioxidant property. Oxidative stress can be encountered due to the elevated levels of the reactive oxygen species (ROS), during the pathogen invasion. TXNs regulate the oxidative stress in cells by maintaining the dithiol/disulphide balance in proteins via its disulphide reductase activity. In the present study, genes coding for two isoforms were identified from the cDNA database of black rockfish Sebastes schlegelii designated as SsTXN-like 1 and SsMtTXN-like respectively. SsTXN-like 1 cDNA had an 867 bp open reading frame (ORF), which encoded 289 amino acids, while SsMtTXN-like cDNA contained a 525 bp ORF, encoding for 175 amino acids. The estimated molecular weights of SsTXN-like 1 and SsMtTXN-like were 32.0 kDa and 19.2 kDa, respectively. Merely the N-terminus of SsMtTXN-like was found to have a mitochondrial localization signal of 67 amino acids.

Spatial distribution of both SsTXN-like 1 and SsMtTXN-like mRNAs was determined by quantitative real-time PCR using different tissues of healthy individuals. Results indicated that SsTXN-like 1 mRNA was highly expressed in muscle and peripheral blood cells (PBCs), while the SsMtTXN-like mRNA was extremely expressed in PBCs. To study the temporal expression of these genes in vivo, fish were challenged with poly I:C, LPS and Streptococcus iniae. Upon all these immune challenges, SsTXN-like 1 mRNA was significantly up-regulated with the peak at 3 h, 48 h post-injection (p.i) in PBCs, meanwhile at 48 h, 72 h p.i in head kidney. SsMtTXN-like mRNA was significantly up-regulated at later phase of p.i in liver, meanwhile fluctuated expression pattern was observed in PBCs.

This study intimates that both TXNs are ubiquitously distributed in rockfish, and up-regulation of both gene expressions during the immune challenge suggests their vital role in redox balance during the pathogen invasion. In order to confirm the functional properties of these TXNs, we have cloned them into expression vector to produce the recombinant proteins to employ them in activity assays and experiments are in progress.