Thioredoxin of golden pompano involved in the immune response to Photobacterium damselae.

Abstract

Thioredoxin (TRX) is one of the key systems responsible for keeping the intracellular environment in a highly reduced state. In this study, a full-length TRX cDNA sequence (ToTRX) from golden pompano Trachinotus ovatus was identified after pyrosequencing of golden pompano cDNA library. ToTRX cDNA is comprised of 786 bp, and contained a 324 bp open reading frame (ORF) encoding a 107 amino acid polypeptide, a 5' untranslated region (UTR) of 116 bp, and a long 3'- UTR of 346 bp. Multiple sequence alignment revealed that ToTRX contained the highly conserved redox active disulphide/dithiol site (CGPC) of the thioredoxin active family, and phylogenetic tree showed that ToTRX had a closer evolution relationship with TRX from Oplegnathus fasciatus and Anoplopoma fimbria. ToTRX mRNA is ubiquitously expressed in all detected tissues with the higher expression levels in the stomach, gill and fin tissues. The expression of ToTRX mRNA was significantly up-regulated in liver, kidney, intestine and spleen of golden pompano injected with Photobacterium damselae. The recombinant ToTRX protein (rToTRX) was expressed in Escherichia coli BL21 (DE3), and then purified and refolded. The insulin disulfides assay was performed to investigate the enzymatic oxidoreductase activity of rToTRX, and the results demonstrated that rToTRX exhibited a high reducing activity in presence of DTT, while no activity was observed in the regroup without DTT and blank control group. Over all, the study provided the useful information to help further understand the functional mechanism of TRX in marine fish immunity.