| Golden pompano(Trachinotus ovatus) has been one of the most important marine fish commercially cultured in South C hina,because of its advantage of rapid growth, less intermuscular bones and delicious taste. Recently, commercial culture of golden pompano is also increasing in the south-east Asian countries such as Malaysia and Singapore. However, the main threat to the industry is infection disease, which results in a huge loss to the commercialization of farming. Therefore, a in-depth study of immunity mechanism must be performed to find ways to improve the disease resistance of golden pompano, and provie some basis for the treatment of diseases of golden pompano. In this study, the full- length c DNA sequences of Prx1 and Trx1 were obtained by randomly sequencing, and the sequence was verified by PCR and sequencing, multiple sequence alignment and phylogenetic trees were performed with the known amino acid sequences of Prxs and Trx, respectively.RT-PCR was performed to to investigate the tissue expression pattern of To Prx1 and To Trx1 m RN A in heart, stomach, brain, eye, gill, fin, skin, muscle, liver, spleen, kidney, intestine and blood,and temporal expression profilesof To Prx1 and To Trx1 Mrna in liver, spleen, kidney and intestine. Meanwhile, r To Prx1 and r To Trx1 fusion proteins activity were tasted to prove some technical means in terms of diseases prevention. The main results are as follows:ToPrx1 from golden pompanoPeroxiredoxin 1(Prx1) is an important antioxidant protein and can protect the organisms against the toxicity of reactive oxygen species(ROS). In this study, a full- length Prx1 c DN A sequence(To Prx1) was identified from golden pompano Trachinotus ovatus.To Prx 1 c DN A was 1049 bp long, and contained a 5’-untranslated region(UTR) of 127 nucleotides, a 3’-UTR of 328 nucleotides and a 594 bp open reading frame(ORF) encodinga 197 amino-acid polypeptide. To Prx1 protein showed strong homology(70-92%) with Prx 1 proteins from other species and contained the conserved Prx domain and the signature of peroxidase catalytic center. Phylogenetic tree revealed that To Prx 1 was in fish Prx 1 subgroup, which suggested that To Prx 1 could belong to the 2-Cys Prx subgroup. To Prx 1 m RNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the fin, spleen, kidney, intestine, eye, gill and blood. The expression levels of To Prx 1 m RN A were significantly up-regulated in liver, spleen, kidney and intestine of golde n pompano injected with P. damselae.Therecombinant To Prx1 protein(r To Prx 1) was expressedand purified through affinity chromatography, and simultaneously refolded using the ion-exchange chromatography. The antioxidant activity assay of r To Prx 1 showed that r To Prx 1 could reduce insulin in the presence of DTT, which suggested that the antioxidant function of r To Prx 1was thiol dependent. This study provided the useful information to help further understand the functional mechanism of Prx 1 inmarine fish immunity.To Trx1 from golden pompanoThioredoxin(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 c DNA sequence(To TRX) from golden pompano Trachinotus ovatus was identified after pyrosequencing of golden pompano c DNA library. To TRX c DNA 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 To TRX contained the highly conserved redox active disulphide/dithiol site(CGPC) of the thioredoxin active family, and phylogenetic tree showed that To TRX had a closer evolution relationship with TRX from Oplegnathus fasciatus and Anoplopoma fimbria. To TRX m RNA is ubiquitously expressed in all detected tissues with the higher expression levels in the stomach, gill and fin tissues. The expression of To TRX m RNA was significantly up-regulated in liver, kidney, intestine and spleen of golden pompano injec ted with Photobacterium damselae.The recombinant To TRX protein(r To TRX) was purified and refolded. The insulin disulfides assay was performed to investigate the enzymatic oxidoreductase activity of r To TRX, and the results demonstrated that r To TRX 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. |
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