Chang-Geng Yang

Signal transducer and activator of transcription 3 (STAT3) homologue in turbot (Scophthalmus maximus): Molecular characterization and expression analysis

Signal transducer and activator of transcription 3 (STAT3) acts as an important mediator in multiple biological processes induced by different cytokines. So far, little information is available in fish STAT3. In this study, turbot (Scophthalmus maximus) STAT3 gene was cloned and characterized for the first time. The turbot STAT3 full-length cDNA consists of 2355 nucleotides encoding a polypeptide of 784 amino acids with four conserved domains including STAT_int, STAT_alpha, STAT_bind and SH2 domain. The phylogenetic tree showed that turbot STAT3 shared the closest relationship with mandarin fish (Siniperca chuatsi) STAT3. The autoactivation experiment in yeast proved that turbot STAT3 was a strong transcription factor. The quantitative RT-PCR experiment indicated that Stat3 mRNA was expressed in widespread tissues with the highest expression levels in the liver. And the further expression patterns analysis revealed that turbot Stat3 expression levels were increased in liver, spleen, kidney of fish infected with Vibrio anguillarum and liver of fish infected with LCDV. Meantime, hepcidin, one of STAT3 target gene, was also up-regulated in liver of fish infected with two pathogens. These results suggested that turbot Stat3 may involved in the immune defense process as a transcription factor.

A new Akirin1 gene in turbot (Scophthalmus maximus): molecular cloning, characterization and expression analysis in response to bacterial and viral immunological challenge.

SmAkirin1, a member of the NF-κB signaling pathway, was isolated from turbot by RACE. Its cDNA was 564 bp and encoded a putative protein of 187 amino acids with a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.05. Amino acid sequence alignments showed that SmAkirin1 was 91% identical to the Salvelinus alpinus Akirin1 protein ACV49694. Transient expression of SmAkirin1-GFP in the turbot kidney cell line SMKC revealed a nuclear localization of the protein, and a typical NLS signal was found at the N-terminal region of the SmAkirin1 protein. Trans-activation assay in yeast demonstrated that SmAkirin1 has no transcriptional activation. Transcriptional analysis showed that SmAkirin1 was expressed in all of the tissues examined, with the highest expression in the spleen and brain. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that the SmAkirin1 transcript was induced by bacterial and viral infection.

Molecular cloning and multifunctional characterization of GRIM-19 (gene associated with retinoid-interferon-induced mortality 19) homologue from turbot (Scophthalmus maximus).

GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), a novel cell death regulatory gene, plays important roles in cell apoptosis, embryogenesis, mitochondrial respiratory chain and immune response. To date, little information is known about fish GRIM-19 characteristics except orange-spotted grouper (Epinephelus coioides). Here a new GRIM-19 gene is identified and characterized from turbot (Scophthalmus maximus), an economic marine fish in China and Europe. Briefly, turbot GRIM-19 is a 595-bp gene encoding a 144 amino acids protein, which shares the closest relationship with Atlantic halibut (Hippoglossus hippoglossus). The expression of turbot grim-19 in liver, spleen and kidney is up-regulated by the infection of Vibrio anguillarum and LCDV (lymphocystis disease virus). Subsequently, a recombinant protein of turbot GRIM-19 is acquired and the anti-bacterial function is proved by liquid culture inhibition experiment. The subcellular location indicates that turbot GRIM-19 is co-localized with STAT3 in the cytoplasm, which is mainly determined by GRIM-19 41-84 amino acids and STAT3 1-321 amino acids. Finally, the involvements of turbot GRIM-19 in cell apoptosis and NF-κB pathway are investigated. All these data help to understand GRIM-19 function in fish, as well as provide the application possibility of GRIM-19 in fish disease resistance breeding.

Cloning, characterization and expression analysis of a cold shock domain family member YB-1 in turbot Scophthalmus maximus

The Y-box proteins are a family of highly conserved nucleic acid binding proteins. In this report we have identified a new member, YB-1 from turbot (Scophthalmus maximus) spleen cDNA library. The full-length cDNA sequence of turbot YB-1 was obtained and then the expression at transcriptional level was researched by qRT-PCR. In normal organs, the expression of YB-1 was higher in liver, brain, gill and heart, respectively. YB-1 had the highest expression level at gastrula stage during the early stages of embryo development. In the liver, kidney and spleen, the turbot YB-1 expression level was the highest at 72 h after challenge with lymphocystis disease virus (LCDV) and the highest at 12 h after challenge with Vibrio anguillarum (V. anguillarum). Furthermore, the expression of turbot YB-1 also distinctly increased in turbot kidney cells (TK) at 24 h after challenge with V. anguillarum and LCDV. These results indicated that the turbot YB-1 protein may play a significant role in the immune response of turbot.