Molecular cloning, characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium, zinc and iron.

A novel selenium-dependent glutathione peroxidase (Se-GPX) was cloned from abalone Haliotis discus hannai Ino (HdhGPx) by homology cloning with degenerate primers and RACE techniques. The full length of HdhGPx cDNA was 963bp with a 669bp open reading frame (ORF) encoding 222 amino acids and a 101bp eukaryotic selenocysteine insertion sequence (SECIS) in 3' untranslated region (UTR). It was showed that HdhGPx has a characteristic codon at (235)TGA(237) that corresponds to selenocysteine (SeC) as U(72). Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 ((96)LGLPCNQF(103)), an active site motif ((179)WNFEKF(184)). In addition, two potential N-glycosylation sites ((112)NGTE(115) and (132)NLTQ(135)) were identified in HdhGPx. 3D modeling analysis showed that the overall structure of HdhGPx monomer had more similarity to human GPx3 than human GPx1. Relatively higher-level mRNA expression was detected in hepatopancreas, mantle and gonad by real-time PCR assays. The relative expression levels of HdhGPx mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15, 1.32 and 48.7mgkg(-1)), zinc (6.69, 33.85 and 710.63mgkg(-1)) and iron (29.17, 65.7 and 1267.2mgkg(-1)) for 20weeks, respectively. The results showed that the expressions of HdhGPx mRNA were statistically higher at adequate dietary selenium (1.32mgkg(-1)), zinc (33.85mgkg(-1)) and iron (65.7mgkg(-1)) than those in low dietary minerals, respectively. But HdhGPx mRNA expression levels were down-regulated by high contents of dietary selenium (48.7mgkg(-1)), zinc (710.63mgkg(-1)) and iron (1267.2mgkg(-1)), respectively. These results indicated that adequate dietary minerals could increase the mRNA expression of HdhGPx, and then to increase the total antioxidant capacities in abalone.