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 963 bp with a 669 bp open reading frame (ORF) encoding 222 amino acids and a 101 bp eukaryotic selenocysteine insertion sequence (SECIS) in 3′ untranslated region (UTR). It was showed that HdhGPx has a characteristic codon at ²³⁵TGA²³⁷ that corresponds to selenocysteine (SeC) as U₇₂. Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 (₉₆LGLPCNQF₁₀₃), an active site motif (₁₇₉WNFEKF₁₈₄). In addition, two potential N-glycosylation sites (₁₁₂NGTE₁₁₅ and ₁₃₂NLTQ₁₃₅) 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 48.7 mg kg^− 1), zinc (6.69, 33.85 and 710.63 mg kg^− 1) and iron (29.17, 65.7 and 1267.2 mg kg^− 1) for 20 weeks, respectively. The results showed that the expressions of HdhGPx mRNA were statistically higher at adequate dietary selenium (1.32 mg kg^− 1), zinc (33.85 mg kg^− 1) and iron (65.7 mg kg^− 1) than those in low dietary minerals, respectively. But HdhGPx mRNA expression levels were down-regulated by high contents of dietary selenium (48.7 mg kg^− 1), zinc (710.63 mg kg^− 1) and iron (1267.2 mg kg^− 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.