Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.

ABSTRACT

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135 bp full-length cDNA contains a 9 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562 bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec4°, or U4°) residue which is encoded by an opal codon, ¹²7TGA¹²9, and forms an active site with residues Q74 and W¹4². Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 (64LAFPCNQF7¹), an active site motif (¹5²WNFEKF¹57), a potential N-glycosylation site (76NTT78), and two residues (R9° and R¹68) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p < 0.05). The SpGPx expression increased and then declined during ovarian and testicular development implying thatnscrpits yowed that SpGPx might play an important role in gonad development by protecting them from oxidative stress. The expression of SpGPx mRNA was induced by lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) in hepatopancreas and haemocytes. The results suggested that SpGPx was implicated in the immune response induced by LPS and H2O2.