Molecular cloning and expression analysis of Sp-PHGPx in Scylla paramamosain

Phospholipid hydroperoxide glutathione peroxidase(PHGPx)is one of the members of GPx family, which has its unique ability to reduce hydroperoxides in complex lipids such as phospholipid, cholesterol and so on.PHGPx is a key enzyme in the antioxidant defense systems of living organisms, including crustaceans, which plays important roles in the effect of antioxidant and sexual maturity of male.In this study, expressed sequence tag(EST)of Scylla paramamosain PHGPx gene was identified from transcriptome sequencing library and the full length cDNA was cloned by SMART-RACE method, which was named Sp-PHGPx.The full-length cDNA of Sp-PHGPx is 1 024 bp, including a 5'UTR of 140 bp, a 3'UTR of 341 bp and an open reading frame of 543 bp, encoded a duduced protein of 180 amino acids.Sequence comparison and phylogenetic analysis showed that Sp-PHGPx was clustered with PHGPx(GPx4)of other species, far from GPx1 and GPx2.Quantitative real time PCR revealed that Sp-PHGPx was expressed in various tissues of the mature female and male crabs with the highest expression level in testis.During the different stages of gonadal development, there was no significant difference in the development of ovary;while Sp-PHGPx was expressed at the highest level in spermatid stage(T2)and was significantly higher than spermatocyte stage(T1)and mature sperm(T3)during the development of testis.Moreover, the expression levels of T2 are higher than these of other stages of ovary development.When under the challenge of LPS, the expression level of Sp-PHGPx was up-regulated significantly at 6 h in gill and at 12 h in hepatopancrea compared with control.The expression of Sp-PHGPx in gill was induced significantly at 3 h, while there was no significant difference in hepatopancrea when exposed to H₂O₂.Above results suggest that Sp-PHGPx may be involved in immune defense and the male reproduction in S.paramamosain.