Full-length CDNA Cloning And Expression Analysis Of Selenium Dependent Glutathione Peroxidase From Macrobrachium Nipponense

glutathione peroxidase (GPx), a key antioxidant enzyme, may protect biomembranes andother cellular components from oxidative damage by catalyzing the reduction of variety of ROS.GPx can be classed into two types: the non-selenium-dependent GPx (non-Se-GPx) and theselenium-dependent GPx (Se-GPx). Investing the GPx gene expression in bacterial challenge canhelp us to understand the defense mechanisms of crustacean.In this study, the commercially important freshwater shrimp Macrobrachium nipponense,obtained from the Yuanyang Huangsi farm with an average length of4.5±0.5cm, were used.Firstly, the total RNA derived from different tissues was extracted, and then the quality of RNAwas check by agarose gel electrophoresis. The full-length cDNA sequence of selenium-dependentglutathione peroxidase (Se-GPx) from M. nipponense was cloned using the reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The immunechallenge test was carried out by injecting of Aeromonas hydrophila into abdominal segment ofeach shrimp at a dose of20μL (5.0×106cells/mL), respectively.The result showed that the length of Se-GPx gene cDNA was908bp, including a91bp of5'-untranslated region (UTR) and a256bp of3'-UTR with one selenocysteine insertion sequence(SECIS), and a polyadenylation signal (AATAAA) with11bp upstream of the PolyA tail. Theopen reading frame (ORF) was561bp, encoding a peptide of186amino acids with an estimatedmolecular mass of21.2kD and a theoretical isoeletric point of6.74. The putative Se-GPx aminoacid sequence contained a selenocysteine (Sec) residue which was encoded by the unusual stopcodon TGA, Homology analysis of the deduced amino acid sequence of the Se-GPx from M.nipponense with other known species revealed that the Se-GPx was the most similar toMacrobrachium rosenbergii in crustacean, and the Se-GPx was more similar to GPx1and GPx2than GPx3and GPx4in vertebrate.The Se-GPx gene was expressed in many tissues such as haemocyte, hepatopancreas, muscle,ovary, epidermis and mandibular organ, but the level of the Se-GPx gene was the highest inhaemocyte and higher in hepatopancreas and ovary comparing to other tissues. The Se-GPx geneexpression was significantly increased at stage D3-4,and then gradually decreased until stage C, indicating that the Se-GPx was important to molt stage. The immune challenge test revealed thatthe expression level of the Se-GPx gene in haemocyte was significantly up-regulated (P<0.05) at3and6h after bacterial challenge, indicating that the Se-GPx was involved in the crustaceanimmune system.