doi:

DOI: 10.3724/SP.J.1035.2012.01113

Acta Hydrobiologica Sinica (水生生物学报) 2012/36:6 PP.1113-1118

FULL-LENGTH cDNA CLONING AND EXPRESSION ANALYSIS OF SELENIUM DEPENDENT GLUTATHIONE PEROXIDASE FROM MACROBRACHIUM NIPPONENSE


Abstract:
Reactive oxygen species (ROS) produced in cells contribute to the pathogenesis of several diseases. Particularly large amounts are produced in inflamed tissues, where they are released by activated macrophages during respiratory bursts that are a defense mechanism against microbial infection. But ROS may be a double-edged sword, its over-expression will cause cellular damage and immune dysfunction. To maintain suitable low levels of ROS, aerobic organisms including crustacean are equipped with an elaborate enzymatic antioxidant defense system, in which glutathione peroxidase (GPx), a key antioxidant enzyme, may protect biomembranes and other cellular components from oxidative damage by catalyzing the reduction of variety of ROS. Investing the GPx gene expression in bacterial challenge can help us to understand the defense mechanisms of crustacean. GPx can be classed into two types: the non- selenium-dependent GPx (non-Se-GPx) and the selenium-dependent GPx (Se-GPx). For Se-GPx, four Se-GPx isozymes have been further identified according to cellular location and substrate specificity in vertebrate, named cellular GPx (GPx1), gastrointestinal GPx (GPx2), plasma GPx (GPx3) and phospholipid hydroperoxide GPx (GPx4). In this study, the commercially important freshwater shrimp Macrobrachium nipponense, obtained from the Yuan-yang Huangsi farm with an average length of (4.5±0.5) cm, were used. Firstly, the total RNA derived from different tissues was extracted, and then the quality of RNA was checked by agarose gel electrophoresis. The full-length cDNA sequence of selenium-dependent glutathione peroxidase (Se-GPx) from M. nipponense was cloned using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The immune chal-lenge test was carried out by injecting of Aeromonas hydrophila into abdominal segment of each shrimp at a dose of 20 µL (5.0×106 cells/mL), respectively. The result showed that the length of Se-GPx gene cDNA was 908 bp, including a 91 bp of 5′-untranslated region (UTR), a 256 bp of 3′-UTR with one selenocysteine insertion sequence (SECIS), and a polyadenylation signal (AATAAA) with 11 bp upstream of the PolyA tail. The open reading frame (ORF) was 561 bp, encoding a peptide of 186 amino acids with an estimated molecular mass of 21.2 kD and a theoretical isoeletric point of 6.74. The putative Se-GPx amino acid sequence contained a selenocysteine (Sec) residue which was encoded by the unusual stop codon TGA, a GPx signature motif 63LAFPCNQF70 and an active site motif 151WNFEKF156. The putative N-glycosylation site 75NNT77 and 107NGS109 were observed in the Se-GPx amino acid sequence. 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 to Macrobrachium rosenbergii in crustacean, and the Se-GPx was more similar to GPx1 and GPx2 than GPx3 and GPx4 in vertebrate. The Se-GPx gene was expressed in many tissues such as haemocyte, hepatopancreas, muscle, ovary, epidermis and mandibular organ, the level of the Se-GPx gene was the highest in haemocyte and higher in hepatopancreas and ovary comparing to other tissues. The immune challenge test revealed that the expression level of the Se-GPx gene in haemocyte was significantly up-regulated (P<0.05) at 3h and 6h after bacterial challenge, indicating that the Se-GPx was involved in the crustacean immune system.

Key words:Macrobrachium nipponense,Selenium-dependent glutathione peroxidase,Full length clone,Expression analysis

ReleaseDate:2014-07-21 16:42:39



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