Cloning And Expression Of Epididymis Specificity Glutathione Peroxidase (GPx5) Gene In Sheep

GPx5gene is the fifth enzyme in the glutathione peroxidase family, its active center is the substitution of cysteine for selenocysteine with secretory protein. GPx5is important to protect integrality and no oxadation sperm, and play an important role in the sperm mature of epididymis. Because of the important of GPx5, sheep caput epididymidis were selected to be experimental model, and RT-PCR was performed to clone sheep GPx5gene to analysis the physical and chemical properties of GPx5protein on the basis of cDNA. The expression profile of GPx5mRNA at different nutrition levels in the caput epididymidis were determined by using real-time PCR. The localization of GPx5in the caput epididymidis were investigated by immunofluorescence technique, we used these two means to detect the level of its gene and protein expression in the different nutrition levels. Our study were designed to research the relationship of GPx5and reproductive performance of buck animals at the molecular level, and to provide a theoretical basis for further study of GPx5in the control of mechanism in the buck reproductive system development and reproductive function. The main results of the experiment are as follows:1. The partial length cDNA of sheep GPx5gene was obtained firstly and submitted to the GenBank (GenBank number JQ320282.1). The1384bp of sheep contained an coding region of660bp. It encoded a polypeptide of219bp amino acidsand, with25kD molecular weight and7.723isoelectric point.2. The result of alignment of nucleic acid of sheep GPx5with cattle, pig, dog, mouse, human showed that the similarity were96.67%,88.94%,82.43%,80.18%,32.13%respectively and homology of sheep and cattle was the highest with the similarity. The deduced GPx5amino acid sequences showed that the polypeptide chain of GPx5was hydrophilicity. The GPx5amino acid sequences showed to be present signal peptide locus between21and22.22amino acid of GPx5protein had one N-glycosylation site, and no O-glycosylation site.24,163,168,203of GPx5protein sited serine,21,37,200,204of GPx5protein sited threonine,31,119,126of GPx5protein sited tyrosine, those were phosphorylation. The secondary structure of GPx5protein contained α-helix (41.1%), β-sheet (36.1%), Turn corner (10.0%) and Coil curl (12.8%).Homology of sheep and cattle was the highest in the protein phyletic evolution.3. Sheep GPx5mRNA appeared to be differentially expressed at different nutrition levels by Real-time PCR. The characteristics of GPx5mRNA expression:VFI group>65%VFI>35%VFI. The immunofluorescence localization of GPx5in the caput epididymidis showed that the expression signal was checked in epithelium cells of sheep caput epididymidis and increased along with rise of nutrition levels.