硒和FSH对羊睾丸PHGPx基因表达调控的分子机制

项目名称： 硒和FSH对羊睾丸PHGPx基因表达调控的分子机制

项目编号： No.30871794

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 岳文斌

作者单位： 山西农业大学

项目金额： 35万元

中文摘要： 本项目应用RNAi技术、RACE技术、Real-time PCR、Western blot等分子生物技术通过体内外试验研究硒和FSH对羊睾丸PHGPx表达调控的分子机制。试验结果如下：（1）成功克隆了山羊844bp PHGPx和1183bp CREMτ22522;因cDNA（GQ 302986和HM802260）；（2）PHGPx和CREMτ22312;山羊睾丸组织中表达量极显著高于其他组织（P<0.01）；（3）PHGPx和CREMτ34920;达水平随山羊周龄增加而增加，PHGPx在8周龄后表达量显著增加而CREMτ22312;12周龄表达量显著升高（P<0.05），免疫组化结果也证实了这一结果；在12周龄时山羊血液FSH浓度显著增加（P<0.05）。（4）日粮添加0.3mg/kg纳米硒显著增加了睾丸PHGPx和CREMτ34920;达水平（P<0.05）；（5）成功建立了山羊睾丸生精细胞共培养系；（6）体外培养山羊睾丸生精细胞共培养系的214位点PHGPx和927位点CREMτ30340;RNAi质粒的干扰效果显著，为进一步研究PHGPx表达调控提供了依据。

中文关键词： 硒；FSH；PHGPx；信号通路；CREMτ65307;

英文摘要： The project was conducted to investigate the effects selenium status and FSH on the regulation of expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and the signal transduction pathway of expression regulation of PHGPx at the different stage of the spermatogenesis. The results showed: (1)The full length of goat CREMτnd PHGPx cDNA was successfully cloned (GenBankNo. HM802260 and GQ 302986 )by RACE technology,which had 1183bp and 844bp respectively; (2) The expression of PHGPx mRNA and CREMτRNA in goat testes assayed by quantitative real-timen PCR technology were significantly higher than any other tissue (P&lt;0.01);(3) The expression of PHGPx mRNA and CREMτRNA increased with age increasing. The level of PHGPx mRNA in 8-wk was notably higher than the level during 1 to 6-wk. however, The level of CREMτRNA and serum FSH concentration in 12-wk was notably higher than the level during 1 to 8-wk （P&lt;0.05）. The pattern of PHGPx protein expression was similar with the mRNA expression by immunohistochemical technology;(4) The expression of PHGPx mRNA and CREMτRNA increased with 0.3 mg/kg Nano-Se supplementation in the diet; (5) Co-culture system of goat spermatogenic cells was established by using two steps of combination enzymatic digestion method; (6) The 214-site siRNA of PHGPx and 927-site siRNA of CREMτignificantly inhibited the expression of mRNA and protein of PHGPx and CREMτespectively (P&lt;0.01),which provided a powerful tool for the further study on the trace element Se or the mechanism of PHGPx involved the spermatogenesis.

英文关键词： selenium; FSH; PHGPx; Signal transduction;CREMτ