小鼠过氧化物酶体增殖物激活受体α下游新靶基因PTG的克隆

Molecular Cloning of Mouse PTG(Peroxisome Proliferator Activated-Receptor α Target Gene)

为了进一步研究过氧化物酶体增殖物激活受体α(PPARα)下游靶基因,应用基因芯片技术比较经过氧化物酶体增殖物(peroxisomeproliferators,PPs)处理和未处理鼠肝RNA,获得经PPs处理而上调的一个EST(expressedsequencetag),C3f.用5′RACE法获得其全长cDNA,并命名为PTG.Northern法检测PTG的组织分布.结果显示,在小鼠心脏、肝脏、肾脏、睾丸等组织,PTG有高表达.Northern印迹检测AOX敲除的小鼠发现,PTG转录水平明显升高,而在Wy14643处理的AOXPPARα双敲除的小鼠没有发现升高.实时监测PPs处理小鼠肝脏PTG表达水平的变化,发现处理2h后,PTG表达水平提高了2倍,这说明PTG是PPARα的早期反应基因.利用PTGEGFP表达质粒转染NIH3T3细胞,显示PTG蛋白定位于质膜下.进一步用Rab5aEGFP和PTGDsRed双定位载体共转染,结果表明,PTG蛋白定位于早期内吞体.

To investigate the new target gene of PPARα（peroxisome proliferator activated-receptor α）, an EST （expressed sequence tag） named C3f was selected which was up-regulated by PPs （peroxisome peroliferators） treatment. By using 5＇-RACE, we cloned the full-length of PTG cDNA and found that PTG is expressed in many tissues by Northern blot with high level in heart, liver, kidney and tesis. Furthermore, the expression pattern of PTG was detected in mouse with different phenotypes and the results indicated that PTG was markedly elevated in PPs-treated wild-type mice compared with untreated mice. Expectively, there was no change of PTG expression in PPARα knockout and PPARα/AOX double knockout mouse. As a definitive test, a time-course experiment was carried out and the result was demonstrated that the level of PTG transcript increased about two folds within two hours after treatment, reenforcing the argument that PTG is an immediately-early response gene for PPARα. By transfecting NIH3T3 cells with pPTG-EGFP plasmid,we found that PTG protein is localized under membrane. Further co-localization experiment with pRabSa-EGFP and pPTG-DsRed fusion protein indicated that PTG is localized in early endosome.