【摘要】 采用RT-PCR技术获得了拟南芥多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]PARP1基因的全长cDNA,转入原核表达载体pET32a并转化宿主菌Origami(DE3),加入终浓度为0.3mmol/L IPTG,在16℃下诱导可获得较多的可溶重组蛋白。纯化TRX-PARP1,在反应液中加入NAD+和断裂DNA,通过SDS-PAGE凝胶电泳和Western blotting分析,TRX-PARP1分子量可随着时间的延长逐渐增大,产生向上的弥散,表明蛋白质连上了ADP核糖分子;与此对比,作为参照的标签蛋白TRX无此现象。实验结果显示原核表达拟南芥PARP1能够催化自身多聚ADP核糖化修饰,为深入研究植物多聚ADP核糖聚合酶的功能奠定了基础。更多还原

【Abstract】 In order to study the enzymatic activity of Arabidopsis poly(ADP-ribose)polymerase 1(PARP1),the cDNA fragment of PARP1was amplified using Reverse-Transcription PCR method from Arabidopsis seedlings treated by bleomycin.The cDNA sequence was cloned into prokaryotic expression vector pET32and the resultant pET32a-PARP1plasmid was transformed into Escherichia coli host strain Origami(DE3).IPTG was added into the culture to a final concentration of 0.3mmol/L to induce expression at 16℃for 20h.The recombinant protein TRX-PARP1was purified and then assayed for enzymatic activity with the substrate NAD+and broken DNA in the reaction buffer.TRX-PARP1protein band shifted upwardly in a time course on SDS-PAGE gel.Western blotting using anti-PARP1antibody further confirmed that the molecular mass of TRX-PARP1increased gradually,due to the linkage of ADP ribose molecules onto the protein.In contrast,the fusion tag protein Thioredoxin(TRX)did not show any change in mobility.These data demonstrated that Arabidopsis PARP1produced in E.coli has auto-modification activity,which facilitates the functional study of PARP1in Arabidopsis.更多还原