【摘要】 根据锤头状核酶的作用模式,设计、合成并克隆了特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)中国分离株(PLRV-Ch)复制酶基因负链RNA的二价核酶序列。将该核酶基因克隆到pGEM-4Z中,构建成体外转录载体pGEM-4ZDR;同时将包含核酶切割识别位点的PLRV-Ch复制酶基因cDNA近5’端的874 bp片段反向插入到pGEM-4Z中,构建了体外转录载体pGEM-4ZR5。以线性化的重组质粒pGEM-4ZDR和pGEM-4ZR5为模板,在T7 RNA聚合酶的作用下,分别转录获得核酶RNA和PLRV-Ch复制酶基因5’端负链RNA底物片段。将以上2种RNA转录物混合并经37℃保温后,检测结果表明,所得核酶RNA对PLRV-Ch复制酶基因负链RNA在体外具有较强的特异切割活性。更多还原

【Abstract】 Based on the functional model of the hammerhead RNAs,a divalent ribozyme was designed for cleavage of Potato leafroll virus(PLRV)RNA,targeting at two sites on the negative strand of PLRV Chinese isolate replicase gene and cloned in vector pGEM-4Z.The recombinant plasmid pGEM-4ZDR was useed for in vitro transcription of the ribozyme RNA.The plasmid pGEM-4ZR5 for in vitro transcription of the substrate viral RNA was synthesized by cDNA subcloning of a PLRV Chinese isolate(PLRV-Ch)into plasmid pGEM-4Z,in which an 874 bp cDNA fragment at the 5′ end of PLRV-Ch replicase gene was reversely inserted downstream of the T7 promoter in pGEM-4Z.By using T7 DNA polymerase,the plasmids of pGEM-4ZDR and pGEM-4ZR5 were linearized with EcoR I and used as templates for in vitro transcription of the divalent ribozyme or the negative strand RNA of PLRV-Ch replicase gene,respectively.After mixture of the RNA transcripts and incubation at 37℃,results showed that the ribozyme has highly catalytic activity to the PLRV-Ch negative strand RNA in vitro.更多还原