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...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。展开更多
With designed specific primers based on Potato leafroll virus(PLRV) coat protein(CP) gene sequence,one PCR fragment(about 0.6 kb) was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the tota...With designed specific primers based on Potato leafroll virus(PLRV) coat protein(CP) gene sequence,one PCR fragment(about 0.6 kb) was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA as template extracted from virus infected potato plant collected in Zhaotong,Yunnan province.The fragment was cloned into pGEM-T easy vector and sequenced.The sequence was compared with that of homologous gene of other PLRV isolates.The results showed it had high homology with the other isolates(the highest homology reached 99.2% of nucleic acid).Based on CP amino acid sequence the phylogenic tree of PLRV was established and the isolates were clustered into many groups.展开更多
文摘With designed specific primers based on Potato leafroll virus(PLRV) coat protein(CP) gene sequence,one PCR fragment(about 0.6 kb) was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA as template extracted from virus infected potato plant collected in Zhaotong,Yunnan province.The fragment was cloned into pGEM-T easy vector and sequenced.The sequence was compared with that of homologous gene of other PLRV isolates.The results showed it had high homology with the other isolates(the highest homology reached 99.2% of nucleic acid).Based on CP amino acid sequence the phylogenic tree of PLRV was established and the isolates were clustered into many groups.
