二价核酶基因构建及对PLRV负链靶序列的体外切割研究  

Construction of a Divalent Ribozyme Gene Specific to Potato Leafroll Virus Repelicase Gene and Cleavage of Negative Strand RNA in vitro by Ribozyme

作　　者：张剑峰[1] 张鹤龄[2] 于嘉林[3] 迟胜起[1] 贺秀芳[1] 郝文胜[2] 杨静华[2] 

机构地区：[1]青岛农业大学农学与植保学院,山东青岛266109 [2]内蒙古大学生命科学院,内蒙古呼和浩特010021 [3]中国农业大学农业生物技术国家重点实验室,北京100094

出　　处：《华北农学报》2012年第2期12-18,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家科技支撑计划项目(2007BAD49B02-6);青岛农业大学人才基金(630915)

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。Based on the functional model of the hammerhead RNAs, a divalent ribozyme was designed for cleav- age of Potato leafroll virus（PLRV） RNA,targeting at two sites on the negative strand of PLRV Chinese isolate repli- case 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 eDNA subcloning of a PLRV Chinese isolate（PLRV-Ch） into plasmid pGEM-4Z,in which an 874 bp eDNA frag- ment 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.

关 键 词：二价核酶 马铃薯卷叶病毒 复制酶基因 体外切割 

分 类 号：Q78[生物学—分子生物学]