葡萄卷叶伴随病毒-3复制酶基因克隆及其在大肠杆菌中的表达  

Cloning and expression in E. coli of grapevine leaf-roll associated virus-3 RdRp gene

作　　者：徐章逸[1] 王国平[1] 洪霓[1] 

机构地区：[1]华中农业大学植物科学技术学院,武汉430070

出　　处：《果树学报》2006年第2期252-255,共4页Journal of Fruit Science

基　　金：科技部863课题资助项目(2001AA241142)。

摘　　要：以感染有葡萄卷叶伴随病毒-3(Grapevineleafroll-associatedvirus-3,GLRaV-3)的葡萄韧皮部组织中提取到的dsRNA为模板,利用RT-PCR和重叠延伸PCR技术,对GLRaV-3的复制酶(RNA-dependentRNApolymerase,RdRp)基因进行克隆,获得了预期大小的目标片段。将此片段克隆到载体pBluescriptSKⅡ,酶切鉴定后测序。测序结果表明:RdRpcDNA全长为1618bp。经BLAST分析推导其可能编码538个氨基酸,与报道的GLRaV-3美国分离物NY1RdRp核苷酸相似性为99.3%,氨基酸相似性为99.6%;推导的氨基酸序列与其同属的PMWaV-2、GLRaV-1和LChV-2的RdRp氨基酸序列相似性分别为53%,50%和38%,包含了GDD在内的8个保守基元序列。将该段RdRp连接到表达载体pET22b(+)上,获得的重组子pET-RdRp转化大肠杆菌BL21(DE3)plysS后,用1mmol/L的IPTG进行诱导。SDS-PAGE分析表明,RdRp在大肠杆菌中被诱导表达,融合蛋白分子量约为61kDa。The dsRNA was extracted from scraped phloem tissue of grapevine canes infected by grapevine leafroll-associated virus-3（GLRaV-3） and used as template for cDNA synthesis. The RNA-dependent RNA polymerase （RdRp） gene of GLRaV-3 was cloned and sequenced. The complete gene consists of 1618 nucleotides and encodes a polypeptide consisting of 538 amino acids（aa）. Comparison of the sequences with those of the isolate NY1 of GLRaV-3 showed 99.3% similarity in nucleotide sequence and 99.6% similarity in amino acid sequence. Alignment of the polypeptide with RdRp of pineapple medybug wilt associated virus-2（PMWaV-2）, grapevine leaf roll associated virus-1（GLRaV-1） and little cherry virus-2 （LCV-2） which were in the same genus with GLRaV-3 revealed the presence of eight conserved motifs including GDD. The similarities between RdRp of GLRaV-3 and that of PMWaV-2, GLRaV-1 and LCV-2 were 53%, 50% and 38%, respectively. The recombinant plasmid pET22b containing RdRp was transformed into E. coli BL21 （DE3） plysS. Results of SDS-PAGE showed that specific expression of a 61 kDa fusion protein was achieved by the inducing of 1mmol/L IPTG.

关 键 词：葡萄卷叶伴随病毒-3 复制酶 反转录-聚合酶联反应 原核表达 

分 类 号：S663.1[农业科学—果树学]