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当前位置: 首页 > 文章 > 马铃薯纺锤块茎类病毒山西分离物侵染性克隆的构建 山西农业科学 2012,40 (7) 701-704
Position: Home > Articles > Construction of Infectious Clone for Potato Spindle Tuber Viriod Shanxi Isolate Journal of Shanxi Agricultural Sciences 2012,40 (7) 701-704

马铃薯纺锤块茎类病毒山西分离物侵染性克隆的构建

作  者：
白云凤;闫建俊;张耀;张忠梁;冯瑞云;张维锋
单  位：
农业部黄土高原作物基因资源与种质创制重点实验室;山西省农业科学院作物科学研究所
关键词：
马铃薯纺锤块茎类病毒;山西分离物;序列分析;体外转录;侵染性克隆
摘  要：
马铃薯纺锤块茎类病毒(Potato spindle tuber viriod,PSTVd)侵染马铃薯会严重影响薯块的产量和品质。根据PSTVd的同源序列设计引物,以山西省马铃薯感病块茎中提取的总RNA为模板,经RT-PCR方法扩增出全长cDNA片段,将其克隆到质粒pBS-T载体上,进行序列分析。结果表明,该类病毒全长359 nt,能通过分子内序列互补形成致密的二级结构。以该序列作为种子序列进行Blast搜索,该序列与PSTVd荷兰分离物(GenBank登陆号:AY372400.1)的序列一致性为100%。将该序列与东北株系、河北株系及已公布的弱株系(GenBank登陆号:M14814.1)、强株系(GenBank登陆号:U23058.1)、中间株系(GenBank登陆号:AY937179.1)比对,有12个核苷酸具有多态性,其中有5个发生在致病区。将线性化的质粒pBS-PSTVd及其PSTVd的体外转录产物接种番茄矮红宝的无毒苗,20 d后接种株轻微显症,RT-PCR检测均呈阳性。将RT-PCR产物测序,其结果与接种的PSTVd原序列完全一致,证实构建的PSTVd山西分离物的克隆具有侵染性。
译  名：
Construction of Infectious Clone for Potato Spindle Tuber Viriod Shanxi Isolate
作  者：
BAI Yun-feng,YAN Jian-jun,ZHANG Yao,ZHANG Zhong-liang,FENG Rui-yun,ZHANG Wei-feng(Key Labotary of Loess Plateau Crop Gene Resources & Germplasm Creation of Agriculture Ministry,Institute of Crop Sciences,Shanxi Academy of Agricultural Sciences,Taiyuan 030032,China)
关键词：
potato spindle tuber viriod;Shanxi isolate;nucleotide sequence analysis;external transcription;infectious clone
摘  要：
Potato spindle tuber viriod(PSTVd) could cause serious impact on yield and quality in potato.A pair of primers were designed and synthesized based on PSTVd homologue sequences.Using the total RNA of infected potato tubers from Shanxi Province as a template,full length cDNA fragments was amplified by RT-PCR method.The fragment was cloned into pBS-T vector and sequenced.It showed that cloned PSTVd-SX isolate was 359 nt in size and could produce a self-complementary rod-shaped secondary structure.Blast search showed that PSTV-SX isolate shared 100% identity with PSTVd-H isolate(GenBank accession:AY372400.1).Sequence alignments showed that 12 single nucleotide polymorphisms(SNPs) existed among PSTVd-SX and other five strains,including PSTVd-DB,PSTVd-HB,PSTVd-M(GenBank accession:M14814.1),PSTVd-S(GenBank accession:U23058.1),PSTVd-I(GenBank accession:AY937179.1),five of which existed in pathogenicity region.The viroid-free tomato plants were inoculated with the PSTVd-SX transcripts in vitro and the plasmid pBS-PSTVd linearized with BamHI respectively.PSTVd was detected by RT-PCR in all inoculated plants 20 d later.It showed that the sequences of RT-PCR products shared 100% identity with PSTVd-SX inoculations.

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