马铃薯纺锤块茎类病毒山西分离物侵染性克隆的构建

Construction of Infectious Clone for Potato Spindle Tuber Viriod Shanxi Isolate

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。

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 homologne 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 serf-complementary rod-shaped secondary structure. Blast search showed that PSTV-SX isolate shared 100% identity with PSTVd-H isolate（ GenBank accession： A^372400.1 ）. 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： M 14814.1 ）, 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 insulated plants 20 d later. It showed that the sequences of RT-PCR products shared 100% identity with PSTVd-SX inoculations.