福建省马铃薯纺锤块茎类病毒快速检测及序列分析

Detection and Sequence Analysis of Potato Spindle Tuber Viroid Found in Fujian

为检测福建省马铃薯纺锤块茎类病毒及了解其分子变异情况,以感染马铃薯纺锤块茎类病毒的植株为试验材料,提取RNA,反转录cDNA,利用设计合成的特异性引物进行RT-PCR检测,扩增产物经回收纯化后克隆和测序。结果表明,PCR扩增出359bp的特异片段,将扩增产物经回收和纯化并进行克隆与测序,测序结果经BLAST比对后,该片段为PSTVd基因序列,PSTVd分离物命名为Fujian PTSTd(KM588065.1),与国内外报道的PSTVd分离物同源性达98%以上。该分离物与弱毒株系(M14814.1)、中间株系(AY492084.1)和强毒株系(U23058.1)比对,有11个核苷酸具有多态性,其中5个发生在致病区。不同国家和地区序列进化树分析表明,该分离物与我国北方及欧美国家分离物亲缘关系最近,与新西兰分离物亲缘关系最远。

A new methodology was developed for etection and determination of the molecular variation of the potato spindle tuber viroid found in Fujian.A pair of primers was designed and synthesized based on the STVd gene isolated and amplified from the extracted viral RNA with RT-PCR.The specific fragment of 359 bp was isolated from the infected plants,but none from the healthy tissue samples.The fragment was amplified and subjected to validation by agarose gel electrophoresis.Then,the purified PCR product was cloned,sequenced and Blast searched to be identified as PSTVd isolate KM588065.1.The isolate shared 98% homogeneity with that from NCBI data bank.Comparing the sequence alignments of 3 other strains,the isolate appeared to contain 11 nucleotide polymorphisms with 5in the pathogenicity region.Based on the phylogenetic tree analysis on the DNA sequences of PSTVd from different countries and regions,this virus isolate was determined to be closely related to those from northern China,Europe and America,and only remotely related to that from New Zealand.