陕北地区马铃薯Y病毒的RT-PCR检测及其序列分析

以陕北地区8个县(区)种植2~3代疑似带毒的马铃薯叶片为材料,Trizol法提取马铃薯叶片总RNA,以已公布的马铃薯Y病毒外壳蛋白(coat protein,CP)基因序列设计1对特异引物,反转录合成cDNA,RT-PCR扩增目的 DNA片段,1.5%的琼脂糖凝胶电泳检测扩增产物。回收纯化CP基因片段并进行克隆和测序,采用DNAstar软件分析序列一致性。结果显示,所有马铃薯叶片中均扩增到与预期大小一致的、长度为400 bp的目的片段,表明这些马铃薯均感染了Y病毒;陕北8个县(区)马铃薯Y病毒CP基因与国内外其他地区12个样品之间的序列一致性为88.4%~99.8%,表明马铃薯Y病毒的CP基因序列比较保守,不易发生变异。RT-PCR方法可快速、准确地检测马铃薯Y病毒,从而为马铃薯茎尖剥离脱毒生产脱毒种苗(薯)提供依据。

RT-PCR Detection and Sequence Analysis of Potato Virus Y in Northern Shaanxi

Taking suspected poison potato leaves of 2-3 generation as materials of eight counties/prefectures in northern Shaanxi and the total RNA were extracted by Trizol method, cDNA was synthesized and the DNA fragment was amplified by RT-PCR, the amplified product was detected by 1.5% agarose gel electrophoresis. then the purified PCR products was cloned and sequenced, and sequence identity analysis was conducted with DNAstar software. The electrophoresis results show that, eight samples of RT-PCR in line with the expected size of 400 bp fragments amplified and these potatoes are infected with potato virus Y. The sequence of CP gene of potato virus Y is more conversation, not so easy to variation, and sequence identity have been reported in the 88.4% to 99.8% and other12 areas at home and abroad. The potato virus Y could be detected by RT-PCR rapidly and accurately to provide a reference for the promotion of detoxification potato by meristem stripped.