应用焦磷酸测序鉴定马铃薯感染的马铃薯Y病毒株系

Identification of Potato virus Y Strains in Infected Potato Plants by Pyrosequencing

为了检验是否可以通过焦磷酸测序法进行短序列测序,来鉴别马铃薯Y病毒(PVY)株系,从NCBI库中选取了代表9个PVY株系的全基因组序列30个,通过比对P1和CP蛋白的氨基酸序列,分别找出一个差异区段,针对这2个区域设计了反转录引物、PCR引物和测序引物。分别从黑龙江省克山县和讷河市各采集了一份马铃薯病叶样品,通过提取总RNA,反转录,再进行PCR扩增获得扩增产物,并对扩增产物进行了焦磷酸测序。测序结果显示,尽管2份马铃薯病叶症状不同,但均感染了PVY(N-Wi株系)(PVYN-Wi)。上述研究表明,利用本试验设计的引物,通过焦磷酸测序法进行短序列测序,可以鉴定马铃薯感染的PVY株系。

In order to test whether pyrosequencing of short sequences can be applied to identify Potato virus Y （PVY） strains, thirty whole PVY genome sequences representing nine PVY strains were retrieved from the NCBI database. Sequences of their P1 and CP proteins were compared and a distinctly different region was found out from the sequence of each protein. Both regions were used to design primers for reverse transcription, PCR and pyrosequencing. Two potato leaf samples were collected from Keshan and Nehe of Heilongjiang Province of China. Total RNA was extracted. Reverse transcription and PCR were carried out. The PCR products were applied as templates in pyrosequencing. The pyrosequencing results showed that both leaf samples were infected by PVYN-w＇. This research confirmed that the primers we designed could be utilized to identify PVY strains in infected potato plants by pyrosequencing of short sequences.