一步法RT-PCR扩增PRSV海南分离物全长基因组cDNA

摘要

以植物总RNA提取试剂盒提取的高纯度的总RNA为模板,使用Oligo dT-Adaptor和Random9引物合成了番木瓜环斑病病毒海南分离物(PRSV HN-2)的cDNA第一链,基于已报道的PRSV全长基因组序列,设计合成了一对引物,一步法RT-PCR扩增出PRSV海南分离物全长基因组cDNA.为了验证获得的全长基因组cDNA的正确性,并以扩增出的全长cDNA为模板,将PRSV全长基因组分成A、B2个大片段进行扩增,应用T载体进行克隆和测序以验证所获得的全长基因组cDNA序列的正确性.测序结果显示,该cDNA序列与国内外报道的PRSV各分离物全长核苷酸序列的相似性很高,表明本文建立的一步法RT-PCR扩增PRSV全长基因组cDNA的方法正确、可行.%To determine the complete nucleotide sequence of Papaya ringspot virus of Hainan isolate, an efficient method was developed to amplify the full-length cDNA of PRSV. Total RNA was extracted by RNAprep Pure Plant Kit from susceptible papaya leaves. The first strand synthesis using Oligo-dT and random nine primers was carried out, which catalyzed by AMV Reverse Transcriptase. The full-length cDNA of PRSV was amplified by using an optimized PCR. Both A and B segment were generated and cloned using the amplified full-length cDNA segment as the template. The sequence of the full-length of PRSV of Hainan isolate was given in this paper.