一步法RT-PCR扩增PRSV海南分离物全长基因组cDNA  

Amplification of Full-length cDNA of Papaya Ringspot Virus of Hainan Strain

作　　者：庹德财[1,2] 沈文涛[2] 高乐[1,2] 言普[2] 周鹏[2] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《热带作物学报》2011年第7期1347-1351,共5页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(No.30760134;31000844;31171822);国家科技支撑计划课题(No.2009BADA2B02-04);中央级公益性科研院所基本科研业务费(ITBB110211)

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、B 2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.

关 键 词：番木瓜环斑病病毒 全长基因组cDNA 克隆与测序 

分 类 号：Q78[生物学—分子生物学]