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.
出处
《热带作物学报》
CSCD
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)
作者简介
庹德财（1986年-）男，硕士研究生。研究方向：农业生物技术。
通讯作者：沈文清．E—mail：Swtdna@126．com。
通讯作者：周鹏，E-mail：zhp6301@126．com
