柑橘黄化脉明病毒基因组的长链RT-PCR扩增、克隆及序列分析  

Long RT-PCR,Cloning and Sequencing of Full-length Genome of Citrus yellow vein clearing virus

作　　者：崔甜甜[1] 王艳娇[1] 宾羽 李中安[1] 周常勇[1] 宋震[1] 

机构地区：[1]西南大学/中国农业科学院柑桔研究所,国家柑橘工程技术研究中心,重庆400712

出　　处：《园艺学报》2017年第5期944-952,共9页Acta Horticulturae Sinica

基　　金：国家公益性行业(农业)科研专项(201203076);重庆市两江学者计划项目;重庆市基础及前沿研究项目(CSTC2014jcyj A80033);中央高校基本科研业务专项(XDJK2017B024)

摘　　要：为了建立柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)基因组长链RT-PCR扩增体系,构建其全长cDNA克隆,为深入了解CYVCV分子特性及其致病机理奠定基础。根据GenBank中CYVCV基因组序列和5′RACE扩增结果设计引物。以感染CYVCV的代代酸橙(Citrus aurantium‘Daidai’)植株总RNA为模板进行长链RT-PCR扩增,得到CYVCV全基因组cDNA,克隆至pGEM-Teasy载体,并进行序列测定与分析。结果显示,建立了一步扩增CYVCV全基因组的长链RT-PCR方法,扩增出约7.5 kb的目标片段;所获得的4个CYVCV全基因组cDNA序列与GenBank中已登录的相关毒株的核苷酸序列同源性为93%~99%。In order to clone full-length cDNA of Citrus yellow vein clearing virus （CYVCV）, further understand the molecular characteristics of CYVCV and its pathogenic mechanism, a long RT-PCR system was established with optimizing conditions and specific primer designed according to the sequences of CYVCV in GenBank and the results of 5＇RACE （rapidamplification of eDNA ends） . After extraction of total RNA from the Citrus aurantium ＇Daidai＇ of the infected CYVCV and amplification the full-length genome of CYVCV by the long RT-PCR, the full-length of CYVCV cDNA was obtained and cloned into pGEM-T easy vector and sequenced. The results showed that the 7.5 kb full-length genome of Citrus yellow vein clearing virus was amplified successfully by the long RT-PCR. The homology of nucleotide sequences of the four amplified full-length eDNA were 93% - 99% to the strains in GenBank.

关 键 词：柑橘黄化脉明病毒 RACE 全长CDNA 长链RT-PCR 

分 类 号：S666[农业科学—果树学]