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

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%。

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

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 cDNA 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 cDNA were 93％-99％ to the strains in GenBank.