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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 establish a long-chain RT-PCR amplification system of the genome of Citrus yellow vein clearing virus (CYVCV) and construct its full-length cDNA clone, it lays the foundation for further understanding of the molecular characteristics of CYVCV and its pathogenesis. Primers were designed based on the CYVCV genome sequence in GenBank and the results of 5’RACE amplification. The whole genome cDNA of CYVCV was amplified by RT-PCR from total RNA of Citrus aurantium ’Daidai’ infected with CYVCV. The full-length cDNA of CYVCV was cloned into pGEM-Teasy vector and sequenced and analyzed. The results showed that a full-length PCR amplification of CYVCV genome was established and a target fragment of about 7.5 kb was amplified. The four CYVCV genome sequences obtained were identical to those of the related nuclei in GenBank The nucleotide sequence homology is 93% ~ 99%.