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葡萄卷叶伴随3型病毒(Grapevine leafroll associated virus-3,GLRaV-3)和葡萄A病毒(Grapevine virus A,GVA)常常复合侵染部分主栽欧美杂交葡萄品种。以半定量RT-PCR与qRT-PCR方法研究了‘希姆劳特’葡萄(Vitis vinifera L.×V.labrusca L.‘Himrod’)中不同组织内病毒的相对积累量,结果表明叶脉和韧皮部内的病毒相对积累量显著高于其他组织。以成熟枝条韧皮部组织的cDNA为模板,优化了RT-PCR多重扩增体系,可以同时扩增待测样本中两种病毒的目标基因。从一株复合感染GLRaV-3与GVA的‘藤稔’葡萄(Vitis vinifera L.×V.labrusca L.‘Fujiminori’)中,分别克隆了GLRaV-3外壳蛋白(coat protein,CP)基因全长序列和GVA部分基因组区域序列,后者包括CP基因的部分序列与病毒RNA沉默抑制子(viral suppressor of RNA silencing,VSR)基因全长序列。病毒核酸同源性分析与系统进化研究表明,不同GLRaV-3分离物的CP高度保守;而‘藤稔’葡萄的GVA分离物包含多种变异株,具有准种病毒的特点。
Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) often co-infected part of the main European and American hybrid grape varieties. The relative accumulation of viruses in different tissues of ’Vitis vinifera L. × V. labrusca L.’Himrod’ was studied by semi-quantitative RT-PCR and qRT-PCR. The results showed that both the veins and phloem Within the relative accumulation of virus was significantly higher than other organizations. Using the cDNA of mature phloem tissue as a template, RT-PCR multiplex amplification system was optimized, which can simultaneously amplify the target genes of two viruses in the sample to be tested. The full length of the GLRaV-3 coat protein (CP) gene was cloned from a strain of Vitis vinifera L. × V. labrusca L.’Fujiminori ’co-infected with GLRaV-3 and GVA Sequence and the GVA partial genomic region sequence, which includes the partial sequence of the CP gene and the full-length sequence of the viral suppressor of RNA silencing (VSR) gene. Viral nucleic acid homology analysis and phylogenetic analysis indicated that the CPs of different GLRaV-3 isolates were highly conserved, whereas the GVA isolates of ’Fujiminori’ grape contained many variants and had the characteristics of quasispecies viruses.