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2004和2005年夏季,在上海郊区露天栽培的番茄地出现典型的病毒病危害,并造成严重的产量损失。分别以32P标记的CMV基因组RNA3部分序列和卫星RNA的cDNA探针对自然感病的番茄叶组织和提纯的病毒粒子中提取的总RNA进行杂交检测,确定以上植物组织和病毒粒子中均具有CMV及卫星RNA。dsRNA分析确定自然感病叶组织中具有典型黄瓜花叶病毒(CMV)基因及其卫星RNA条带。以常规引物对感病植物的总RNA进行RT-PCR扩增,获得CMV RNA3全长克隆,经测序显示该RNA3序列属于CMV亚组Ⅱ;通过在卫星dsRNA两端分别加上15nt的单链DNA接头,以接头互补序列为引物进行扩增获得一个383nt的卫星RNA的全长序列。同源性分析结果显示其与已报道的CMV卫星RNA同源性为72.6%~99.5%,但其3′末端存在多个碱基的突变。根据同位素杂交检测及分子鉴定,CMV及其卫星RNA变异类型在上海自然感病番茄上发生和普遍存在,可能对病害流行和新的症状出现产生影响。
In the summer of 2004 and the summer of 2005, a typical virus disease occurred in open-air tomato plants in the suburbs of Shanghai and resulted in serious yield losses. The 32P-labeled CMV genomic RNA3 partial sequences and satellite RNA cDNA probes were used to detect the total RNA extracted from the naturally infected tomato leaf tissue and the purified virions respectively. The results showed that all of the above plant tissues and virions had CMV And satellite RNA. dsRNA analysis identified a typical cucumber mosaic virus (CMV) gene and its satellite RNA band in naturally infected leaf tissue. The total RNA of CMV RNA3 was amplified by RT-PCR using conventional primers. The full-length cDNA of CMV RNA3 was sequenced and showed that the RNA3 sequence belonged to CMV subgroup Ⅱ. A 15 nt single-stranded DNA Linker, and the complementary sequence of the linker was used as a primer to amplify the full length sequence of a 383nt satellite RNA. Homology analysis showed that the homology with the reported CMV satellite RNA was 72.6% ~ 99.5%, but there were multiple base mutations at the 3 ’end. According to the isotope hybridization detection and molecular identification, CMV and its satellite RNA variant types occur and prevail in naturally infected tomato in Shanghai, which may affect the epidemic and new symptoms.