侵染上海番茄的黄瓜花叶病毒及其卫星RNA的32P分子标记杂交检测及分子鉴定

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。

ISOTOPIC DIAGNOSIS AND MOLECULAR IDENTIFICATION OF CUCUMBER MOSAIC VIRUS AND SATELLITE RNA INFECTING TOMATO IN SHANGHAI

In summer of 2004 and 2005,typical viral disease symptoms were found on field tomato from Shanghai,which remarkably reduced the yield of tomato.Total RNA of tomato leaves and purified virions were detected by hybridization with()~(32)P probes conducted with partial sequence of CMV RNA3 and full cDNA of CMV satRNA.Viruses were also confirmed by analyzing dsRNA extracted from tomato leaves.Full sequence of CMV RNA3 was gained by RT-PCR and the result of sequencing indicated that genomic RNA3 belongs to subgroup Ⅱ.Two 15nt complementary ssDNA primers were ligated into double stranded satRNA.Complete sequence of satRNA was obtained by RT-PCR using these 15nt ssDNA as amplification primers.Phylogenetic analysis showed that the identity of this 383nt satellite and some documented satRNAs was 72.6 to 99.5% at the nucleotide level.Several mutation sites were found at the 3′terminus of the newly discovered satRNA.By Isotopic diagnosis and molecular Identification,variation of CMV and its satRNA were found in tomato from Shanghai,which may influence the viral disease prevalence and the emergence of new symptom.