黄瓜花叶病毒自然重组株Tsh基因组RNA的功能研究

Indentificating Genomic RNA Functions of a Cucumber Mosaic Virus Intraspecific Reassortant Strain Tsh

对一株黄瓜花叶病毒(cucumber mosaic virus,CMV)亚组IA和亚组II间的天然假重组体Tsh-CMV的RNA2、RNA3和亚组I标准株Fny-CMV的RNA1、RNA2及RNA3进行人工交叉重组获得一系列人工假重组体,分别为F1F2F3-CMV,F1F2T3-CMV,F1T2F3-CMV和F1T2T3-CMV。各个假重组体接种于心叶烟(Nicotiana glutinosa.)后,表型存在明显差异,其中F1F2T3-CMV致病性最严重,严重矮化,黄化及丛簇;F1T2T3-CMV致病性次之,矮化,局部性枯斑及丛簇;继而为F1F2F3-CMV;F1T2F3-CMV症状最轻,仅表现为轻微花叶。Northern杂交结果显示:各假重组体病毒侵染心叶烟7 d,14 d,21 d和28 d后,F1F2F3-CMV,F1F2T3-CMV,F1T2F3-CMV和F1T2T3-CMV的基因组含量在7 d时含量较高,在14 d时含量最高,此后21 d和28 d检测到CMV基因组含量相对较少且呈逐渐下降趋势。F1F2F3-CMV和F1T2F3-CMV的基因组含量比F1F2T3-CMV和F1T2T3-CMV高,杂交结果显示其症状的轻重与CMV基因组含量无关。本研究重现了CMV不同亚组之间基因组RNA的假重组过程。

Tsh-CMV,an intraspecific reassortant between Cucumber mosaic virus（CMV） subgroup IA and Ⅱ,was obtained from natural-infected tomato collected from Shanghai,China.Infectious cDNA clones of Tsh-CMV RNA2 and RNA3 were been constructed and infectious RNA transcripts of Tsh-CMV RNA2 and RNA3 were generated by in vitro transcription.Along with the RNA transcripts of Fny-CMV,Tsh-CMV RNA transcripts were combined to reform wild type F1F2F3-CMV and to form the pseudorecombinants between Fny-CMV and Tsh-CMV RNA2 and RNA3,namely F1T2T3,F1F2T3,F1T2F3.Each pseudorecombinant was inoculated onto seedings of Nicotiana glutinosa.Symptoms developed on N.glutinosa indicated that F1F2T3 was the most severe pathogenically,inducing stunt,severe mosaic,chlorosis and rosette,while F1T2T3 caused less severe symptoms including stunt,local necrotic spot and rosette,and F1F2F3 caused typical mosaic,distortion and leaf linearization,but F1T2F3 only caused mild mosaic.The result of Northern blot indicat-ed that F1F2F3-CMV and F1T2F3-CMV showed similar levels of viral RNA accumulation at 7 and 14 days postinoculation（dpi）,the accumulation of RNA declined at 21 and 28 dpi.In leaf tissues of N.glutinosa,the accumulation levels of genomic RNAs for Fny-CMV and F1T2F3-CMV were higher than those of F1F2T3-CMV and F1T2T3-CMV.Thus,it could be concluded that the pathogenicity of CMV strains was not correlated with the accumulation of genomic RNAs.Our present report has presented the artificial pseudorecombination between subgroups of CMV.