中文摘要：

【目的】鉴定引起辣椒产生褪绿黄化症状的病原物,构建侵染性克隆。【方法】大田辣椒样品通过ELISA检测,结合病毒外壳蛋白SDS-PAGE及病毒RNA分析,初步确定辣椒中病原物为黄瓜花叶病毒（CMV）Phy株系。以辣椒病毒粒子RNA为模板,采用含T7启动子的不同正向引物通过RT-PCR扩增CMV-Phy全长基因组RNA1、RNA2和RNA3。PCR产物经过双酶切后连接到pUC118载体,并分别比较5种（DH5α、HB101、JM109、LE392和NM522）感受态细胞的转化效率。体外转录CMV-Phy的基因组cDNA克隆（pUC-P1、pUC-P2和pUC-P3）成RNA分子（P1P2P3）,分析其转录效率和侵染活性。P1P2P3与CMV的卫星RNA进行假重组,进一步确定CMV-Phy侵染性克隆的成功构建。【结果】引起辣椒产生褪绿黄化症状病原物为CMV,携带卫星RNA;心叶烟接种辣椒病毒粒子后同样产生褪绿黄化症状。HB101感受态细胞最适合CMV-Phy基因组转化。CMV-Phy基因组及其卫星RNA的大小如下：RNA1为3356nt、RNA2为3048nt和RNA3为2220nt,卫星RNAPz-satRNA为384nt（序列登陆号分别为：DQ402477,DQ412731,DQ412732EF363688）。CMV-Phy的cDNA克隆体外转录在5′端添加G有利于提高转录效率,但影响其侵染活性;P1P2P3在苋色藜和心叶烟产生的症状与其病毒粒子产生的症状相一致。除了Pz-satRNA,P1P2P3还能作为T1-satRNA、Rs-satRNA和Tsh-satRNA辅助病毒;T1-satRNA可加重CMV-Phy在心叶烟症状反应,而其它3个卫星RNA则对此起减弱作用。【结论】以病毒粒子RNA为模板,采用touch-upPCR扩增参数在1个反应管中同时获得CMV-Phy基因组RNA1、RNA2和RNA3;CMV-PhyRNA1、RNA2和RNA3的5′端添加1个G最有利于侵染性克隆构建。

英文摘要：

[ Objective ] In this study, the pathogen which caused chlorosis symptom on Capsicumfrutescens was confirmed as CMV-Phy, and infectious clones for CMV-Phy were constructed. [Method] Serological ELISA, SDS-PAGE and viral RNAs analysis were adopted to identify the virus in the seedlings of C.frutescens. With different forward primers containing T7 promoter at 5′ terminal, full length of RNA1, RNA2 and RNA3 or satRNA were amplified by RT-PCR, using CMV-Phy viral RNA as template. PCR products of CMV-Phy RNA1, RNA2 and RNA3 were ligated into pUC 118 vector, respectively. Transformation efficiency among DH5α, HB101, JM109, LE392, and NM522 E. coli competent cell was compared, cDNA clones of CMV-Phy genomic RNAs （pUC-P1, pUC-P2 and pUC-P3） were transcribed into RNA in vitro and RNA transcripts were mechanically inoculated onto Nicotiana glutinosa. In order to further prove that infectious clones were successfully constructed, psuedorecombination between CMV-Phy RNA transcripts and CMV-satRNA was analyzed. [Result] The virus in C. frutescens tissues was CMV-Phy, which harboring a satRNA and N. glutinosa also expressed chlorosis symptom when inoculated with CMV-Phy viroin isolated from the C. frutescens. RNA1, RNA2 and RNA3 of CMV-Phy were obtained by RT-PCR. Only HB101 competent cell was suitable to pUC-P1 transformation, but all the five kinds ofE. coli were fit for pUC-P2 and pUC-P3 transformation. Full length of RNA1, RNA2, RNA and Pz-satRNA was 3356, 3048 2220, and 384 nt, respectively （accordingly Accession Number： DQ402477, DQ412731, DQ412732） In vitro transcription efficiency improved but RNA transcripts was no activity when guanosine was added to 5′ end of RNA 1, RNA2 and RNA3 cDNA clones. Biological assays of CMV-Phy infectious RNA （P1P2P3） showed that symptom induced by P1P2P3 was the same as that induced by CMV-Phy virion. Besides Pz-satRNA, P1P2P3 could support replication of TlsatRNA, Rs-satRNA and Tsh-satRNA in N. glutinosa. The presence of TI-satRNA aggravated symptom induced by P1P2P3 w