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甜菜黄脉坏死病毒RNA3和RNA4全长cDNA的合成是分别以Oligo-d(T)_(15)为引物合成第一链cDNA,cDNA的第二链的合成则分别应用RNA3和RNA4特异引物完成。全长双链cDNA分别克隆在pGEM3Zf(+)载体中,置于噬菌体Sp6启动子控制下。在体外以质粒DNA为模板应用“run off”系统及Sp6RNA Polymerase成功地合成了大量的具有高度生物活性的转录产物RNA3和RNA4。在两种转录反应系统中,以第一种方法即转录与加帽(capping)分步进行转录效率高,第二种方法(转录与加帽同时进行)转录效率较低,但转录产物侵染性更高。虽然在RNA3和RNA4转录产物的5′末端和3′末端分别含数目不同的非病毒核酸序列,但转录产物的活性并未受到显著的影响。应用这种具有高度生物活性的体外转录产物与该病毒Rg1分离物机械接种甜菜幼苗根毛,首次阐明了RNA3是导致甜菜丛根病的主要因素。
The synthesis of the full-length cDNA of RNA3 and RNA4 of Beet Huang vein necrosis virus was the synthesis of the first strand cDNA with Oligo-d (T) _ (15) as the primer, and the synthesis of the second strand of the cDNA by RNA3 and RNA4 specific primers . The full-length double-stranded cDNA was cloned separately in the pGEM3Zf (+) vector and placed under the control of the bacteriophage Sp6 promoter. A large number of highly biologically active transcripts, RNA3 and RNA4, were successfully synthesized using in vitro plasmid DNA as a template using the “run off” system and Sp6 RNA Polymerase. In both transcriptional response systems, the transcription efficiency was high in the first method, ie capping transcription and capping. The second method (transcription and capping simultaneously) was less efficient in transcription, but the transcripts were infested Sex is higher. Although different numbers of non-viral nucleic acid sequences were present at the 5 ’and 3’ ends of RNA3 and RNA4 transcripts respectively, the activity of the transcripts was not significantly affected. Using this highly biologically active in vitro transcript and mechanical inoculation of the root beard of sugar beet seedlings with Rg1 isolate of the virus, it was first demonstrated that RNA3 is a major contributor to root disease of sugar beet.