大麦黄矮病毒(BYDV) cDNA的合成、克隆及初步应用

 以大麦黄矮病毒二叉蚜和麦长管蚜专化株的病毒核酸为模板,以小牛胸腺DNA为引物,合成cDNA的第一条链,再用缺口翻译法合成第二条链,然后采用加装BamH₁人工接头的方法将ds-cDNA插入到质粒载体pUC₈中,重组质粒于大肠杆菌JM—83中进行克隆,以克隆的颜色变化选择含有外源DNA的克隆,再用病毒核酸制备的探针筛选真正病毒cDNA插入的克隆。重组质粒中ds-DNA的插入长度在300—1600bp之间。用缺口翻译法制备质粒DNA分子探针检测同源病毒液,反应灵敏度在100pg-1ng之间。应用cDNA探针检测不同病毒和病毒株系,从中筛选出黄矮病毒株系专化克隆系,黄矮病毒专化克隆系和黄矮病毒组专化克隆系.

SYNTHESIS,CLONING AND APPLICATION OF cDNA FROM BYDV-RNA

The first strand of the cDNA was synthesized by using BYDV-RNA as temolate and calf thymus DNA hydrolysate as random primer.The second strand was obtained according to nick-translation procedure with slight modification.ds-cDNA With a BamHl linker was inserted into a plasmid pUC₈.The plasmid was then used to transform E.coli strain JM-83 and colonies containing cDNA inserts were identified by screening on α-gal substrate.Colony and blot hybridizations using ³²P-labelled BYDV-RNA fragments confirmed the inserts were of viral origin.Analysis of some clones suggested that the length of the inserted fragments ranges from 300bp to 1600bp.The sensitivity of dot blot assay of plasmid DNA was between 100pg-lng.Some viruses of luteovirus group and some strains of BYDV could be identified by using cDNA probe.