【摘要】 本文采用改进的新技术从大麦条纹花叶病毒核酸合成互补脱氧核糖核酸,重组质粒及选择特异性克隆。由于技术的改进,简化了操作程序,提高了工作效率。我们以大麦条纹花叶病毒三个组分核酸混合物为模板,寡（dT）8为引物,合成cDNA第一条链,用Gubler和Hoffman缺口翻译法合成第二条链,采用加HindⅢ人工接头的方法将cDNA插入pUC 9质粒载体上,于大肠杆菌JM-103中进行克隆,并以克隆颜色变化表示有无β-半乳糖苷酶活性的直观法,选择含有外源DNA的克隆。再以克隆原位分子杂交法检查克隆对病毒RNA各组分的特异性。仅对RNA₁或RNA₂有特异性的克隆,分别称为pBV₁和pBV₂,另有相当数量的克隆既与RNA₂也与RNA₃杂交,所以称为pBV2+3。用HindⅢ内切酶对一部分RNA₃杂交阳性的PBV2+3进行分析表明,插入的外源基因长度在500～1,000碱基对之间。文中讨论了RNA₂与RNA₃存在同源的可能性。更多还原

【Abstract】 A simple and efficient method for synthesizing and cloning the ds-cDNA of barley strip mosaic virus RNAs ( BSMV-RNA ) is presented.The first strand of the cDNA was synthesized by using the mixture of BSMV-RNA 1, 2, 3 as emplatte and oligo ( dT ) as primer.The second strand was obtained according to Gubler and Hoffman’s nick-translation procedure with slight modification.cDNA with a Hindi linker was inserted into the single HindⅢ site of pUC9.The white, clones containing foreign DNA fragment were picked out, monitored and selected by hybridization with the probe, a mixture of 32P-labelled RNA 1, 2, 3. All the selected clones were sorted further by respective hybridization with individual 32P-labelled RNA probe purified by PAGE.The pBV1 and pBV2 hybridized with RNA1 and RNA2 respectively, while in the case of pBV2 + 3, with both RNA2 and RNAS.The possible partial homology existance between RNA2 and RNA3 is discussed .Analysis of some clones hybridized with RNA3 suggested that the length of the inserted fragments ranges from 500 to 1;000bp.更多还原