摘要
从接种芜菁花叶病毒后发病芥菜(Brassica Juneaen)中提取病毒,然后提取其核酸(TuMV-RNA)。以纯化的TuMV-RNA为模板,以Oligo(dT)_(12-18)及小牛胸腺DNA水解物为引物,合成双链cDNA,双链cDNA长度约500—4300bp。将双链cDNA补齐后,钝端连接到pUC19质粒的Smal位点,转化E.coli DH,获得500多个白色克隆。菌落原位杂交及酶切分析表明,重组质粒中的插入片段大多数为芜菁花叶病毒RNA的互补DNA,长度为500—4000bp。
TuMV particles were purified from artificially infected leaves of Brasslca juneaen, and TuMV-RNA was extracted from these particles. The virus thus purified showed typical nucleo-protein absorbent peak under UV light scanning, and the ratio of A260/A280 was 1.21. The RNA thus prepared showed typical ribonucleic acid absorbent peak, and the ratio of A260/A230 was 2.2. The RNA was then used as template and oligo (dT) 12-18 together with the end product of DNase digested calf thymus DNA were used as primer for the synthesis of cDNA. The length of double stranded cDNAs synthesized were distributed continuously with the sizes between 500-4300bp. The ds-cDNA repaired by Klenow fragment was inserted into the Smal site of pUC19 by blunt-end ligation. Then the recombinant molecules were used to transform E. coli strain DH5α. Rapid electrophoresis of plasmid prepared by alkali method, EcoRI-HindⅢ digestion and in situ hybridization with32P labeled TuMV-RNA showed ihat inserted fragments were with various sizes and complement to TuMV-RNA.
关键词
芜菁花叶病毒
CDNA
分子克隆
TuMV, cDNA, Molecular cloning, in situ hybridization
