[Synthesis and molecular cloning of the cDNA of TuMV-RNA]

TuMV particles were purified from artificially infected leaves of Brassica juneaen, and TuMV-RNA was extracted from these particles. The virus thus purified showed typical nucleoprotein 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/A280 was 2.2. The RNA was then used as template of 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 500-4300 bp. The ds-cDNA repaired by Klenow fragment was inserted into the Small site of pUC19 by blunt-end ligation. Then the recombinant molecules were used to transform E. coli strain DH5 alpha. Rapid electrophoresis of plasmid prepared by alkali method, EcoRI-HindIII digestion and in situ hybridization with 32P labeled TuMV-RNA showed that inserted fragments were with various sizes and complement to TuMV-RNA.