Restriction endonuclease Mapping of the homologous chromosomal region of nicotiana tabacum with the rolC gene (TL-DNA ORF12) in agrobacterium rhlC Gene (TL-DNA ORF12) in agrobacterium rhizogenes Ri plasmid

Abstract

The rolC gene in Agrobacterium rhizogenes Ri plasmid behaves as oncogene in plant. As compared with the viral oncogenes, host genomic counterpart of the rolC gene was searched for in Nicotiana tabacum. The rolC gene contained in HindIII-EcoRI fragment of pPCV002-C in Agrobacterium tumefaciens (GV3101) was transferred into the corresponding site of pUC19 and constructed a new plasmid pYTS-C. The 1kb HpaI-EcoRI fragment excised from pYTS-C included the rolC gene and was used as a probe in hybridization analysis and in screening libraries. Before screening libraries, the existence of homologous region in several plant chromosomal DNAs with the rolC gene was demonstrated by slot blot hybridization. One of samples shown positive signals, Nicotiana tabacum``s lambada genomic library was obtained. Screening of recombinant bacteriophage was then carried out by in situ plaque hybridization with $^{32}P$-labeled rolC-containing fragment. As a result of first screening, 15 positive recombinant plaques were obtained. Ten positive plaques were randomly selected and were performed up to tertiary screeing. Two of them were restriction endonuclease mapped.