Cloning, characterization and gene regulation studies with Rol genes of Ri plasmid of Agrobacterium rhizogenes.

摘要

The rol loci of the root-inducing (Ri) plasmid of Agrobacterium rhizogenes that control the root induction and growth response in hairy root disease were identified, and their biological effects in transgenic plants were studied. RolB was required for root induction on Kalanchoe diagremontiana and Nicotiana tabacum var. xanthi leaves. When rolB and rolC genes were present together, more roots were produced than with rolB alone. RolC or rolA alone were unable to direct root induction. However, rolA influenced root development. Tobacco plants containing rol genes were analyzed for hairy root syndrome and rooting response. Plants containing rolA, B and C genes displayed the typical symptoms of hairy root syndrome. Among single genes, rolB was primarily associated with increased adventitious rooting and floral hyperstyly, while rolC was required for the vigorous in vitro growth of transformed tobacco roots (hairy roots). RolC also directed distinct abnormalities in transgenic tobacco plants (wrinkled leaves, short plants, small flowers with hyperstyly, and reduced fertility) compared with normal plants or plants with rolB. Although each of the three genes induce independent developmental abnormalities, their combined effects were needed to obtain complete hairy root phenotype.; Kalanchoe leaf cells require both auxin-biosynthetic genes (TR-DNA) and rolB gene with an extended upstream region (1180 bp) at the 5{dollar}spprime{dollar} end of the gene for root induction. The rolB gene with a short 5{dollar}spprime{dollar} end (305 bp) including TATA and CCAAT boxes was not sufficient to trigger rooting. Both 1180 bp and 305 bp upstream fragments were placed in front of the GUS coding sequence. Transformed tobacco roots with the fusion constructs showed the greatest expression with the 1180 bp fragment compared to the 305 bp upstream fragment end. The results indicate that certain level of rolB expression was required for rooting.