Hairy root-activation tagging: a high-throughput system for activation tagging in transformed hairy roots.
Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in "transformed hairy roots". The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.
Seki
Hikaru
H
RIKEN Plant Science Center, Kanagawa, Tsurumi-ku, Yokohama 230-0045, Japan.
Nishizawa
Tomoko
T
Tanaka
Nobukazu
N
Niwa
Yasuo
Y
Yoshida
Shigeo
S
Muranaka
Toshiya
T
D017360:Arabidopsis / Q000235:genetics; D015139:Blotting, Southern; D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D018517:Plant Roots / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D012231:Rhizobium / Q000235:genetics; D011198:Solanum tuberosum / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA