Detection of gene regulatory signals in plants revealed by T-DNA-mediated fusions.
A binary vector, pPRF120, was designed to detect T-DNA insertions within transcriptionally active areas of the plant genome. Linked to the right-border repeat, the vector contains a promoterless beta-glucuronidase (GUS) gene which can, upon integration into chromosomes, be activated by cis-acting regulatory elements. The vector also incorporates a chimeric marker gene conferring resistance to kanamycin to ensure recovery of gene fusions regardless of the extent of their tissue-specific or developmentally regulated expression, and to permit analysis of the frequency of plants which express the promoterless reporter. Approximately 1000 transgenic tobacco plants harboring pPRF120 were regenerated. Analysis of 52 individuals indicated that more than 80% contain single, intact copies of the T-DNA, regardless of their ability to express the promoterless GUS gene. Screening of leaf tissue from the 1000 pPRF120 transformants revealed that ca. 5% of the plants contained GUS activity. Fluorogenic and histological GUS assays were used to visualize and quantify tissue- and cell-specific gene expression. The potential usefulness of pPRF120 in comparison to other vectors designed to generate in vivo gene fusions is discussed.
Fobert
P R
PR
Department of Biology, Carleton University, Ottawa, Ontario, Canada.
Miki
B L
BL
Iyer
V N
VN
D016960:Agrobacterium tumefaciens / Q000235:genetics; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003227:Conjugation, Genetic / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D010947:Plants, Toxic; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D007612:Kanamycin; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D005966:Glucuronidase