| A sensitive and nondestructive method is required to study the regulation of gene expression and T-DNA insertional mutagenesis in higher plants. In an attempt to accomplish this goal, a bacterial luciferase gene-based binary plant transformation vector was modified to construct two new vectors with enhanced capacity for cloning and expression of a foreign gene in higher plants. Furthermore, a promoterless bacterial luciferase lux A gene from Vibrio harveyi was used to construct a specialized T-DNA insertional vector to monitor the activation of plant genes in vivo and to study the regulation of gene expression in a native environment. Using this strategy, large numbers of transformed tobacco plants (253) were created by Agrobacterium-mediated transformation containing the plant insertional vector pPCVG lux A&B. Following in vivo screening, insertional mutants (45) were obtained that expressed the luciferase marker gene during different stages of plant growth. Of these, one transgenic plant that expressed the luciferase marker gene predominantly in flowers was identified and studied in detail. A putative promoter element which controls the organ specific gene expression of the luciferase was identified and rescued directly using inverse PCR. DNA sequence analysis of this fragment revealed the location of a TATA-like box and three CAAT boxes in the region closely linked to the 5... |
