Identification of plant genes in vivo by tagging with T-DNA border-linked luciferase genes followed by inverse polymerase chain reaction amplification

We describe a method for the isolation of transcriptional control regions of developmentally regulated plant genes. This method is based on T-DNA mediated random insertion into the plant genome of a promoterless luciferase (lux) marker gene fused to the right border of the T-DNA. Insertion of the coding region of the luciferase gene downstream of a transcriptional regulatory element of a plant gene allows detection of luciferase gene expression by low-light image analysis. Using these screening methods, transgenic plants with constitutive or regulated lux gene expression were identified in vivo at different developmental stages. Genomic DNa is isolated from transgenic plants with luciferase expression in one organ only. Amplification of the tagged plant DNA sequences upstream of the lux insertion site by the inverse polymerase chain reaction method facilitates cloning of the plant promoter region. The promoterless luciferase marker gene system as part of a functional insertional element can be used for easy screening of insertional mutants, followed by isolation of developmentally regulated genes and their control elements from plants and other multicellular organisms.