Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping

Abstract

The technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thaliana. A population of transgenic A. thaliana was generated containing the promoter trap vector pdeltagusBin19, following Agrobacterium tumefaciens-mediated transformation. This vector contains at the T-DNA left border, a promoterless gusA gene, which is activated following integration downstream of a native gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric assay. Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T-DNA inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border. Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was identified, demonstrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.