In order to identify and isolate genomic sequences that direct gene expression in the vascular tissues a 'promoter trapping' approach was adopted. Populations of transgenic Arabidopsis thaliana have been generated that contain a promoter trap vector, comprising a promoterless gusA gene. The rationale is that the inserted gusA sequence is only expected to be activated when integrated downstream of a native gene promoter, creating a functional gene fusion. Five transgenic Arabidopsis lines that were previously demonstrated to exhibit GUS fusion activity in vascular tissues were selected for this study. The number of T-DNA inserts in these lines was determined, and three of the five lines were found to contain single T-DNA copies. One line containing a single T-DNA insert, designated AtVS-1, was selected for further study.;Promoterless gusA expression in AtVS-1 was analysed throughout development, and a temporal and spatial regulation of gusA gene expression was observed. A putative aberrant roof phenotype was also revealed. An inverse PCR strategy was used to amplify genomic sequences flanking the T-DNA left border in AtVS-1, to yield an IPCR product of 1.45kb. The IPCR product was used as a molecular probe to identify homologous clones in wild-type genomic and cDNA libraries, which have been partially sequenced and show no similarity with other known genes. AtVS-1 was also used as a marker line to analyse cellular organization in two embryonic morphogenesis mutants. These results demonstrate the potential of promoter trapping as a means of creating functional tags to analyse genomic sequences direction transgene expression in the vascular tissues, and to facilitate the isolation of those sequences.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:696238 |
| Date | January 1997 |
| Creators | Muskett, Paul Raymond |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/29757 |
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