Towards the ultimate goal of deciphering the function of every gene in the Arabidopsis genome, it is paramount to generate high-resolution spatial and temporal maps of gene expression patterns and sub-cellular localization of the corresponding gene products. Translational fusions between a reporter gene, such as GFP, and a gene of interest in its chromosomal context provide the most accurate information on that gene's expression pattern. In the absence of efficient homologous recombination system in Arabidopsis, large Arabidopsis artificial chromosomes can be precisely tagged with fluorescence proteins by homologous recombination in E. coli and then transferred to the Arabidopsis genome. The main objective of this project is to examine the efficiency and effectiveness of this bacterial recombination system for large-scale gene function analysis in Arabidopsis. The accuracy of the obtained information and, therefore, the utility of the system will be evaluated generating GFP-tagged Arabidopsis lines for genes with well-characterized expression patterns, as well as with well-defined mutant phenotypes. The efficiency of each step in the process and the "scalability" to a whole-genome level will be examined. Finally, the utility of using additional reporter gene tags such as those based on GAL4 will be investigated, exploring the potential of employing the generated tagged clones to confer specific well-defined expression patterns to any gene of interest and to test the functional relevance of a particular expression pattern. Finally, the transferability of this technology to other plant species will be investigated.
Broader Impacts: All biological materials generated in this project will be made available, upon testing, to the research community from the Arabidopsis Biological Resource Center. The availability of these resources will be announced through the Arabidopsis News Group (www.bio.net/biomail/listinfo/arab-gen). List of genes, position of the tags, and project progress will be posted in a project-dedicated webpage. Importantly, the long-term potential impact of this project is the production of essential tools for generating the Arabidopsis "localizome", high-resolution expression maps of all Arabidopsis genes. This project will also provide an excellent opportunity for attracting and introducing the field of functional genomics to high school and undergraduate students, particularly those from underrepresented groups and especially Hispanics.
