An Integrated Approach to Functional Genomics: Construction of a Novel Reporter Genomic Library for Osmium baselicum Plant

Abstrsct: As a means of investigating gene function, we developed a vigorous transcription fusion reporter vector to measure gene expression in plant. The vector, plasmid, was used to construct a haphazard insert library for the Osmium bsilicum genome. plasmid replicates in Escherichia coli and can be transferred to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA fragments cloned in plasmid into the Osmium bsilicum genome generates transcriptional fusions to either the reporter genes gfp and lacZ or gusA and rfp, depending on the orientation of the cloned section. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed. Sequence study and the plasmid clones were recombined into Osmium bsilicum. Reporter enzyme activities following growth of these recombinants in complex medium (LB) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those uttered at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an in capacity to recover recombinants from plasmid clones that carried fragments internal to gene or operon transcripts.