Poster: Tropisms
Abs #
673: Advances toward cloning the gps genes in arabidopsis
The gps (gravity persistent signal) mutants of Arabidopsis have an abnormal response when gravistimulated at 4oC and returned to room temperature. gps1 does not respond, gps2 bends in the wrong direction, gps3 over responds. These mutants were obtained by a T-DNA insertion that included a kanamycin resistance gene. Three strategies have been employed to clone the gps genes. Kanamycin plasmid rescue technique involves complementation of the Presc 38 plasmid. Presc 38 contains the first 183 nucleotides of the kanamycin resistance (nptII) coding sequence up to a PstI restriction site. A restriction digest was performed on the mutant genomic DNA and ligated into the Presc 38 plasmid at the PstI site and transformed into E.Coli. Colonies selected for kanamycin resistance should contain a fragment of the T-DNA tag and genomic DNA flanking it representing the gps gene. Several colonies have been isolated using the genomic DNA from gps1 and gps3. The second approach is thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Nested primers were designed based on the known T-DNA sequence and used with a degenerate primer to amplify the regions flanking the left border sequence of gps1 and gps3 mutants. gps2 does not maintain a strong kanamycin resistance phenotype and both kanamycin rescue and TAIL PCR have proven inefficient. Molecular and visual mapping strategies have been employed to obtain the gene sequence of gps2. (Supported by grants from United States Department of Agriculture and National Aeronautics and Space Administration)
