ISOLATION OF AN ASPARAGUS INTRACELLULAR PR GENE (AOPR1) WOUND-RESPONSIVE PROMOTER BY THE INVERSE POLYMERASE CHAIN-REACTION AND ITS CHARACTERIZATION IN TRANSGENIC TOBACCO

The Asparagus officinalis intracellular PR1 (AoPR1) gene is expressed in response to wounding and pathogen attack. We utilized the inverse polymerase chain reaction (IPCR) to isolate the cis-acting regulatory sequences of the AoPR1 gene following unsuccessful attempts to identify hybridizing clones in genomic libraries. Sequence analysis of two IPCR products revealed that a 347 bp intron was present in the AoPR1 gene and that it was probable that the AoPR1 regulatory sequence had been amplified. To test the AoPR1 cis-acting sequences for biological function a translational fusion was constructed with the beta-glucuronidase (GUS) reporter gene and tested in tobacco. These data demonstrated that sequences 982 bp from the probable start of transcription are sufficient to direct wound-inducible transcription and that there is no signal peptide encoded by the first 31 residues of the predicted AoPR1 protein. Histochemical localization of GUS activity in transgenic tobacco demonstrated strong activity localized to wound and pathogen invasion sites. GUS activity was also found in mature pollen grains.