TRANSGENIC ANALYSIS OF THE 5'-FLANKING AND 3'-FLANKING REGIONS OF THE NADH-DEPENDENT HYDROXYPYRUVATE REDUCTASE GENE FROM CUCUMIS-SATIVUS L

The 5'- and 3'-flanking regions of HPRA, a cucumber gene that encodes hydroxypyruvate reductase, were evaluated for regulatory activity with respect to light responsiveness and organ specificity. To define the functional regions of the 5'-flanking region of HPRA, a series of deletions was generated and the remaining portions fused to the beta-glucuronidase (GUS) reporter gene (uidA) containing a minimal 35S promoter truncated at -90. The region from -66 to + 39 was found to be necessary for light-regulated expression of the uidA reporter gene, while the region from -382 to -67 was found to be necessary for its leaf-specific expression. Further deletion of the HPRA 5' flanking region to -590 resulted in high levels of root expression, suggesting the presence of a negative regulatory element responsible for silencing root expression of the HPRA gene between -590 and -383. The 3'-flanking region of the HPRA gene downstream of the polyadenylation site contains several sequence motifs resembling regulatory elements present in the promoters of several light-responsive genes. An 823 bp portion of the HPRA 3'-flanking region containing these putative regulatory elements enhanced GUS expression in leaves when placed downstream of the uidA reporter gene in the forward orientation, but not in the reverse orientation. When placed 5' of the -90 355 promoter, the 823 bp fragment enhanced slightly, independently of orientation, the root tip-specific expression pattern intrinsic to the -90 35S promoter, indicating that in some cases this region can act as a transcriptional enhancer.