HIGH-LEVELS OF RIPENING-SPECIFIC REPORTER GENE-EXPRESSION DIRECTED BYTOMATO FRUIT POLYGALACTURONASE GENE-FLANKING REGIONS

The 1.4 kb 5' polygalacturonase (PG) gene-flanking region has previous ly been demonstrated to direct ripening-specific chloramphenicol acety l transferase (CAT) expression in transgenic tomato plants. The steady state level of CAT mRNA in these plants was estimated to be less than 1% of the endogenous PG mRNA. Further constructs containing larger PG gene-flanking regions were generated and tested for their ability to direct higher levels of reporter gene expression. A 4.8 kb 5'-flanking region greatly increased levels of ripening-specific reporter gene ac tivity, while a 1.8 kb 3' region was only shown to have a positive reg ulatory role in the presence of the extended 5' region. Transgenic pla nts containing the CAT gene flanked by both of these regions showed th e same temporal pattern of accumulation of CAT and PG mRNA, and steady -state levels of the transgene mRNA were equivalent to 60% of the endo genous PG mRNA on a per gene basis. The proximal 150 bp of the PG prom oter gave no detectable CAT activity. However, the distal 3.4 kb of th e 4.8 kb 5' PG promoter was shown to confer high levels of ripening-sp ecific gene expression when placed in either orientation upstream of t he 150 bp minimal promoter. The DNA sequence of the 3.4 kb region reve aled a 400 bp imperfect reverse repeat, and sequences which showed sim ilarity to functionally significant sequences from the ripening-relate d, ethylene-regulated tomato E8 and E4 gene promoters. The possible ro les of the flanking regions in regulating PG gene expression are discu ssed.