DELETION ANALYSIS OF A 2S SEED STORAGE PROTEIN PROMOTER OF BRASSICA-NAPUS IN TRANSGENIC TOBACCO

The promoter and upstream region of the Brassica napus 2S storage prot ein napA gene were studied to identify cis-acting sequences involved i n developmental seed-specific expression. Fragments generated by succe ssive deletions of the 5' control region of the napA gene were fused t o the reporter gene beta-glucuronidase (GUS). These constructs were us ed to transform tobacco leaf discs. Analyses of GUS activities in matu re seeds from the transformed plants indicated that there were both ne gatively and positively acting sequences in the napin gene promoter. D eletion of sequences between -1101 and -309 resulted in increased GUS activity. In contrast, deletion of sequences between -309 and -211 dec reased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between -152 and +44. Further 5' del etion of the fragment to -126 abolished this activity. Sequence compar ison showed that a G box-like sequence and two sequence motifs conserv ed between 2S storage protein genes are located between -148 to -120. Histochemical and fluorometric analysis of tobacco seeds showed that t he spatial and developmental expression pattern was retained in the de letion fragments down to -152. However, the expression in tobacco seed s differed from the spatial and temporal expression in B. napus. In to bacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main ex pression of GUS was localized to the embryo. No significant GUS activi ty was found in either root or leaf.