A 68 BP ELEMENT OF THE BETA-PHASEOLIN PROMOTER FUNCTIONS AS A SEED-SPECIFIC ENHANCER

In beans, expression of the beta-phaseolin gene (phas), encoding the m ajor seed storage protein of bean (Phaseolus vulgaris) is confined to the cotyledons of developing embryos. Phaseolin has not been detected in the endosperm, which remains liquid and is lost early in developmen t. However, fusion constructs between the phas promoter and the gus-co ding region yield expression in both embryo and endosperm of developin g seeds from transgenic tobacco (Nicotiana tabacum) plants. Although e lements extending 1470 bp upstream of the transcription start site are known to modulate phas expression, the proximal 295 bp (p295) are suf ficient to drive high levels of seed-specific GUS activity. This regio n was dissected into three elements: a 68 bp element (seed specific en hancer, SSE: -295 to -227), a middle region (-227 to -109) and a basal phas promoter(-109 to +20: p109). Different promoter constructs conta ining the SSE or middle region upstream of p109 or a CaMV 35S basal pr omoter (-64 to +6) were fused to gus. Each construct was expressed in seed, but not in vegetative tissues. Use of the various phas promoter regions yielded notable differences in relative GUS activity in embryo or endosperm. Addition of both the SSE and middle region resulted in higher activity than the sum of adding either element alone to p109, i ndicating synergistic interaction between these elements. Seeds from p lants transformed with the proximal 227 bp of promoter (p227) showed e mbryo-specific GUS activity. In contrast, constructs containing two co pies of the SSE element were preferentially expressed in the endosperm . These results illustrate the modular nature of the proximal phas pro moter, where distinct elements contribute to high levels of expression in different parts of the seed.