FUNCTIONAL-STUDIES ON A SEED-SPECIFIC PROMOTER FROM A BRAZIL NUT 2S GENE

The 5' upstream regulatory region of the Brazil nut 2S albumin gene was studied in order to identify sequences involved in gene expression. Restriction fragments from the regulatory region were fused to the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS). The CAT constructs were used to transform tobacco leaf discs via Agrobacterium and the GUS-promoter fusions were introduced into bean embryos by particle bombardment. With both systems all promoter deletions conferred expression of the GUS and CAT genes. In the biolistically transformed bean embryos reporter gene activity dropped at position -460 base pairs upstream from the transcription initiation site. CAT activity in stably transformed tobacco plants was less markedly affected at this site but declined sharply after the deletions reached -210 bp upstream from the transcription initiation site. The temporal expression of the CAT gene controlled by the Brazil nut 2S promoter in developing, transgenic tobacco seeds parallels that of 2S albumin expression in Brazil nut. DNA retardation assays and footprint analysis using Brazil nut 2S promoter fragments and nuclear extracts from Brazil nut seeds allowed the identification of at least two sequence motifs, ACGT and CCAC, that might be involved in promoter activity.