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 wa s studied in order to identify sequences involved in gene expression. Restriction fragments from the regulatory region were fused to the rep orter genes chloramphenicol acetyl transferase (CAT) and beta-glucuron idase (GUS). The CAT constructs were used to transform tobacco leaf di scs via Agrobacterium and the GUS-promoter fusions were introduced int o bean embryos by particle bombardment. With both systems all promoter deletions conferred expression of the GUS and CAT genes. In the bioli stically transformed bean embryos reporter gene activity dropped at po sition -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 i n developing, transgenic tobacco seeds parallels that of 2S albumin ex pression in Brazil nut. DNA retardation assays and footprint analysis using Brazil nut 2S promoter fragments and nuclear extracts from Brazi l nut seeds allowed the identification of at least two sequence motifs , ACGT and CCAC, that might be involved in promoter activity.