CIS-ACTING REGULATORY REGIONS OF THE SOYBEAN SEED STORAGE 11S GLOBULIN GENE AND THEIR INTERACTIONS WITH SEED EMBRYO FACTORS

A 2.2 kb fragment containing the 5'-flanking region of the soybean gly cinin A2B1a gene and its successive deletions with a shorter 5'-flanki ng sequence were fused, in frame, to the beta-glucuronidase (GUS) repo rter gene. The resultant fusions were introduced into tobacco plants v ia Agrobacterium tumefaciens. Assays of the GUS activity in seeds of t ransgenic tobacco showed that the upstream region, -657 to -327 (relat ive to the transcription initiation site [+1]), of the glycinin gene i s required for optimal expression of the transformed gene. Interaction s between embryo nuclear factors and DNA fragments covering the downst ream region of -326, in which are included the TATA box and legumin bo xes, were not apparent. The embryo factors capable of binding specific ally to three subregions, -653 to -527, -526 to -422, and -427 to -321 , of the upstream regulatory region were detected. Such factors appear ed to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowe ring). Evidence obtained by characterizing the nature of the binding p roteins and by gel mobility shift assays established that the same fac tor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' re gulatory region of another glycinin A1aB1b gene. Thus it is likely tha t the observed interaction between the nuclear factor and the conserve d motifs would lead to activation of transcription from the glycinin g enes in maturing soybean seeds.