THE RY SEQUENCE IS NECESSARY BUT NOT SUFFICIENT FOR THE TRANSCRIPTIONACTIVATION OF A WINGED BEAN CHYMOTRYPSIN INHIBITOR GENE IN DEVELOPINGSEEDS

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restrict ed to the period between the mid- and late-maturation stage. To unders tand the mechanisms that regulate the expression of WCI genes, we anal yzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By us ing a series of constructs with 5' deletions in the upstream sequences , the region between -882 and -623, relative to the transcription star t site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promot er in both orientations, the chimeric promoters showed only a weak tra nscription activity in transgenic tobacco plants. Further analyses usi ng internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of t he RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seed s. These results suggest that sequences necessary for high level induc tion of the WCI-3b gene transcription in developing seeds are disperse d in the region between -882 and -174, and that the RY sequence is one of these sequences.