CHARACTERIZATION OF COMMON CIS-REGULATORY ELEMENTS RESPONSIBLE FOR THE ENDOSPERM-SPECIFIC EXPRESSION OF MEMBERS OF THE RICE GLUTELIN MULTIGENE FAMILY

Glutelin is the most abundant storage protein in rice, which is expres sed specifically in the endosperm of maturing seed. Glutelin is encode d by about 10 genes per haploid genome, which are clearly divided into two subfamilies (GluA and GluB). Most of them are coordinately expres sed during seed maturation in spite of the remarkable divergence in th e 5'-flanking regions between members of two subfamilies. In order to identify the common regulatory mechanisms responsible for the endosper m-specific expression, various cis-regulatory elements in the 5'-flank ing region of the glutelin GluB-1 gene were characterized by studying the expression of chimeric genes that consisted of the sequentially de leted or mutagenized promoter and a beta-glucuronidase (GUS) reporter gene in transgenic tobacco seeds. The essential cis-regulatory element s governing the spatially and temporally specific expression of the gl utelin gene expression were located within the first 245 bp of the pro moter region of the GluB-1 gene from the site of initiation of transcr iption. The AACA motif between positions -73 and -61 common to all the six genes for glutelin sequenced to date and is repeated between posi tions -212 and -200 is implicated in the seed-specific expression. The GCN4 motif between positions -165 and -158 and between positions -96 and -92 that is conserved at homologous sites in all the members of gl utelin gene family is also involved in the seed-specific regulation. H owever, both are required for the high level of seed-specific expressi on, because deletion of the region containing one set of both elements or substitution mutation of the AACA or GCN4 motif substantially redu ced the activity. As a whole, our results suggest the combinatorial in teraction of the elements in regulation of the glutelin gene expressio n.