Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1

Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5' deletion analysis using transgenic tobacco revea led that the minimum 5' region necessary for endosperm specificity was with in -245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5'-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specif ic expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promo ter. Base substitution analysis shows that the proximal AACA motif between -73 and -61, and the GCN4 motif between -165 and -158 act as critical eleme nts. An ACGT motif between -81 and -75, and Skn-I-like elements between -17 3 and -169 also play important roles in controlling the seed-specific expre ssion. When the distal region between -245 and -145 containing the AACA and the GCN4 motifs or the proximal region between -113 and -46 containing the ACGT and AACA motifs is fused to a truncated promoter (-90 to +9) of the C aMV 35S gene fused to the beta-glucuronidase (GUS) reporter gene, high leve ls of seed-specific expression are observed in these fusions, thereby indic ating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (-46 to +1), seed expression is abolished, suggesting that the sequence be tween -90 and -46 of the CaMV 35S promoter containing G-box-like motif (as- 1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory ele ments, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene.