Identification of positive and negative regulatory cis-elements of the rice glutelin Gt3 promoter

A series of 3' promoter deletions of the rice glutelin Gt3 gene (-181 bp, -278 bp, -300 bp, -320 bp, -482 bp, -580 bp, -775 bp and -835 bp from the transcriptional initiation site) was obtained and linked to the -90 core promoter fragment of the 35S CaMV gene located upstream of the P-glucuronidase protein coding region of pBI 101. After transfer into tobacco via Agrobacterium tumefaciens-mediated transformation, the spatial and temporal of the Gt3 promoter was analyzed in 16- and 24-day-old seeds, as well as leaves and roots of the transgenic tobacco plants. Other than constitutive expression in roots contributed by the CaMV 35S -90 core promoter sequences, significant expression of the intact Gt3-CaMV 35S hybrid promoter was evident only during seed development where maximum expression was evident in 16-day-old seeds. Analysis of hybrid promoters containing various 3' deletions of the rice promoter revealed two regions, +7 to -181 and -278 to -320, that were essential for maximum Gt3 expression. Removal of the proximal region, +7 to -181, resulted in a 50% decrease of GUS expression with an additional 27% decrease upon removal of the distal region. A negative regulatory element that suppressed expression of the chimeric promoter in leafs is located between -320 bp and -482 bp. These results together with those from an earlier study (Zhao et al., Plant Mel. Bioi. 325 (1994) 429-436) from this laboratory demonstrate that the developmental control of the rice Gt3 promoter requires at least three regions governing spatial, temporal and quantitative expression in seeds and at least one region suppressing expression in leaves.