A maize α-zein promoter drives an endosperm-specific expression of transgene in rice

An alpha-zein promoter isolated from maize containing P-box, E motif sequence TGTAAAGT, opaque-2 box and TATA box was studied for its tissue-specific expression in rice. A 1,098 bp promoter region of alpha-zein gene, fused to the upstream of gusA reporter gene was used for transforming rice immature embryos (ASD 16 or IR 64) via the particle bombardment-mediated method. PCR analysis of putative transformants demonstrated the presence of transgenes (the zein promoter, gusA and hpt). Nineteen out of 37 and two out of five events generated from ASD 16 and IR 64 were found to be GUS-positive. A histological staining analysis performed on sections of mature T-1 seeds revealed that the GUS expression was limited to the endosperm and not to the pericarp or the endothelial region. GUS expression was observed only in the following seed development stages : milky (14-15 DAF), soft dough (17-18 DAF), hard dough (20-23 DAF), and mature stages (28-30 DAF) of zein-gusA transformed (T-0) plants. On the contrary a constitutive expression of GUS was evident in CaMV35S-gusA plants. PCR and Southern blotting analyses on T-1 plants demonstrated a stable integration and inheritance of transgene in the subsequent T-1 generation. GUS assay on T-2 seeds revealed that the expression of gusA gene driven by alpha-zein promoter was stable and tissue-specific over two generations. Results suggest that this alpha-zein promoter could serve as an alternative promoter to drive endosperm-specific expression of transgenes in rice and other cereal transformation experiments.