5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco.

The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.