The endosperm-specific expression of a rice prolamin chimaeric gene in transgenic tobacco plants

The 5' upstream region (-680 to +40), containing the potential promoter and complete signal peptide coding sequence of the rice seed storage prolamin gene was amplified in vitro using the polymerase chain reaction from the genome of Chinese rice cultivar Zhonghua 8. The physical map and DNA sequence analysis show strong homology with the 5' flanking region of the rice prolamin gene published by Kim and Okita (1988a). No change in the signal peptide coding sequence and a long leader sequence with several small open reading frames were found. The chimaeric gene containing the 5' flanking region of the prolamin gene (-680 to -18) was transcriptionally fused with the beta-glucuronidase (GUS) reporter gene and the fusion junction was confirmed by both physical mapping and DNA sequence analysis. The resultant chimaeric gene was used to transform tobacco explants, using the Ti binary system of Agrobacterium tumefaciens LBA4404. Three transgenic tobacco plants with as many as 20 copies of the chimaeric GUS gene (confirmed by dot and Southern hybridization) were analysed further. Histochemical analysis revealed GUS activity in the endosperm tissue of tobacco seed at the developmental stage about 20 days after flowering (DAF). No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we conclude that the 5' upstream region from -680 to -18 was sufficient to confer the endosperm-specific expression of the rice prolamin gene.