5'-FLANKING REGION RESPONSIBLE FOR ENDOSPERM-SPECIFIC EXPRESSION OF RICE PROLAMIN CHIMERIC GENE IN TRANSGENIC TOBACCO

The 5' upstream region (-680-+40) containing the complete signal pepti de coding sequence of the rice seed storage prolamin gene was amplifie d in vitro with polymerase chain reaction from the genome of Chinese s uper rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamin ge ne reported by Kim in 1988. No changes in the signal peptide coding se quence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamin gene has b een transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequen ce analysis. The resultant chimeric gene was used to transform the tab acco explants by Ti binary system of Agrobacterium tumefaciens LBA4404 . With dot and Southern blotting hybridization, three transgenic tobac co plants with the copies of chimeric GUS genes as many as 20 were obt ained. Histochemical analysis revealed that the GUS activity in the en dosperm 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 endos perm-specific and temporal expression of rice prolamin gene.