5'-flanking Region Responsible for Endospermspecific Expression of Rice Prolamin Chimeric Gene in Transgenic Tobacco  

作　　者：周先锦 范云六 

机构地区：[1]Laboratory of Molecular Biology, Biotechnology Research Centre, Chinese Academy of Agricultural Sciences, Beijing 100081, PRC

出　　处：《Science China Chemistry》1993年第11期1307-1314,1409,共9页中国科学（化学英文版）

基　　金：Project supported by Rockefeller Foundation for International Program on Rice Biotechnology.

摘　　要：The 5' upstream region (—680—+40) containing the complete signal peptide coding sequence of the rice seed storage prolamin 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 prolamin 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 prolamin gene has been transcriptionally fused with the β-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 tabacco 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 prolamin gene.The 5’ upstream region (—680—+40) containing the complete signal peptide coding sequence of the rice seed storage prolamin 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 prolamin 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 prolamin gene has been transcriptionally fused with the β-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 tabacco 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

关 键 词：β glueuronidase ENDOSPERM PROMOTER TRANSGENIC TOBACCO riee prolamin. 

分 类 号：O6[理学—化学]