Zhou X J, Fan Y L
Laboratory of Molecular Biology, Biotechnology Research Centre, Chinese Academy of Agricultural Sciences, Beijing.
Sci China B. 1993 Nov;36(11):1307-14.
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.
利用聚合酶链式反应从中国超级稻品种“中华8号”的基因组中体外扩增出包含水稻种子贮藏醇溶蛋白基因完整信号肽编码序列的5′上游区域（-680-（+）40）。物理图谱和DNA序列分析表明，其与Kim于1988年报道的水稻醇溶蛋白基因的5′侧翼区域具有高度同源性。未发现信号肽编码序列有变化，且发现了一个带有几个小开放阅读框的长前导序列。含有醇溶蛋白基因5′侧翼区域的嵌合基因已与β-葡萄糖醛酸酶报告基因进行转录融合。通过物理图谱和DNA序列分析对融合接头进行了确认。所得嵌合基因通过根癌农杆菌LBA4404的Ti二元系统用于转化烟草外植体。通过斑点杂交和Southern杂交，获得了3株含有多达20个嵌合GUS基因拷贝的转基因烟草植株。组织化学分析显示，在发育阶段烟草种子胚乳组织中的GUS活性约在授粉后20天出现。在转基因烟草植株的叶、茎、根和花中未发现GUS活性。因此，我们得出结论，从-680到-18的5′上游顺式元件足以赋予水稻醇溶蛋白基因胚乳特异性和时间特异性表达。
