Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium.
EMBO J. 1983;2(6):987-95. doi: 10.1002/j.1460-2075.1983.tb01532.x.
Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.
Opin 合成酶是双子叶植物在自然基因转移现象的作用下产生的酶。农杆菌含有 Ti 质粒,可指导植物中许多基因的转移、稳定整合和表达,包括编码章鱼碱或胭脂碱合成酶的基因。这一事实被用于构建许多嵌合基因,将胭脂碱合成酶 (nos) 基因的 5'上游启动子序列和大部分非翻译前导序列与两个细菌基因的编码序列结合起来:Tn5 的氨基糖苷磷酸转移酶 (APH(3')II) 基因和 R67 质粒的氨甲蝶呤不敏感二氢叶酸还原酶 (DHFR Mtx)。APH(3')II 酶使许多氨基糖苷类抗生素如卡那霉素、新霉素和 G418 失活。卡那霉素、G418 和氨甲蝶呤对植物非常有毒。当使用 Ti 质粒作为基因载体将嵌合 NOS-APH(3')II 基因转移到烟草细胞中时,该基因被表达,并赋予植物细胞对卡那霉素的抗性。卡那霉素抗性烟草细胞中含有典型的 APH(3')II 磷酸化酶活性。该嵌合基因可用作植物中有效的显性选择标记。NOS-DHFR Mtx 基因也得到了类似的结果。我们的结果表明,当使用已知在植物细胞中具有活性的启动子进行适当的构建时,外源基因不仅可以转移,而且可以功能性表达。