McBride K E, Schaaf D J, Daley M, Stalker D M
Calgene Inc., Davis, CA 95616.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7301-5. doi: 10.1073/pnas.91.15.7301.
Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.
T7噬菌体RNA聚合酶已在大肠杆菌中广泛用于在噬菌体T7基因10启动子控制下的特定基因的高水平表达。我们构建了一个类似的系统用于高等植物的质体。通过核转化将一个含有花椰菜花叶病毒35S启动子和烟草核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基叶绿体转运肽序列的T7 RNA聚合酶嵌合基因导入烟草。用T7启动子/β-葡萄糖醛酸酶（GUS）报告基因构建体对表达T7 RNA聚合酶活性的烟草进行稳定的质体转化，结果在所有检测的组织中均有GUS mRNA的表达和酶活性。GUS活性在成熟叶中极高，在幼叶和花瓣中适中，在茎、根和发育中的种子中较低。用相同的嵌合GUS基因对野生型烟草进行质体转化，未检测到GUS mRNA水平和酶活性。遗传杂交表明，沉默的T7/GUS报告基因可通过来自父本的活性核T7 RNA聚合酶基因的传递在F1代中被激活。
