Locatelli F, Vannini C, Magnani E, Coraggio I, Bracale M
Istituto di Biologia e Biotecnologia Agraria, CNR, via E. Bassini 15, 20133, Milan, Italy.
Plant Cell Rep. 2003 Jun;21(9):865-71. doi: 10.1007/s00299-003-0593-x. Epub 2003 Mar 7.
We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.
我们描述了一种优化的聚乙二醇（PEG）介导的烟草原生质体瞬时转化方案。正如预期的那样，由pCaMVGus驱动的定量β-葡萄糖醛酸酶（Gus）活性取决于所用质粒的量。然而，我们通过免疫检测方法证明，转化效率并不取决于所用质粒的量，而是取决于细胞感受态所施加的限制。事实上，我们使用广泛的质粒浓度范围（每次测试0.1 - 10微克）获得了相同比例的转化细胞（约60%）。最后，我们表明，当我们以每种浓度范围为0.1至10微克的两种质粒类型混合使用时，所有转化细胞都表达了两种质粒编码的蛋白质。瞬时表达和共转化实验是常规使用的方法，并且可能该领域的许多研究人员都假定了这项工作的主要结果，但我们的数据通过实验支持了这一假设。
