Rolland Vivien
Commonwealth Scientific and Industrial Research Organisation (CSIRO), Agriculture & Food, Canberra, 2601, ACT, Australia.
Methods Mol Biol. 2018;1770:263-283. doi: 10.1007/978-1-4939-7786-4_16.
In plants, stable expression is arguably the method of choice to test transgene function but it is a slow and labor-intensive process. This bottleneck generally limits the number of transgenes that can be tested, and as such hinders construct optimization. In the face of this challenge, transient expression in Nicotiana benthamiana leaves has emerged as a powerful screening platform to test gene expression, as well as subcellular distribution and function of many proteins within a week. This system relies on the infiltration of Agrobacterium tumefaciens (Agrobacterium) carrying DNA of interest into the leaf air spaces of N. benthamiana plants. Agrobacterium rapidly transforms the plant cells and the leaves can be analyzed within a few days. Investigating the subcellular localization of a protein of interest often relies on its fusion to a fluorescent tag. While the amount of accumulation of such fusion proteins can often be gauged by observing the fluorescence of the tag at the whole-leaf level, subcellular protein distribution is best determined in protoplasts extracted from transformed leaves. Here I present a simple and effective method to transform N. benthamiana leaves with Agrobacterium and to prepare protoplasts from these leaves to characterize the subcellular localization of proteins of interest.
在植物中，稳定表达可以说是测试转基因功能的首选方法，但它是一个缓慢且 labor-intensive 的过程。这个瓶颈通常限制了可测试的转基因数量，因此阻碍了构建体的优化。面对这一挑战，本氏烟草叶片中的瞬时表达已成为一个强大的筛选平台，可在一周内测试基因表达以及许多蛋白质的亚细胞分布和功能。该系统依赖于将携带感兴趣 DNA 的根癌农杆菌（农杆菌）渗入本氏烟草植物的叶片气腔。农杆菌迅速转化植物细胞，叶片可在几天内进行分析。研究感兴趣蛋白质的亚细胞定位通常依赖于将其与荧光标签融合。虽然此类融合蛋白的积累量通常可以通过在全叶水平观察标签的荧光来衡量，但亚细胞蛋白质分布最好在从转化叶片中提取的原生质体中确定。在这里，我介绍一种简单有效的方法，用农杆菌转化本氏烟草叶片，并从这些叶片中制备原生质体，以表征感兴趣蛋白质的亚细胞定位。
