Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy.
Plant Methods. 2008 Jan 22;4:3. doi: 10.1186/1746-4811-4-3.
Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.
Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations.
This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.
最近的发展，包括一些植物基因组的测序，大大增加了科学家可获得的数据量，并使得高通量研究成为可能，同时研究许多基因。为了进行这些研究，已经将重组克隆方法（如 Gateway 系统）适应于植物转化载体，以利于大规模创建过表达、标记和沉默载体。
这里我们提出了一种杂交克隆策略，它结合了重组和传统克隆的优点，特别适合于使用分子生物学和细胞生物学技术进行蛋白质功能的低至中等通量研究。该系统由一系列 12 个 Gateway Entry 盒组成，通过传统克隆方法可以将感兴趣的基因插入其中，从而生成与表位标签和荧光蛋白的 N 或 C 末端融合。所得的基因-标签融合物然后可以重组到基于 Gateway 的目的载体中，从而提供广泛的选择抗性标记物、启动子和表达系统。这种改良的 Gateway 克隆策略的优点在于，感兴趣的标记蛋白的整个可读框都包含在 Entry 载体中，因此在重组后，标签和蛋白之间不再添加任何可能干扰蛋白功能和表达的额外连接序列。我们展示了该系统在瞬时和稳定的农杆菌介导的植物转化中的应用。
这种改良的 Gateway 克隆策略与更传统的基于 Gateway 的系统互补，因为它扩展了标签和更高阶组合的选择，并允许对感兴趣的蛋白和表位标签之间的连接序列进行更多的控制，这对于蛋白功能的研究尤为重要。
