Department of Botany, Miami University, Oxford, OH 45056, USA.
Plant Methods. 2008 Jan 22;4:4. doi: 10.1186/1746-4811-4-4.
In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway(R) cloning technology (Invitrogen Co.) has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem.
An integrated protocol for cloning genes of interest from PCR to Agrobacterium transformants via the Gateway(R) System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections.
The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to Agrobacterium via the Gateway(R) System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications.
在植物功能基因组研究中，将基因克隆到用于植物转化的二元载体中是一个常规程序。传统上，基因克隆依赖于限制性内切酶消化和连接。然而，近年来，Gateway（R）克隆技术（Invitrogen Co.）开发了一种快速可靠的替代克隆方法，该方法使用噬菌体重组策略。虽然有许多兼容 Gateway 的载体可用，但我们经常遇到重组载体之间细菌选择抗生素抗性基因相同的问题。在这些条件下，使用抗生素抗性来选择所需的转化体是困难的，如果不是不可能的话。因此，我们开发了一种实用的方法来解决这个问题。
开发了一种从 PCR 到农杆菌转化体通过 Gateway（R）系统克隆目的基因的综合方案。该方案利用了所使用质粒复制起点的独特特性，并消除了在质粒选择中需要限制性内切酶消化的必要性。
这里提出的方案是一种简化的程序，用于通过 Gateway（R）系统从 PCR 快速可靠地克隆目的基因到农杆菌。该方案克服了两个重组载体携带相同抗生素选择标记的关键问题。此外，该方案可以适应高通量应用。
