Chen Songbiao, Songkumarn Pattavipha, Liu Jianli, Wang Guo-Liang
Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, USA.
Plant Physiol. 2009 Jul;150(3):1111-21. doi: 10.1104/pp.109.137125. Epub 2009 Apr 29.
With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.
随着近期许多生物体完整基因组序列的可得，对用于基因克隆和功能分析的高通量且经济高效的系统需求巨大。尽管基于位点特异性重组的克隆系统，如Gateway克隆技术，对于将DNA片段高效转移到多个目的载体中极为有用，但两步克隆过程既耗时又昂贵。在此，我们报告一种零背景TA克隆系统，该系统可实现对PCR扩增的DNA片段进行简单且高效的直接克隆，几乎不存在自连接现象。改进后的T载体系统利用限制性内切酶XcmI在消化后产生T突出端，并利用负选择标记基因ccdB消除转化后的自连接背景。我们通过开发一组用于植物组成型基因表达、基因沉默、蛋白质标签、蛋白质亚细胞定位检测和启动子片段活性分析的瞬时和稳定转化载体，证明了该技术的可行性和灵活性。由于该系统可轻松适用于为其他生物体开发专用表达载体，零背景TA提供了一个通用、经济高效且高通量的平台，可作为Gateway克隆系统在基因克隆和功能基因组学方面的补充。
