T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体，然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是，BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现，attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此，设计含有core-attL序列的引物，通过PCR克隆得到DNA片段并连入pMD18-T载体，然后进行LR反应，可成功得到目标表达载体，并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草，能够正常表达该蛋白质。结果表明，通过将含有attL核心位点基因片段连接到pMD18-T载体上，可以省略BP反应而将目标片段连接到终载体上，节约了反应时间和成本。

Rapid,T-vector Mediated,Directional Cloning of Short Core-attL Attached PCR Products into the Gateway System

The widely-used Gateway cloning technology is site-specific and recombination-based. It standardly includes two steps. First, a DNA fragment is inserted into a donor vector in a BP reaction, creating an entry vector. Then the fragment is transferred to a destination vector through an LR reaction, producing the expression vector. Compared with classical restriction enzyme-based cloning methods, it is easy and rapid. However, the enzymes used in two reactions are expensive. In this study, we identified a short conserved attL sequence(21-22 bp) by aligning the atts recombination sites of three Gateway vectors, and named it “core attL”. The “core attL” was attached to gene-specific sequences to design primers for amplification of DNA fragments. Then, the PCR fragments containing genes of interest, flanked by “core attL” sequences were cloned into a commercial pMD18-T vector to generate “entry clones”, which can be directly used to recombine with any Gateway destination vectors (LR reaction) to create expression clones. This cloning method was stable and efficient as indicated by high proportion of positive clones obtained each time. Furthermore, an expression construct containing the eGFP gene was introduced into tobacco leaf cells and could be transiently expressed. Our results clearly indicated that the “core attL” is sufficient for site specific recombination during the LR reaction to generate the expression vectors for functional studies. Moreover, TA cloning with pMD18-T vectors can be used to efficiently create entry clones instead of using BP reactions and entry vectors. Thus, we developed a simple, BP-reaction-free Gateway cloning strategy for rapid directional cloning of PCR products into destination vectors.