利用TA克隆的方法简便构建入门克隆

Gateway技术是一种通用型克隆方法，其基于λ噬菌体位点特异性重组，将目的DNA快速克隆到各种与Gateway技术兼容的目的载体上，不需要进行酶切和连接反应。但存在获得入门克隆过程中相关反应酶制剂价格昂贵，且药品订购时间较长等问题。通过对入门载体pDONR207的改造，使之产生3’端具有单个T 末端的线性化的入门载体，采用TA克隆的方法替代BP反应，从而简便、经济和快速地获得入门克隆。利用改造后的Gateway技术构建拟南芥SOS2基因的原核表达载体和真核表达载体，通过原核表达和原生质体瞬时表达证明通过此方法构建的表达载体在原核细胞和真核细胞中都得到了很好的表达。

Simplification of Entry Vector by TA Approach

Gateway technology is a universal cloning approach that enables rapid cloning of DNA fragments into multiple Gateway-compatible destination vectors using λ phage site-specific recombination, eliminating the requirement to work with restriction enzymes and ligase. But a problem using this system for making entry clone is the expensiveness and long time to buy the enzyme. To solve this problem, we created the TA cloning entry vector that contained a T-tail in each 3’ -end through modification of pDONR207. The TA cloning approach can construct entry clones simply, economically and rapidly. Using Gateway T vectors prepared by this improved method, prokaryotic expression vector and eukaryotic expression vector for SOS2 gene were constructed. Through the methods of prokaryotic expression and transient gene expression in Arabidopsis protoplasts, it proved that the SOS2 gene expressed well in both prokaryotic cells and eukaryotic cells.