利用 TA 克隆的方法简便构建入门克隆  

Simplification of Entry Vector by TA Approach

作　　者：殷宪伦[1,2] 王春涛[1] 孔祥翔[1] 杨永平[1] 胡向阳[1] 

机构地区：[1]中国科学院昆明植物研究所,云南昆明650201 [2]中国科学院研究生院,北京100049

出　　处：《植物分类与资源学报》2012年第4期397-402,共6页Plant Diversity

基　　金：The Major Science and Technology Program (110201101003-TS-03;2011YN02 和 2011YN03)

摘　　要：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。Gateway technology is a universal cloning approach that enables rapid cloning of DNA fragments into mul- tiple Gateway-compatible destination vectors using A 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 expensive- ness 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 expres- sion vector and eukaryotic expression vector for SOS2 gene were constructed. Through the methods of prokaryotic ex- pression and transient gene expression in Arabidopsis protoplasts, it proved that the SOS2 gene expressed well in both prokaryotic cells and eukaryotic cells.

关 键 词：GATEWAY技术 TA克隆 入门克隆 原核表达 拟南芥原生质体瞬时表达 

分 类 号：Q78[生物学—分子生物学]