【目的】构建方便快捷的植物表达载体.【方法】应用Isothermal in vitro recombination system or “Gibson Assembly”方法设计包含片段间20~25 bp互补重叠序列的引物，通过PCR扩增出带有首尾重叠的目的DNA片段，可将1个或多个片段和线性化的载体一步组装成表达载体.【结果和结论】利用该方法快速构建了多个水稻基因的全长以及部分缺失编码区表达载体.此外，还通过对Gibson Assembly连接产物进行PCR扩增后再连接到载体，提高多DNA片段组装的效率.本方法不受目的片段内部限制性酶切位点的限制，可广泛应用于各种载体的构建.
【Objective】 To construct a convenient and efficient plant expression vectors.【Method】 Gibson et al developed an isothermal in vitro recombination system or Gibson Assembly for ligation of multiple DNA fragments. The primers were designed to amplify target DNA sequences that contained 20-25 bp overlapping ends. The target DNA segments， linearized vector and the enzyme mixture were mixed in one tube to perform the assembly reaction.【Result and conclusion】 Using this method, several vectors for expressing rice genes were assembled. To increase the efficiency for cloning multiple fragments, the assembled primary products by PCR were amplified and then ligated into the vector. The Gibson Assembly method is faster and simpler without the limitation of internal restriction endonuclease sites of target genes, and it can be applied widely to construction of various vectors.
戢志呈,江雁翔,刘耀光,张群宇.应用 Gibson Assembly 方法构建植物表达载体[J].华南农业大学学报,2014,35(5):112-116复制