应用Gibson Assembly方法构建植物表达载体  

Application of Gibson Assembly method for plant expression vector construction

作　　者：戢志呈 江雁翔 刘耀光[1] 张群宇[1] 

机构地区：[1]亚热带农业生物资源保护与利用国家重点实验室/华南农业大学生命科学学院,广东广州510642

出　　处：《华南农业大学学报》2014年第5期112-116,共5页Journal of South China Agricultural University

基　　金：国家自然科学基金(30871331);973计划项目(2011CB100204)

摘　　要：【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 Assem-bly”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] Gib-son 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 o-verlapping 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 ex-pressing rice genes were assembled .To increase the efficiency for cloning multiple fragments , the assem-bled 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 .

关 键 词：GibsonAssembly 体外重组 植物表达载体构建 

分 类 号：Q754[生物学—分子生物学]