一种载体构建的新方法:一步克隆法  

A novel method of constructing vectors:one-step cloning

作　　者：欧阳平[1] 李玉[1] 严红[1] 马立新[1] 

机构地区：[1]湖北大学微生物与基因工程省重点实验室,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2007年第2期178-181,共4页Journal of Hubei University：Natural Science

基　　金：湖北省科技攻关项目(2003AA101C22)资助

摘　　要：报道一种载体构建的新方法,在PCR引物设计时,相连片断设计15 bp同源序列,PCR克隆目的片段后,产生多个首尾具同源末端的片段,然后进行多片段定向连接、转化和重组子鉴定.以4片段拼接的叶绿体单交换质体载体pSMGA(pBluescriptⅡSK(+)-man-gfp-aadA)的构建为例,应用上述方法构建仅需做一次连接、转化即可.该方法设计操作相对简便,无需中间载体的构建,减少连接和转化次数.利用该法构建了10多个6 kb以内的载体,证明这是一种简便、通用、高效并值得推广的构建小载体的方法.A novel method of constructing vectors was developed. There are several steps as follows： first adding 15 bp homologous fragments when designing the PCR primers, then cloning purpose fragments by PCR ,after that the fragments were put together, they would recombine in an orientation , the next step is to transform the mix and finally identifying the transformants. Let＇s take the Oryza sativa single-cross chloroplast vector pSMGA （pBluescript Ⅱ SK （＋）-man-gfp-aadA） which was constructed with four fragments as an example, if constructed the vector using the method mentioned above, it would do only one recombination and one transformation . Compared with the classic method, this new method could make less medium vectors, do less recombinations and transformations. Scores of experiments has proved that it is a simple universal high-throughput method that is worth popularizing to construct the vectors within 6 kb.

关 键 词：载体构建 一步克隆 新方法 同源片段 水稻 

分 类 号：Q786[生物学—分子生物学]