利用一步克隆方法快速高效构建Gateway入门载体  

A quick and efficient method to construct Gateway entry vector by one-step cloning

作　　者：张艳军[1] 赵利[1] 李雨佳 赵江哲[1] 张可伟[1] ZHANG Yanjun;ZHAO Li;LI Yujia;ZHAO Jiangzhe;ZHANG Kewei(Institute of Plant Genetics and Developmental Biology,College of Chemistry and Life Sciences,Zhejiang Normal University,Jinhua 321004,China)

机构地区：[1]浙江师范大学化学与生命科学学院植物遗传发育研究所

出　　处：《浙江师范大学学报（自然科学版）》2020年第1期72-76,共5页Journal of Zhejiang Normal University:Natural Sciences

基　　金：国家自然科学基金资助项目(31470370;31670277);浙江省公益技术应用研究计划项目(2015C32043)

摘　　要：Gateway技术是一种非常便捷的载体构建方法,但目前构建入门克隆的方法成本较高,且存在一定的局限性.利用限制酶Eco RⅠ对pCR8-AtS5H入门克隆进行单酶切,获得入门载体pCR8片段,然后利用不依赖序列和连接的一步克隆方法(one-step sequence-and ligation-independent cloning,One-SLIC)将纯化后水稻异分支酸合成酶基因的启动子(ICS1 pro)片段连接至pCR8入门载体,并通过菌落PCR和测序方法鉴定得到阳性入门克隆,最后通过LR重组反应将ICS1 pro片段转移至Gateway目的载体pMDC163上.这种利用One-SLIC法快速高效构建入门克隆的方法操作简便,成功率高且成本低,是一种具有广泛应用前景的Gateway入门载体构建方法.Gateway technology was a very convenient method to construct vectors,but the existed methods to construct the entry vector were relatively expensive and had some limitations.The entry vector pCR8 fragment was released by a single restrictive enzyme Eco RⅠfrom the constructed vector pCR8-AtS5H,the ICS1 pro fragment was amplified and cloned to the pCR8 fragment by one-step sequence-and ligation-independent cloning(SLIC).Based on the colony PCR and sequencing results,the positive clone was identified and then the ICS1 pro fragment was recombined to the Gateway destination vector pMDC163 via the LR reaction.The method to construct entry vector by one-step sequence-and ligation-independent cloning was simple,efficient and cost-effective.Therefore,it would be a very useful method in constructing the entry vector for Gateway cloning.

关 键 词：入门克隆 SLIC技术 GATEWAY LR重组反应 

分 类 号：Q785[生物学—分子生物学]