利用Cre/loxP系统和rd29A启动子构建诱导型植物标记基因删除载体

Construction of Inducible Deletion System of Selectable Marker Gene by Using Cre/loxP System and rd29A Promoter

出于对转基因作物中标记基因的安全性考虑,植物转基因育种中标记基因的删除将成为未来研究的热点之一。位点特异性重组系统之一Cre/loxP系统是目前在植物遗传转化中应用较多,较成熟的一个标记基因删除系统。为了利用Cre/loxP系统构建一种可调控的标记基因删除系统,本研究首先从拟南芥中克隆了逆境诱导型的启动子rd29A,同时从质粒pCre上克隆了Cre基因,构建了含有rd29A:Cre:Tnos表达元件的中间载体,将这一表达元件插入到另一植物双元表达载体pKsb中,最终构建了含有loxP-Pnos-nptⅡ-Tocs-rd29A-Cre-Tnos-loxP的可诱导型删除标记基因nptⅡ的植物双元表达载体。

In view of the biosafety of the marker gene in genetically modified crops, deletion of marker gene m transgenic breeding would be become one of the research focuses in the near future. In this study, we put forward a strategy for building a kind of inducible deletion system of selectable marker gene by employing the stress inducible rd29A promoter instead of constitutive 35S promoter and combining with Cre/loxP system, one of the site-specific recombination systems, which would be known as a comparatively sophisticated system, widely used in marker gene deletion. We cloned the stress inducible rd29A promoter from A rabidopsis thaliana and Cre gene from plasmid pCre firstly. And then we built the bridge plasmid harboring rd29A ：Cre：Tnos fusion unit by using common DNA manipulation, i.e., enzyme digestion, ligation and transformation. The rd29A：Cre ：Tnos fusion unit was further integrated into a binary vector, pKsb. An inducible deletion system of selection marker gene harboring loxp-Pnos-npt Ⅱ -Tocs-rd29A-Cre-Tnos-loxP fusion unit was finally constructed, which would provide an idea for building safety expression construct without fearing about marker gene risks.