Wang Yong, Chen Bojun, Hu Yuanlei, Li Jingfu, Lin Zhongping
Department of Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China.
Transgenic Res. 2005 Oct;14(5):605-14. doi: 10.1007/s11248-005-0884-9.
In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This auto-excision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.
在植物转化过程中，有必要使用能够用于筛选再生转基因植物的标记基因。然而，一旦完整的转基因植物建立起来，选择标记基因通常就不再需要了。此外，它们可能会给转基因作物的释放和商业化审批带来监管方面的困难。我们构建了一个带有Cre/lox系统的二元表达载体，以便方便地从转基因植物中去除标记基因。在该载体中，热激启动子控制下的重组酶基因cre和CaMV35S启动子控制下的选择标记基因nptII以正向排列的方式置于两个loxP位点之间，而目的基因则插入到loxP位点之外。通过使用该载体，经过热激处理后，cre和nptII基因在大多数初代转化烟草的再生植株中都被去除了，而目的基因得以保留并稳定遗传。这种由Cre/lox系统介导并经过热激处理以去除选择标记基因的自动切除策略易于采用，为培育无标记转基因植物提供了一种有前景的方法。
