Wen Mengling, Gao Yuan, Wang Lijun, Ran Lingyu, Li Jiahui, Luo Keming
Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, China; Chongqing Key Laboratory of Transgenic Plant and Safety Control, Southwest University, Chongqing, China.
Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, China; Chongqing Key Laboratory of Transgenic Plant and Safety Control, Southwest University, Chongqing, China; Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China.
PLoS One. 2014 Oct 17;9(10):e110290. doi: 10.1371/journal.pone.0110290. eCollection 2014.
The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ''split-Cre'' fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.
Cre/loxP系统越来越多地被用于体外和体内DNA的基因操作。此前有报道称，当重叠共表达由两个不同启动子控制时，无活性的“分裂型Cre”片段可在转基因小鼠中恢复Cre活性。在本研究中，我们分析了分裂型Cre蛋白的重组活性，发现在体外重组反应中，仅表达分裂型Cre蛋白的N端结构域（NCre）时未检测到重组酶活性，而当提供C端（CCre）或NCre和CCre片段时则获得了重组活性。我们还测定了在转基因烟草发根中共表达的分裂型Cre蛋白的重组效率。单独表达NCre或CCre基因时，在转基因烟草发根中未观察到Cre重组事件。相反，在共表达两个无活性分裂型Cre基因的转基因毛根中发现了高效的重组事件。此外，在转基因株系中，与核定位序列（NLS）融合的分裂型Cre蛋白恢复后的重组效率高于完整Cre。因此，分裂型Cre蛋白介导的DNA重组为转基因植物中基因表达的时空调控提供了一种替代方法。
