Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Plant J. 2011 Dec;68(5):929-37. doi: 10.1111/j.1365-313X.2011.04741.x. Epub 2011 Oct 4.
Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.
靶向基因组修饰是一种重要的遗传工具，可以通过同源、非同源或位点特异性重组来实现。尽管已经做出了许多努力，但在植物中，这种工具还没有用于常规应用。本研究描述了一种简单而有用的方法，用于靶向诱变或基因靶向，通过在卵母细胞中特异性表达蛋白质，专门针对拟南芥的花浸转化。在卵器特异性增强子（EASE）下稳定或瞬时表达的蛋白质成功地定位于卵母细胞区域。此外，在 EASE 控制下表达的锌指核酸酶诱导了靶向诱变。在生殖系中特异性发生的 EASE 控制下获得的突变与遗传上独立的事件相对应。此外，与野生型植物相比，EASE 控制下的 RAD54 表达使基因靶向效率提高了约 10 倍。EASE 控制的基因表达通过时间和空间控制的蛋白质表达为精确工程化拟南芥基因组提供了一种方法。该系统可作为拟南芥基础研究以及作物中靶向遗传修饰工具优化的有用方法。
