Nontransgenic Genome Modification in Plant Cells
Nontransgenic Genome Modification in Plant Cells
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DOI：
10.1104/pp.110.164806
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发表时间：
2010-11-01
期刊：
影响因子：
7.4
通讯作者：
Vainstein, Alexander
中科院分区：
文献类型：
--
作者：
Marton, Ira;Zuker, Amir;Vainstein, Alexander
锌指核酸酶（ZFN）是真核细胞中基因组编辑的有力工具。ZFN已用于模型和作物物种中的靶向诱变。在动物和人类细胞中，瞬时ZFN表达通常通过直接基因转移到靶细胞中来实现。然而，稳定转化是在植物物种中进行基因表达的优选方法，并且表达ZFN的转基因植物已经用于恢复由于使用直接基因转移方法进入靶细胞而可能被分类为转基因的突变体。在这里，我们提出了一种替代的，非转基因的方法ZFN交付和生产突变体植物使用一种新的烟草脆裂病毒（TRV）为基础的表达系统，间接瞬时交付ZFN到各种组织和细胞的完整的植物。TRV系统感染其宿主和病毒ZFN介导的靶向诱变可以清楚地观察到在新开发的感染组织中，如通过烟草（Nicotiana tabacum）和矮牵牛（Petunia hybrida）植物中突变的报告基因的激活所测量的。TRV移动到发育中的芽和再生组织的能力使得突变的烟草和矮牵牛植物能够恢复。序列分析和突变传递到下一代证实了ZFN诱导的遗传变化的稳定性。由于TRV是一种RNA病毒，可以感染广泛的植物物种，它提供了一个可行的替代生产ZFN介导的突变体，同时避免使用直接的植物转化方法。
Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.