Stimulation of homologous recombination in plants by expression of the bacterial resolvase ruvC.
Stimulation of homologous recombination in plants by expression of the bacterial resolvase ruvC.
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DOI：
10.1073/pnas.96.13.7398
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发表时间：
1999-06
影响因子：
11.1
通讯作者：
G. Shalev;Y. Sitrit;N. Avivi-Ragolski;C. Lichtenstein;A. Levy
中科院分区：
文献类型：
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作者：
G. Shalev;Y. Sitrit;N. Avivi-Ragolski;C. Lichtenstein;A. Levy
靶向基因破坏利用同源重组（HR）作为强大的反向遗传工具，例如在细菌、酵母和转基因敲除小鼠中，但由于HR的低频率和缺乏重组突变体，其尚未应用于植物。为了增加植物中HR的频率，我们构建了携带与植物病毒核定位信号融合的大肠杆菌RuvC基因的转基因烟草系。我们表明，RuvC，编码一种核酸内切酶，结合并解决重组中间体，我们观察到基因组序列之间的体细胞交叉刺激12倍，染色体内重组刺激11倍，通过粒子轰击共转化到幼叶中的质粒之间的染色体外重组频率增加了56倍。这种刺激作用可以转移到任何植物物种以获得重组植物，因此构成了基因靶向的重要一步。
Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.