Plant Genetic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.
Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):12034-9. doi: 10.1073/pnas.1000234107. Epub 2010 May 27.
Site-directed mutagenesis in higher plants remains a significant technical challenge for basic research and molecular breeding. Here, we demonstrate targeted-gene inactivation for an endogenous gene in Arabidopsis using zinc finger nucleases (ZFNs). Engineered ZFNs for a stress-response regulator, the ABA-INSENSITIVE4 (ABI4) gene, cleaved their recognition sequences specifically in vitro, and ZFN genes driven by a heat-shock promoter were introduced into the Arabidopsis genome. After heat-shock induction, gene mutations with deletion and substitution in the ABI4 gene generated via ZFN-mediated cleavage were observed in somatic cells at frequencies as high as 3%. The homozygote mutant line zfn_abi4-1-1 for ABI4 exhibited the expected mutant phenotypes, i.e., ABA and glucose insensitivity. In addition, ZFN-mediated mutagenesis was applied to the DNA repair-deficient mutant plant, atku80. We found that lack of AtKu80, which plays a role in end-protection of dsDNA breaks, increased error-prone rejoining frequency by 2.6-fold, with increased end-degradation. These data demonstrate that an approach using ZFNs can be used for the efficient production of mutant plants for precision reverse genetics.
在高等植物中，定点突变仍然是基础研究和分子育种的一个重大技术挑战。在这里，我们利用锌指核酸酶（ZFNs）展示了拟南芥中内源性基因的靶向基因失活。针对应激反应调节剂 ABA 不敏感 4 型（ABI4）基因设计的工程化 ZFNs 在体外特异性切割其识别序列，并且由热休克启动子驱动的 ZFN 基因被引入拟南芥基因组。在热休克诱导后，通过 ZFN 介导的切割在体细胞中观察到 ABI4 基因的缺失和取代突变，频率高达 3%。ABI4 基因的 ZFN 介导突变的纯合突变体系 zfn_abi4-1-1 表现出预期的突变表型，即 ABA 和葡萄糖不敏感。此外，我们还将 ZFN 介导的突变应用于 DNA 修复缺陷型突变体植物 atku80。我们发现，AtKu80 的缺失，其在 dsDNA 断裂的末端保护中起作用，增加易错连接频率 2.6 倍，并增加末端降解。这些数据表明，使用 ZFN 的方法可用于高效生产用于精确反向遗传学的突变体植物。
