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Targeted gene disruption of ATP synthases 6‐1 and 6‐2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs
The Plant Journal ( IF 6.2 ) Pub Date : 2020-10-24 , DOI: 10.1111/tpj.15041 Shin‐ichi Arimura 1 , Hiroki Ayabe 1 , Hajime Sugaya 1 , Miki Okuno 2 , Yoshiko Tamura 1 , Yu Tsuruta 1 , Yuta Watari 1 , Shungo Yanase 1 , Takaki Yamauchi 1 , Takehiko Itoh 2 , Atsushi Toyoda 3 , Hideki Takanashi 1 , Nobuhiro Tsutsumi 1
The Plant Journal ( IF 6.2 ) Pub Date : 2020-10-24 , DOI: 10.1111/tpj.15041 Shin‐ichi Arimura 1 , Hiroki Ayabe 1 , Hajime Sugaya 1 , Miki Okuno 2 , Yoshiko Tamura 1 , Yu Tsuruta 1 , Yuta Watari 1 , Shungo Yanase 1 , Takaki Yamauchi 1 , Takehiko Itoh 2 , Atsushi Toyoda 3 , Hideki Takanashi 1 , Nobuhiro Tsutsumi 1
Affiliation
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We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)‐associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6‐1 and atp6‐2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6‐1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single‐molecule mito‐compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral‐dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb‐size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.
中文翻译：
mitoTALENs对拟南芥线粒体基因组中ATP合酶6-1和6-2的靶向基因破坏
我们最近通过使用线粒体靶向的转录激活因子样效应子核酸酶（mitoTALENs）实现了水稻和油菜籽线粒体基因组中细胞质雄性不育（CMS）相关基因的靶向破坏。这是植物线粒体基因组稳定且可遗传的靶向基因修饰的第一份报告。在这里，我们尝试使用mitoTALENs使用三种不同的启动子和两种类型的TALENs破坏模型植物拟南芥（Arabidopsis）中的两个线粒体基因。目标是ATP合酶亚基6基因的两个同工型atp6-1和atp6-2。这些基因中的每一个都被成功删除，线粒体基因组以同质状态被回收。核基因组也有一个atp6-1，我们能够确定敲除的是线粒体基因，而不是核假基因。在尝试的三个mitoTALEN启动子中，RPS5A启动子是最有效的。常规的mitoTALENs比单分子mito-compactTALENs更有效。靶向线粒体基因的缺失是通过杂交以及通过将mitoTALEN构建体引入细胞核的花浸转化来实现的。基因破坏是由大（kb大小）的缺失引起的。其余序列的末端主要通过重复之间的非法同源重组连接到远处的基因座。
更新日期：2020-12-22
中文翻译：
mitoTALENs对拟南芥线粒体基因组中ATP合酶6-1和6-2的靶向基因破坏
我们最近通过使用线粒体靶向的转录激活因子样效应子核酸酶（mitoTALENs）实现了水稻和油菜籽线粒体基因组中细胞质雄性不育（CMS）相关基因的靶向破坏。这是植物线粒体基因组稳定且可遗传的靶向基因修饰的第一份报告。在这里，我们尝试使用mitoTALENs使用三种不同的启动子和两种类型的TALENs破坏模型植物拟南芥（Arabidopsis）中的两个线粒体基因。目标是ATP合酶亚基6基因的两个同工型atp6-1和atp6-2。这些基因中的每一个都被成功删除，线粒体基因组以同质状态被回收。核基因组也有一个atp6-1，我们能够确定敲除的是线粒体基因，而不是核假基因。在尝试的三个mitoTALEN启动子中，RPS5A启动子是最有效的。常规的mitoTALENs比单分子mito-compactTALENs更有效。靶向线粒体基因的缺失是通过杂交以及通过将mitoTALEN构建体引入细胞核的花浸转化来实现的。基因破坏是由大（kb大小）的缺失引起的。其余序列的末端主要通过重复之间的非法同源重组连接到远处的基因座。
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