Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew
Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew
复制标题
DOI：
10.1038/nbt.2969
复制
发表时间：
2014-09-01
影响因子：
46.9
通讯作者：
Qiu, Jin-Long
中科院分区：
文献类型：
--
作者：
Wang, Yanpeng;Cheng, Xi;Qiu, Jin-Long
序列特异性核酸酶已被应用于在多倍体基因组中设计靶向修饰(1)，但多个同源等位基因的同时修饰尚未见报道。在这里，我们使用转录激活物样效应核酸酶(TALEN)(2，3)和成簇的，规则间隔的短回文重复序列(CRISPR)-Cas9。4，5)在六倍体面包小麦中引入编码抗白粉病基因(MLO)蛋白的三个同源等位基因的定向突变技术(6)。遗传冗余阻碍了对面包小麦中所有三个MLO等位基因的突变是否可能赋予对白粉病的抗性的评估，这是自然群体中没有的一个特征。我们表明，在同一植物中，TALEN诱导的所有三个TaMLO同源物的突变赋予了对白粉病可遗传的广谱抗性。我们进一步使用CRISPR-Cas9技术来产生携带TaMLO-A1等位基因突变的转基因小麦植株。我们还证明了通过TALENS引起的双链断裂的非同源末端连接来工程定向DNA插入面包小麦的可行性。我们的发现为改良多倍体作物提供了一个方法学框架。
Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes(1), but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator like effector nuclease (TALEN)(2,3) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins(6). Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations(7). We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.