CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening
CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening
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DOI：
10.1016/j.bbrc.2015.09.117
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发表时间：
2015-11-06
影响因子：
3.1
通讯作者：
Toki, Seiichi
中科院分区：
文献类型：
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作者：
Ito, Yasuhiro;Nishizawa-Yokoi, Ayako;Toki, Seiichi
利用遗传学方法进行的定点突变可以为作物育种以及生物学研究提供丰富的资源。成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关9核酸内切酶（CRISPR/Cas9）系统是一种用于诱导特定基因组区域突变的新策略;该系统在多种生物体中发挥作用，包括植物。在这里，我们报告了CRISPR/Cas9系统在番茄基因组有效诱变中的应用。在这项研究中，我们针对番茄RIN基因，它编码一个MADS盒转录因子调节果实成熟。靶向基因内的三个区域，并且在To再生植物中的Cas9切割位点处发现由单个碱基插入或多于三个碱基的缺失组成的突变。RIN蛋白缺陷突变体产生的不完全成熟果实中，红色色素沉着显着低于野生型，而异源突变体表达其余野生型基因达到完全成熟的红色，证实了RIN在成熟中的重要作用。在三个独立的靶位点产生的几个突变在T-1后代中遗传，证实了该诱变系统在番茄中的适用性。(C)2015 Elsevier Inc. All rights reserved.
Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in To regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wildtype gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T-1 progeny, confirming the applicability of this mutagenesis system in tomato. (C) 2015 Elsevier Inc. All rights reserved.