Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
Gene Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
Sci Rep. 2017 Aug 1;7(1):7057. doi: 10.1038/s41598-017-06400-y.
γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1-5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.
γ-氨基丁酸（GABA）是一种非蛋白氨基酸，具有降血压作用。番茄（Solanum lycopersicum L.）是世界上种植和消费最广泛的蔬菜之一，其 GABA 含量高于其他主要作物。增加这些水平可以进一步增强番茄果实的降血压功能。谷氨酸脱羧酶（GAD）是 GABA 生物合成的关键酶；它具有 C 端自动抑制结构域，调节酶的功能，删除该结构域会增加 GAD 活性。番茄基因组有五个 GAD 基因（SlGAD1-5），其中两个（SlGAD2 和 SlGAD3）在番茄果实发育过程中表达。为了增加番茄中的 GABA 含量，我们使用成簇规律间隔短回文重复（CRISPR）/CRISPR 相关蛋白（Cas）9 技术删除了 SlGAD2 和 SlGAD3 的自动抑制结构域。在自动抑制结构域之前引入一个终止密码子可使 GABA 积累增加 7 到 15 倍，同时对植物和果实大小和产量有不同的影响。这是首次描述 CRISPR/Cas9 系统在增加番茄果实 GABA 含量方面的应用。我们的研究结果为通过基于 CRISPR/Cas9 的遗传修饰来改善其他类型的作物提供了基础。
