Background

Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca²⁺/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice.

Results

In this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca²⁺/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T₁), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T₂) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca²⁺/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1.

Conclusions

We have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

中文翻译：

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水稻谷氨酸脱羧酶3（Os GAD3）体内钙调蛋白结合域的体内靶向删除增加了谷物中γ-氨基丁酸的含量

背景

γ-氨基丁酸（GABA）是存在于所有生物中的一种非蛋白质氨基酸。GABA主要由谷氨酸通过谷氨酸脱羧酶（GAD）合成。在植物中，GAD的酶活性被Ca ²⁺激活。/ calmodulin结合（CaMBD）在C端响应各种压力，从而使GABA在细胞中快速积累。GABA不仅在胁迫响应中起着核心作用，而且在植物生长和发育的许多方面都作为信号分子发挥着重要作用。此外，已知是促进健康的功能性物质，其改善了与生活方式有关的疾病，例如高血压，糖尿病，高脂血症等。先前的报道表明CaMBD发现植物GAD具有自抑制功能，因为GAD的截短导致植物细胞中GABA的极度积累。因此，我们尝试通过基因组编辑技术对水稻GAD进行遗传修饰，以增加水稻可食部分中的GABA水平。

结果

在这项研究中，我们集中研究了GAD3（水稻基因组中存在的5个GAD基因之一），因为如前所述，GAD3主要在种子中表达。我们确认GAD3具有可作为自抑制域的真实Ca ²⁺ / CaMBD。进行修剪CaMBD的编码区关闭从CRISPR / Cas9介导的基因组编辑OsGAD3的基因，然后使用所有功能于一个载体携带导RNA和CRISPR / Cas9导入该转基因导入水稻盾片衍生的愈伤组织通过农杆菌到再生水稻。24种转化稻中（T ₁），分别在编码Os GAD3-CaMBD的N端位置和终止密码子下游40 bp的N端位置进行了两次独立的切割和连接后，经基因组编辑的水稻品系（＃8_8）在C-中显示了AK NQDAAD肽推定的Os GAD3的末端区域代替其完整的CaMBD（粗体表示真实CaMBD的N末端二肽的痕迹）。从CaMBD的N端编码区中的一个碱基对缺失获得了非常相似的，在Os GAD3中带有AK NRSSRRSGR的水稻系（＃8_1）。种子的游离氨基酸分析（T ₂）表明前一系的GABA含量比野生型高7倍，而后一系的含量与野生型相似，尽管基于GAD3氨基酸序列的重组GAD蛋白的体外酶活性在没有Ca ²⁺ /牛CaM的情况下，这两条线均高于野生型对应物。除了＃8_8中的GABA含量高外，每粒平均种子重量和蛋白质含量均优于野生型和＃8_1。

结论

我们已经使用CRISPR / Cas9基因组编辑技术成功建立了GABA强化大米。改良稻米的GABA含量比野生型糙米高7倍，并且谷物和蛋白质含量也高得多。这是通过基因组编辑生产富含GABA的水稻的首次报道。