BACKGROUND AND AIMS Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare L.) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. METHODS Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. KEY RESULTS Mutagenesis efficiency among T0 regenerated plantlets was 50-65 % as a result of two simultaneously expressed gRNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. CONCLUSIONS Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley..

中文翻译：

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通过启用 CRISPR/Cas9 的基因组编辑对大麦维生素 E 生物合成中的 HGGT 和 HPT 进行功能剖析

背景和目的 维生素 E（tocochromanol）是一种脂溶性抗氧化剂，是人类健康必不可少的营养素。在谷类作物中，大麦 (Hordeum vulgare L.) 含有大量维生素 E，其中包括生育酚和生育三烯酚。尽管维生素 E 生物合成途径已在双子叶植物（如拟南芥）中表征，其仅积累生育酚，但由于缺乏功能突变体，关于单子叶植物中维生素 E 生物合成的知识有限。本研究旨在获得基因敲除突变体，以阐明大麦中维生素 E 成分的遗传控制。方法 大麦中 HvHPT 和 HvHGGT 的靶向敲除突变是通过启用 CRISPR/Cas9 的基因组编辑创建的。进行高效液相色谱 (HPLC) 以分析无转基因纯合 Hvhpt 和 Hvhggt 突变体中生育色胺醇异构体的含量。主要结果 T0 再生植株的诱变效率为 50-65 %，这是因为两个同时表达的 gRNA 靶向每个基因；大多数突变稳定地遗传给下一代。Hvhpt 和 Hvhggt 的无转基因纯合突变体表现出降低的籽粒大小和重量，并且 HvHGGT 突变导致表型缩小并显着降低籽粒中的总淀粉含量。HPLC 分析显示 HvHPT 的靶向突变显着降低了生育酚和生育三烯酚的含量，而 HvHGGT 中的突变完全阻止了大麦谷物中生育三烯酚的生物合成。烟叶中 HvHPT 同系物的瞬时过表达显着增加了 γ- 和 δ-生育酚的产生，这可能部分解释了为什么大麦谷物中 HvHPT 的靶向突变没有消除生育酚的产生。结论 我们的结果在功能上证实了 HvHGGT 是唯一产生生育三烯酚的基因，而 HvHPT 部分负责大麦中生育酚的生物合成。