Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant-environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9-mediated multiplex gene-editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII-1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T0 transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T0 triple-mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T3 generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one-third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T2 triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.

中文翻译：

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多重CRISPR/Cas9介导的代谢工程增加了大豆异黄酮含量和对大豆花叶病毒的抵抗力。


异黄酮类化合物包括多种次生代谢产物，来源于苯丙素途径，主要分布在豆科植物中。这些化合物在植物与环境的相互作用中发挥着关键作用，并且有益于人类健康。异黄酮合酶 (IFS) 是异黄酮合成中的关键酶，与黄烷酮 3-羟化酶 (F3H) 和黄酮合酶 II (FNS II) 具有共同的底物。本研究采用CRISPR/Cas9介导的多重基因编辑技术同时靶向大豆毛状根和植物中的GmF3H1、GmF3H2和GmFNSII-1。在毛状根中观察到各种突变类型和频率。 T0转基因植株的突变效率较高，三基因突变效率为44.44%，且这些定点突变的结果在后代中稳定遗传。 T0 三突变体叶子的代谢组学分析显示异黄酮含量显着提高。与野生型相比，T3代纯合三突变体的叶片异黄酮含量约增加一倍，且感染菌株SC7后，大豆花叶病毒（SMV）外壳蛋白含量显着降低三分之一，表明异黄酮含量增加。增强叶片对SMV的抗性。 T2三重突变体种子中的异黄酮含量也显着增加。该研究不仅为提高大豆异黄酮含量和抗SMV提供了材料，而且为产生大豆多重突变提供了一个简单的系统，这可能有利于进一步的育种和代谢工程。