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级代谢产物，它们是从苯丙烷类途径衍生而来的，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起着至关重要的作用，对人体健康有益。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。在这项研究中，采用CRISPR / Cas9介导的多重基因编辑技术同时针对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。在T0转基因植物中发现了更高的突变效率，其中三基因突变效率为44.44％，这些靶向诱变的结果在子代中稳定地遗传。T0三突变体叶片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代纯合三重突变体的叶片异黄酮含量约为两倍，大豆花叶病毒（SMV）外壳蛋白含量在感染SC7菌株后显着降低了三分之一，表明异黄酮含量增加增强了叶片对SMV的抗性。T2三重突变体种子中的异黄酮含量也显着增加。这项研究不仅提供了改善大豆异黄酮含量和抗SMV的材料，而且还提供了一个简单的系统来在大豆中产生多重突变，