Institute of Crop Science, NARO, Tsukuba, Ibaraki, Japan.
Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan.
Plant Cell Physiol. 2019 May 1;60(5):1082-1097. doi: 10.1093/pcp/pcz025.
Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.
三萜类化合物（C30）是具有广泛应用前景的天然产物的多样化类别之一，可用于食品、化妆品和制药行业。大豆皂甙是广泛存在于豆科植物中的齐墩果烷型三萜类化合物，在大豆种子中尤为丰富。它们与各种药理作用有关，也会给大豆基食品带来不良口感。揭示大豆皂甙的生物合成基因将为控制该途径以造福人类提供新的机会。然而，由于缺乏天然突变体，大豆皂甙生物合成途径尚未完全阐明。在这里，我们应用结构密集的大豆突变体文库进行三萜生物合成的正向遗传学筛选。使用高通量薄层层析和液相色谱串联质谱分析评估突变体文库中种子大豆皂甙的多态性。这种筛选鉴定出了 35 个突变体（909 个突变株系中的 3.85%），具有七种不同的大豆皂甙表型（类别 1-7），这比以前报道的天然突变体数量（22 个突变体，约 12428 个品系中的 0.18%）要多。鉴定出 9 种大豆皂甙生物合成的独特中间体，并根据它们的 MS/MS 碎片模式估计其化学结构。根据已发表的信息，19 个突变体可能与通过表达序列标签挖掘或定位克隆鉴定的四个大豆皂甙生物合成基因的功能丧失有关，而其余 16 个突变体是新的，可能有助于发现大豆皂甙的未知生物合成基因。我们的方法和文库可能有助于鉴定与特殊代谢产物生产和其他性状相关的新表型材料和致病基因。
