High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins
High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins
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DOI：
10.1093/pcp/pcz025
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发表时间：
2019-05-01
影响因子：
4.9
通讯作者：
Ishimoto, Masao
中科院分区：
文献类型：
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作者：
Krishnamurthy, Panneerselvam;Fujisawa, Yukiko;Ishimoto, Masao
三萜（C-30）是一类在食品、化妆品和制药工业中具有潜在应用前景的天然产物。大豆皂苷是广泛存在于豆科植物中的齐墩烷型三萜，在大豆种子中尤其丰富。它们与以大豆为基础的食品的各种药理意义和不良味道特性有关。揭示大豆皂苷的生物合成基因将为控制这一途径提供新的机会，造福人类。然而，大豆皂苷的生物合成途径尚未完全阐明，部分原因是缺乏天然突变体。本研究采用结构化高密度大豆突变体文库对三萜生物合成进行正向遗传筛选。采用高通量薄层色谱和液相色谱串联质谱分析对突变文库中的大豆皂苷多态性进行了分析。该筛选鉴定出35个突变体（占909个突变系的3.85%）具有7种不同寻常的大豆皂素表型（类别1-7），这比之前报道的自然突变体数量（22个突变体，占12428个相似突变体的0.18%）要多。鉴定了9个独特的大豆皂苷合成中间体，并根据它们的MS/MS片段图谱估计了它们的化学结构。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4个大豆皂苷生物合成基因的功能丧失有关，而其余16个突变体是新的，可能有助于发现未知的大豆皂苷生物合成基因。我们的方法和文库可能有助于识别新的表型材料和与特殊代谢物产生和其他性状相关的致病基因。
Triterpenes (C-30) 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 similar to 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.