豆科MBW转录复合体促进花青素类而抑制异黄酮合成的调节机理

Leguminous plants including important crops, such as soybean (Glycine max L) and model legume Medicago truncatula, synthesizes a vast array of flavonoids, majorly anthocyanins, proanthocyanidins (PAs), and isoflavones, which play important roles in regulating plant growth, pollination; soybean symbiosis, biotic and abiotic defense responses, seed color and flower pigmentation in plant. These flavonoids in seeds of legume crops also contribute multiple health benefits to human beings as important daily phytonutrients. While biosynthesis of anthocyanins and PAs is well regulated by MYB-bHLH-WD40 (MBW) transcription regulatory complex, and many quantitative trait loci for pigmentation of soybean seed coat and flower are associated with structural genes, little is known about functions of MBW complex on these traits. We and other groups have shown different MYB-MtTT8-MtWD40-1 ternary complex guided by MtPAR, MtMYB5, and MtMYB5 activates multiple downstream regulators and structural genes to regulate the pigmentation of legume plants. Meanwhile, it was not clear how MBW complex down-regulate isoflavone biosynthesis. MYB activators and repressors with C2 or EAR motif antagonistically act together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs and anthocyanins. By using M. truncatula as model legume and soybean as crop for justification, this project is to investigate how MBW complex differentially regulate the production of anthocyanins/PAs and isoflavones. By employing yeast one hybrid for assay promoter activation, yeast two-hybrid and BiFC for detection protein-protein interaction, Dual-luciferase reporter assay system for transactivation or transrepression assay on target gene’s promoters, transgenic hairy root expression system, and tobacco expression, we will dissect MBW downstream transcription factors including MtGL2, MtTTG2, and MtMYB2 for their function in isoflavone repression by MBW activator complex, This project will also characterize several C2 and EAR motif repressor from M. truncatula and soybean, by expressing them in hairy roots for their effects on isoflavone production. The finding from the project will not only fill up the knowledge gaps in our current understanding of isoflavone regulation, but provide new insights into pigmentation patterning in legume seed coat, trifoliate, and flower. The finding will also pave the way to metabolic engineering of soybean flavonoids and molecular breeding of soybean cultivars for better agronomic traits such as disease resistance and desirable soybean nutrition values for human health.

豆科植物合成丰富的花青素、原花青素及异黄酮等黄酮类化合物。它们影响豆科植物的生物固氮、抗逆抗病性、种皮颜色花纹及种子营养品质等性状。MYB-bHLH-WD40 (MBW)转录复合体可激活蒺藜苜蓿和大豆等原花青素和花青素的合成，但对该MBW同时抑制异黄酮合成的机理仍不清楚。本课题将在前期研究基础上研究豆科模式植物蒺藜苜蓿和大豆黄酮类化合物合成的MBW转录调控网络和其抑制异黄酮合成，调节色素斑纹形成的机理。通过转基因技术，酵母单杂交和酵母双杂交检测技术体系，结合转录活性报告基因系统、BiFC互作验证以及突变体互补、代谢组和转录组分析等手段来解析豆科植物MBW调节不同类型黄酮类化合物合成的机理。目的在于阐明调控花青素、原花青素和异黄酮合成的MBW复合体下游调节因子的功能、调节网络和机理，填补异黄酮类物质合成调节的知识空白，为改良大豆黄酮类营养品质、提高大豆抗逆性等提供理论依据和分子育种手段。

大豆是重要的粮油作物，提供人类约23%的食用油和近40%的食用蛋白质。大豆种子也含有很多高附加值营养物质，如异黄酮，大豆磷脂，维生素E，皂苷等。研究大豆几种主要营养物质的合成代谢对于促进大豆营养品质改良有重要意义。本研究对大豆花青素，异黄酮和油脂合成代谢的转录调控机理及调控网络开展了研究，解析了一些关键调节和代谢基因的分子遗传功能。研究成果包括：克隆鉴定大豆MBW复合体基因TT2a, TT8a, TTG1a, MYB115，及复合体下游调控因子GmMYB7 和GmMYB3。进一步阐明了MBW复合体激活原花青素和花青素的原理，及抑制异黄酮的至少3个层面的机理，即，挤占MYB激活子结合顺式作用元件，抑制可激活异黄酮合成的转录因子基因CsMYB7，及上调可抑制异黄酮合成的转录因子CsMYB3的表达。同时，发现MBW复合体抑制油脂合成转录因子GmWRI1基因，并鉴定其功能，解释了大豆种子原花青素和油脂合成负相关的机理。本研究进一步发现CsMYB7和CSMYB3 也调节丙二酰异黄酮糖苷酰基转移酶（GmIMaT1/3, GmMaT2, 4）。它们是催化合成大豆主要的异黄酮类-丙二酰异黄酮糖苷类-的关键酶。项目鉴定了这些酶的生化功能并阐明了GmMaT2参与大豆结瘤的生理过程。本研究还发现MBW复合体负向调节独脚金内脂信号转导途径，而独脚金内酯和异黄酮积累密切相关。我们阐明了独脚金内脂信号转导蛋白GmMAX2a和GmD14, GmKAI2在调节大豆根系发育和根瘤形成过程中的作用和机理。本研究为大豆营养品质改良提供依据和分子辅助育种手段