Metabolomics and Genome Wide Association Mapping for the Elucidation of Triterpene Saponin Molecular Biochemistry in Medicago

植物化合物的产生和作用，被称为次生化合物，是一个具有许多农业和生态维度的关键科学问题。该项目的基本目标是利用革命性的技术鉴定和表征苜蓿中重要植物次生化合物中负责三萜皂苷生物合成的新基因。药用苜蓿（Medicago truncatula）已成为苜蓿等豆科作物的重要实验模型。三萜皂苷是一类结构多样的植物天然产物，具有化感、抗真菌、抗菌、抗虫、抗癌、抗营养等广泛的生物活性。苜蓿和大豆等豆科牧草中三萜皂苷的抗营养特性具有重要的经济意义。然而，三萜皂苷的生物合成的特征很差，这一途径在大多数教科书中是缺失的。该项目将使用尖端的代谢组学、基因组全关联图谱、相关基因表达谱和传统的分子验证方法来发现和表征与三萜皂苷生物合成相关的基因。该项目的目标将通过以下具体目标来实现：1)对大量不同的松茎草种质进行代谢组学分析，鉴定高（高）和低（低）皂苷积累品系，建立与皂苷含量相关的基因位点全基因组关联图谱；2)对高（高）和低（低）皂苷积累品系进行基因表达比较分析，鉴定可能参与三萜皂苷生物合成和调控的基因；3)基于多重证据优先选择皂素生物合成基因的分子和生化确认。初步数据证明了皂苷积累的代谢多样性，这是成功关联图谱和比较微阵列分析的必要前提。更广泛的影响：该项目将识别和表征参与豆科三萜皂苷生物合成的特定基因。在该项目中鉴定的特定基因有潜力用于未来作物品种的代谢工程：1)提高适应性和防御能力；2)减少腹胀和抗营养特性，从而优化牲畜增重性能；3)与人类和动物健康相关的潜在植物中性药和佐剂。高分辨率生化表型数据将通过匿名FTP服务器（ftp://sumnerftp.noble.org/pub）生成，并通过匿名FTP服务器公开。这些数据将为皂素生物合成的进一步分子和生化解剖提供重要的特殊种质。更重要的是，生成的丰富的代谢组学数据将提供大量的社区价值，因为它将能够与丰富的其他代谢物（已知和未知）和相关适应度性状进行关联映射，包括但不限于共生（类黄酮、异黄酮、二羧酸）、耐旱/耐盐（脯氨酸、甘露醇和其他渗透保护剂）和/或耐铝（苹果酸）。本项目资助培养先进技术、植物分子生物学、大规模集成系统生物学和大规模分析生物化学领域的2名博士后和1名本科生暑期实习生/小时工。博士后研究人员将在科学写作、科学报告、出版、资助申请、建设性同行评议、项目管理、时间管理和人事管理等方面得到指导，以进一步塑造一个伟大的科学头脑，使其更接近现代学术和商业研究的现实和专业期望。目前正在举办一系列以当地高中生为重点的浓缩讲习班。参与小组包括俄克拉何马州向上发展科学/数学计划，阿德莫尔高中AP和科学俱乐部，以及俄克拉何马州南部技术中心生物技术计划。这些一年一度的丰富机会激发了学生对科学、植物生物学和天然产物生物化学的兴趣。在该项目的第三年将举办一次国际专题讨论会。这将为皂素研究的最新进展提供一个论坛，并有助于突出该项目的成果。该计划资助的博士后在组织和执行研讨会方面发挥着关键的领导作用。

The production and role of plant compounds, known as secondary compounds, is a key scientific problem that has many agricultural and ecological dimensions. The fundamental goal of this project is to identify and characterize novel genes responsible for triterpene saponin biosynthesis, in important class of plant secondary compounds, in Medicago truncatula using revolutionary technologies. The plant species Medicago truncatula has emerged as an important experimental model for leguminous crop species such as alfalfa. Triterpene saponins are a class of structurally diverse plant natural products with a wide range of demonstrated bioactivities including allelopathic, antifungal, antibacterial, antiinsect, anticancer, antinutritive activities. The antinutritive properties of triterpene saponins in legume forages such as alfalfa and soybean are of particular and substantial economic importance. However, the biosynthesis of triterpene saponins is poorly characterized and this pathway is absent from most textbooks. This project will use cutting-edge metabolomics, genome wide association mapping, correlated gene expression profiling, and traditional molecular validation approaches for gene discovery and characterization related to triterpene saponin biosynthesis in M. truncatula. The goal of this project will be accomplished through the following specific aims: 1) Metabolome analyses of a large and diverse M. truncatula germplasm collection to identify hyper- (high) and hypo- (low) saponin accumulating lines and to enable genome-wide association mapping between specific loci with saponin content 2) Comparative gene expression analyses of hyper and hypo saponin accumulating lines to identify putative genes involved in triterpene saponin biosynthesis and regulation, and 3) Molecular and biochemical confirmation of select targeted saponin biosynthetic genes prioritized based upon multiple levels of evidence. Preliminary data document substantial metabolic diversity in saponin accumulation which is a necessary prerequisite for successful association mapping and comparative microarray analyses.Broader Impacts: This project will identify and characterize specific genes involved in legume triterpenoid saponin biosynthesis. Specific genes identified in this project have the potential for future metabolic-engineering of crop varieties with: 1) improved fitness and defense, 2) decreased bloat and antinutritive properties resulting in optimized livestock weight gain performance, and 3) potential plant neutraceuticals and adjuvants related to human and animal health. High resolution biochemical phenotype data generated for a large number of M. truncatula accessions core collection will be generated and will be made publicly available via an anonymous FTP server (ftp://sumnerftp.noble.org/pub). This data will highlight specific germplasm critical for further molecular and biochemical dissection of saponin biosynthesis. More importantly, the copious metabolomics data generated will provide substantial community value in that it will enable association mapping with a wealth of other metabolites (both known and unknown) and related fitness traits including, but not limited, to symbiosis (flavonoids, isoflavonoids, dicarboxylic acids), drought/salinity tolerance (proline, manitol, and other osmoprotectants), and/or aluminum tolerance (malate). Funding of this project provides multidisciplinary training for two postdoctoral researchers and one undergraduate summer intern/hourly worker in advanced technologies, plant molecular biology, large-scale integrated systems biology, and largescale analytical biochemistry. A postdoctoral researcher is being mentored in scientific writing, scientific presentations, publishing, grant applications, constructive peer review, project management, time management, and personnel management to further mold a great scientific mind more towards the realistic and professional expectations of modern academic and commercial research. A series of enrichment workshops focused upon local high school students is being provided. Participating groups include Oklahoma Upward Bound Science/Math Program, Ardmore High School AP and Science Club, and Southern Oklahoma Technology Center Biotechnology Program. These annual enrichment opportunities stimulate student interests in science, plant biology, and natural product biochemistry. An international symposium is being be hosted in year three of the project. This will provide a forum for the latest advances in saponin research and serve to highlight the outcomes of this project. Postdocs funded by this proposal are serving key leadership roles in organizing and executing the workshops.