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，是一个关键的科学问题，具有许多农业和生态层面。 该项目的基本目标是利用革命性技术鉴定和表征蒺藜苜蓿中负责三萜皂苷生物合成的重要植物次生化合物的新基因。 蒺藜苜蓿已成为豆科作物如苜蓿的重要实验模型。 三萜皂苷是一类结构多样的植物天然产物，具有化感作用、抗真菌、抗菌、抗昆虫、抗癌、抗营养等多种生物活性。豆科牧草如苜蓿和大豆中的三萜皂苷的抗营养特性具有特殊的和实质性的经济重要性。 然而，三萜皂苷的生物合成的特点很差，这条途径是缺席的大多数教科书。该项目将使用尖端的代谢组学、全基因组关联图谱、相关基因表达谱和传统的分子验证方法来发现和表征与M.蒺藜本项目的主要目标是：1）对一个大型的、多样性的M.蒺藜属种质收集以鉴定高（高）和低（低）皂苷积累系并能够在特定基因座与皂苷含量之间进行全基因组关联作图2）高和低皂苷积累系的比较基因表达分析以鉴定参与三萜皂苷生物合成和调节的推定基因，和3）基于多个证据水平优先选择的靶向皂苷生物合成基因的分子和生物化学确认。初步数据文件大量的代谢多样性皂苷积累，这是一个必要的先决条件，成功的关联映射和比较微阵列analyses.Broader影响：该项目将确定和表征参与豆科三萜皂苷生物合成的特定基因。该项目中发现的特定基因具有未来作物品种代谢工程的潜力，具有：1）提高适应性和防御力，2）减少肿胀和抗营养特性，从而优化牲畜增重性能，以及3）潜在的植物营养保健品和佐剂与人类和动物健康相关。高分辨率的生化表型数据产生了大量的M。将生成蒺藜登记核心收集物，并通过匿名FTP服务器（ftp：//sumnerftp.noble.org/pub）公开提供。这些数据将突出具体种质的进一步分子和生化解剖皂苷生物合成的关键。更重要的是，所产生的丰富的代谢组学数据将提供实质性的社区价值，因为它将使得能够与大量其他代谢物（已知和未知）和相关的适合性性状进行关联映射，包括但不限于共生（类黄酮、黄酮、二羧酸）、干旱/盐度耐受性（脯氨酸、甘露醇和其他植物保护剂）和/或铝耐受性（苹果酸）。该项目的资助为两名博士后研究人员和一名本科生暑期实习生/小时工提供先进技术，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的多学科培训。博士后研究人员正在指导科学写作，科学演讲，出版，资助申请，建设性同行评审，项目管理，时间管理和人事管理，以进一步塑造一个伟大的科学头脑，更接近现代学术和商业研究的现实和专业期望。正在提供一系列以当地高中生为重点的充实讲习班。参与团体包括俄克拉荷马州向上的科学/数学计划，阿德莫尔高中AP和科学俱乐部，以及南俄克拉荷马州技术中心生物技术计划。这些年度丰富的机会激发学生对科学，植物生物学和天然产物生物化学的兴趣。该项目第三年将举办一次国际研讨会。 这将为皂苷研究的最新进展提供一个论坛，并突出该项目的成果。由该提案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.