Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid Pathways

合作研究：分子遗传学和代谢分析，以表征萜类吲哚生物碱途径杰奎琳尚克斯爱荷华州立大学CBET-0729753专业知识在生物化学工程和植物分子生物学将结合起来，采取跨学科的方法来解决一些基本问题所面临的成功工程萜类吲哚生物碱（TIA）和其他复杂的代谢途径。为了增加对TIA途径的基本了解，将开发和实施改进的代谢物分析方法。此外，代谢工程，特别是萜类途径和TIA转录因子，将用于进一步探索这些途径的调节。本工作的具体目的是：（1）获得和鉴定转基因C。过表达萜类化合物和吲哚途径代谢基因的策略性组合的蔷薇属毛状根系，（2）表征过表达TIA基因的转录激活因子和降低TIA基因的转录阻遏物的表达对TIA代谢的影响，以及（3）发展TIAs和途径中间体的代谢谱分析方法，并将该技术应用于转基因毛状根系的分析智力价值：拟议的实验将大大增加对TIA和相关途径的理解。特别是，这些实验将有助于识别和量化代谢途径中不同步骤的代谢中间体。这些实验还将通过检查操纵TIA生物合成或调节基因的不同组合的表达的影响来提供关于这些途径的调节的关键信息。更广泛的影响：从这项工作中获得的工具和知识是设计TIA和其他途径以生产更高水平的商业重要药物的必要先决条件，从而有助于降低处方药的成本。拟议的工作还将为本科生和研究生提供跨学科培训，并作出重大努力，将代表性不足的群体的成员包括在内。

Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid PathwaysJacqueline ShanksIowa State UniversityCBET-0729753Expertise in biochemical engineering and plant molecular biology will be combined to take an interdisciplinary approach to addressing some of the fundamental issues facing successful engineering of terpenoid indole alkaloids (TIAs) and other complex metabolic pathways. To increase basic understanding of TIA pathways, improved methods of metabolite analysis will be developed and implemented. In addition, metabolic engineering, particularly of the terpenoid pathway and of TIA transcription factors, will be used to further explore regulation of these pathways. The Specific Aims to be carried out in this work are to: (1) generate and characterize transgenic C. roseus hairy root lines overexpressing strategic combinations of terpenoid and indole pathway metabolic genes, (2) characterize the effects of overexpressing transcriptional activators of TIA genes and of decreasing expression of transcriptional repressors of TIA genes on TIA metabolism, and (3) develop the methodology for metabolic profiling of TIAs and pathway intermediates and apply this technology to the analysis of the transgenic hairy root lines developed.Intellectual merit: The proposed experiments will significantly increase understanding of TIA and related pathways. In particular, these experiments will help identify and quantify the metabolic intermediates for different steps in the pathways. These experiments will also provide critical information about the regulation of these pathways by examining the effects of manipulating expression of different combinations of TIA biosynthetic or regulatory genes. Broader impacts: The tools and knowledge to be gained from this work represent a necessary prerequisite to engineering TIA and other pathways to produce higher levels of commercially important pharmaceuticals, thereby helping to lower the costs of prescription drugs. The proposed work will also provide interdisciplinary training for undergraduate and graduate students, with significant efforts being made to include members of underrepresented groups.