BIOSYNTHESIS OF ISOFLAVONOID AND FLAVONOID NUTRIENTS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项研究的长期目标是识别和鉴定编码生物合成酶的克隆，这些酶对于在植物中积累有益的天然产物至关重要。这些克隆以后可能被用来分析影响这些化合物在植物中积累的因素，以提高它们在作物物种中的利用率，或促进生物医学营养和生物可用性研究。主要的天然产物是花青素、儿茶素/缩合单宁和异黄酮类化合物。最初，大部分研究工作花在获取和繁殖必要的生物试剂上，包括积累明显不同水平的天然产物的突变植物品系，以及重新组织实验室设施，从严格的化学研究转向更多的生化或生物医学研究环境。克隆的紫花苜蓿β-葡萄糖苷酶的高效表达载体已被构建，并在大肠杆菌和巴斯德毕赤酵母中进行了测试，目的是生产和纯化足够的酶用于动物饲养研究。对已发表的方法进行了改进，用来从紫花苜蓿种子中生产纯化的缩合单宁样品；这将作为分析来自紫花苜蓿突变株的缩合单宁的实验室标准。对元宝枫异黄酮类途径酶的诱导条件进行了研究，并用暗生苗和7 mM的硫酸铜对其进行了诱导。已知的类黄酮类和异黄酮类途径基因在诱导组织或突变株系和野生型植物品系的组织中的相对表达水平正在被测试。一旦获得了令人满意的RNA样本并通过RT-PCR进行了表征，这些样本将使用为元胞杆菌开发的70聚体寡核苷酸阵列进行探测。除了揭示导致突变品系自然产物变异的基因表达差异外，这些实验还应该为未来基于微阵列的基因发现或动物饮食分析做准备。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long term goal of this research is to identify and characterize clones encoding biosynthetic enzymes essential to the accumulation of beneficial natural products in plants. These clones may later be used to analyze factors which effect the accumulation of these compounds in plants, to improve their availability in crop species, or to facilitate biomedical nutrition and bioavailability studies. The main natural product areas are anthocyanins, catechins/condensed tannins, and isoflavonoids. Initially, much of the research effort was spent acquiring and propagating necessary biological reagents, including mutant plant lines accumulating visibly different levels of natural products, and re-organizing laboratory facilities from strictly chemical research to a more biochemical or biomedical research environment. Vectors for microbial over-expression of cloned Medicago truncatula beta-glucosidases active on isoflavonoid glucosides have been constructed, and are being tested in E. coli and Pichia pastoris, with the goal of producing and purifying sufficient enzyme to test in animal feeding studies. A modification of a published method was used to produce a sample of purified condensed tannins from alfalfa seeds; this will serve as a laboratory standard during the analysis of the condensed tannins from alfalfa mutants. Conditions for induction of isoflavonoid pathway enzymes in M. truncatula were investigated, and a usable level of induction has been obtained with dark-grown seedlings and 7 mM copper sulfate. RT-PCR primers are being tested to measure the relative expression levels of known flavonoid and isoflavonoid pathway genes in induced tissues or in tissues from mutant and wild-type plant lines. Once satisfactory RNA samples have been obtained and characterized by RT-PCR, these will be probed using 70-mer oligo arrays developed for M. truncatula. In addition to revealing the gene-expression differences conferring the natural product variations in mutant lines, these experiments should prepare for future microarray-based gene discovery or animal diet analysis.