BIOSYNTHESIS OF ISOFLAVONOID AND FLAVONOID NUTRIENTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。本研究的长期目标是鉴定和表征编码植物有益天然产物积累所必需的生物合成酶的克隆。这些克隆以后可用于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可利用性，或促进生物医学营养和生物利用度研究。主要的天然产物领域是花青素、儿茶素/浓缩单宁和异黄酮。最初，大部分研究工作都花在获取和繁殖必要的生物试剂上，包括突变植物系积累明显不同水平的天然产物，以及重新组织实验室设施，从严格的化学研究转向更生化或生物医学的研究环境。构建了具有异黄酮糖苷活性的克隆苜蓿β -葡萄糖苷酶的微生物过表达载体，并在大肠杆菌和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修改，从苜蓿种子中提取纯化浓缩单宁；这将作为实验室标准，在分析从紫花苜蓿突变体浓缩单宁。研究了长尾草中异黄酮途径酶的诱导条件，在暗生苗和7 mM硫酸铜条件下获得了适宜的诱导水平。目前正在测试RT-PCR引物，以测定已知类黄酮和异黄酮途径基因在诱导组织或突变型和野生型植物株系组织中的相对表达水平。一旦获得满意的RNA样品并通过RT-PCR表征，这些将使用为M. truncatula开发的70-mer寡核苷酸阵列进行探测。除了揭示突变系自然产物变异的基因表达差异外，这些实验还应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.