BIOSYNTHESIS OF ISOFLAVONOID AND FLAVONOID NUTRIENTS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。这项研究的长期目标是鉴定和表征编码对植物中有益天然产物积累至关重要的生物合成酶的克隆。这些克隆随后可用于分析影响这些化合物在植物中积累的因素，以提高其在作物物种中的可用性，或促进生物医学营养和生物利用度研究。主要的天然产品领域是花青素、儿茶素/缩合单宁和儿茶素。 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，包括积累明显不同水平的天然产物的突变植物系，以及将实验室设施从严格的化学研究重新组织到更生物化学或生物医学的研究环境。已经构建了用于微生物过表达克隆的对蒺藜苜蓿糖苷有活性的蒺藜苜蓿β-葡萄糖苷酶的载体，并正在E.大肠杆菌和巴斯德毕赤酵母，目的是生产和纯化足够的酶，以测试在动物饲养研究。一个修改公布的方法被用来生产一个样品的纯化缩合单宁从苜蓿种子，这将作为一个实验室标准的缩合单宁从苜蓿突变体的分析过程中。诱导M.研究了对蒺藜的诱导，并且用黑暗生长的幼苗和7 mM硫酸铜获得了可用的诱导水平。正在测试RT-PCR引物，以测量已知的类黄酮和类黄酮途径基因在诱导组织或突变体和野生型植物系的组织中的相对表达水平。一旦获得了令人满意的RNA样品并通过RT-PCR进行了表征，将使用为M.蒺藜除了揭示基因表达差异赋予突变株系中的天然产物变异外，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好准备。

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.