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.
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