title: A genomic approach to isoflavone biosynthesis in kudzu (Pueraria lobata).
abstract: Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6'-deoxy- and 6'-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.
authors.affiliation: Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA.
authors.forename: XianZhi
authors.initials: X
authors.lastname: He
authors.forename: Jack W
authors.initials: JW
authors.lastname: Blount
authors.forename: Shujun
authors.initials: S
authors.lastname: Ge
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Tang
authors.forename: Richard A
authors.initials: RA
authors.lastname: Dixon
mesh_terms: D053898:Biosynthetic Pathways; D002851:Chromatography, High Pressure Liquid; D016000:Cluster Analysis; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D007529:Isoflavones / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D029908:Pueraria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D007529:Isoflavones; D016695:Glycosyltransferases; C033607:puerarin