Date of Award
1-26-2017
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Fabricio Medina-Bolivar
Committee Members
Brett Savary; Carole Cramer; John Hershberger; Keithanne Mockaitis
Call Number
LD251.A566d 2016 Y36
Abstract
Peanut (Arachis hypogaea) produces prenylated stilbenoids as phytoalexins to fight against various pathogens. In addition, the prenylated stilbenoids exhibit several bioactivities with potential applications in human health. Despite their importance, the availability of prenylated stilbenoids is limited and their biosynthetic pathways remain to be elucidated. To date, stilbenoid prenyltransferase genes have not been identified from any plant species. Based on the prenyl unit position on the prenylated stilbenoids, it was hypothesized that different prenyltransferases are involved in the biosynthesis of distinct prenylated stilbenoids in peanut. To this end, an elicitor controlled stilbenoid bioproduction system was developed using hairy roots of peanut and the transcriptome from the elicitor-treated hairy roots was used to identify genes encoding for stilbenoid prenyltransferases. Hairy root cultures of peanut were induced to produce stilbenoids upon treatment with various elicitors. Co-treatment with methyl jasmonate and methyl-β-cyclodextrin led to sustained high levels of stilbenoids. The elicitation medium was subsequently optimized by addition of hydrogen peroxide and supplementation of magnesium chloride using an orthogonal array design. A two-step method was established for the purification of prenylated stilbenoids from the hairy root cultures that included high performance counter current chromatography followed by semi-preparative high performance liquid chromatography. The peanut hairy roots were further used to study the biosynthesis of prenylated stilbenoids. Using metabolic inhibitors, it was demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. Furthermore, the characterization of a membrane-bound stilbenoid-specific prenyltransferase activity was achieved. Using a combined transcriptomic and targeted metabolic approach, five stilbenoid-specific prenyltransferase genes that catalyze two distinct dimethylallylation steps in the biosynthesis of prenylated stilbenoids were cloned from the cDNA of peanut hairy roots. Functional characterization of two of these prenyltransferases, AhR4DT-1 and AhR3’DT-1, confirmed their specificity for stilbenoid substrates and their subcellular location in the plastid was confirmed by fluorescence microscopy. The strategies for the selection of prenyltransferase candidates can be used for cloning and characterizing other important genes in the biosynthesis of stilbenoids, as well as identifying genes involved in the biosynthesis of other bioactive metabolites in other species.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Yang, Tianhong, "Molecular cloning and characterization of a stilbenoid prenyltransferase gene family in peanut" (2017). Student Theses and Dissertations. 598.
https://arch.astate.edu/all-etd/598
