University of Massachusetts Amherst ScholarWorks@UMass Amherst Masters Theses 1911 - February 2014 2009 Identification of pmt, tr1, and h6h Gene Polymorphism and Tropane Alkaloid Chemotypes in Hyoscyamus Niger L. (black henbane) Lawrence Kramer University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/theses Part of the Genetics Commons, Natural Products Chemistry and Pharmacognosy Commons, and the Plant Breeding and Genetics Commons Kramer, Lawrence, "Identification of pmt, tr1, and h6h Gene Polymorphism and Tropane Alkaloid Chemotypes in Hyoscyamus Niger L. (black henbane)" (2009). Masters Theses 1911 - February 2014. 363. Retrieved from https://scholarworks.umass.edu/theses/363 This thesis is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Masters Theses 1911 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. IDENTIFICTION OF pmt, tr1, AND h6h GENE POLYMORPHISM AND TROPANE ALKALOID CHEMOTYPES IN Hyoscyamus niger L. (Black henbane) . A Thesis Presented by LAWRENCE KRAMER Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE September 2009 Plant Soil & Insect Sciences © Copyright by Lawrence Kramer 2009 All Rights Reserved TITLE OF THESIS: IDENTIFICTION OF pmt, tr1, AND h6h GENE POLYMORPHISM AND TROPANE ALKALOID CHEMOTYPES IN Hyoscyamus niger L. (Black henbane) . A Thesis Presented by LAWRENCE KRAMER Approved as to style and content by: _______________________________________ Lyle E. Craker, Chair _______________________________________ Robert Bernatzky, Member _______________________________________ Daniel J. Fairbanks, Member _______________________________________ David A. Reckhow, Member ____________________________________ Stephen Rich, Department Head Plant, Soil & Insect Sciences DEDICATION To my patient and loving wife Chandra J. Meesig and my sweet and vicious daughter Jane. ACKNOWLEDGMENTS The work would not have been possible to complete without Robert Berntzky, Ph.D. Maiko Inoue, Ph.D., and Daniel Fairbanks, Ph.D. for their help with the genetic analysis methods, David Reckhow, Ph.D for giving me access to his analytic equipment and advice on chromatography, Susan Han Ph.D. for her scientific and personal support, Lyle Craker, Ph.D. for supplies, equipment, and advice, and Scott Ebdon, Ph.D. and Geunhwa Jung, Ph.D. for their help with statistical analysis. I also thank Ahirhudi Dixit, Bibin Palouse and Indrajit Duda, Ph.D. for their help and encouragement, and the greenhouse managers Christopher Joyner and Jeffrey Anderson for many important favors and expert advice about plants and animals. v ABSTRACT IDENTIFICTION OF pmt, tr1, AND h6h GENE POLYMORPHISM AND TROPANE ALKALOID CHEMOTYPES IN Hyoscyamus niger L. (Black henbane) SEPTEMBER 2009 LAWRENCE KRAMER, B.S., KENT STATE UINVERSITY M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professors Robert Bernatzky and Lyle Craker To examine the significance of genetic variation on the tropane alkaloid (TA) constituents of the plant Hyoscyamus niger L. (black henbane), 13 accessions of North American and European origin were propagated in a random block design in greenhouse conditions. A selection of hybrid and F 2 populations were developed to compare to the parental chemotypes. Samples were lyophilized and the scopolamine and hyoscyamine content were quantified following liquid – liquid extraction by high performance liquid chromatography. Primers were designed to amplify the putrescine N-methyltransferase (pmt ), tropinone reductase 1 ( tr1 ), and hyoscyamine 6 β-hydroxylase (h6h ) genes of the TA biosynthetic pathway. Two alleles for the h6h gene locus were identified. The trend of genotype by chemotype comparison of a small number of F 2 samples from a monohybrid cross showed greater activity associated with the 690 bp amplicon of the h6h allele ( b) over the expected 550 bp amplicon ( a). Increased overall TA in root tissue of the bb genotype was accompanied with reduced scopolamine in leaf tissue. As a result, the marker is a good candidate for QTL analysis for linkage between TA biosynthesis and transport genes. vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...................................................................................................v ABSTRACT ....................................................................................................................... vi LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x CHAPTER 1. INTRODUCTION ..........................................................................................................1 1.1 Medicinal plants ................................................................................................1 1.2 Tropane alkaloids ..............................................................................................2 1.2.1 Biosynthesis of TAs ...........................................................................3 1.3 Increasing TA production .................................................................................5 1.3.1 Plant breeding ....................................................................................5 1.3.2 Whole plant regeneration of Hyoscyamus ssp. from cell and organ cultures. ........................................................................................7 1.3.3 Plant cell and organ culture systems ..................................................7 1.3.4 Genetic engineering of Hyoscyamus ssp. and Atropa ssp..................8 1.4 Commercial plant production, processing, and utilization. ..............................9 2. DIRECTION AND PURPOSE OF THE RESEARCH ................................................11 3. MATERIALS AND METHODS ..................................................................................13 3.1 Plant Material. .................................................................................................13 3.1.1 Selection of model plant. .................................................................13 3.1.2 Plant propagation .............................................................................14 3.1.3 Hybridization method. .....................................................................15 3.2 Sampling and preparation of plant material. ...................................................18 3.2.1 Extraction of TAs from lyophilized plant tissue. .............................19 vii 3.2.2 Quantification of TAs in plant tissue by high performance liquid chromatography. ........................................................................20 3.2.3 Chemotype parameters.....................................................................22 3.3 Genetic analysis ..............................................................................................22 3.3.1 Extraction of DNA ...........................................................................23 3.3.2 Determination of pmt , tr1 , h6h polymorphism using polymerase chain reaction....................................................................24 3.3.2.1 Primers ..............................................................................24 3.3.2.2 Conditions for polymerase chain reaction ........................27 3.3.2.3 PCR product separation and analysis ................................27 3.4 Statistical analysis. ..........................................................................................28 4. RESULTS AND DISCUSSION ...................................................................................29 4.1 Detection of Tropane Alkaloids ......................................................................29 4.2 Chemotypes among black henbane accessions and selection of breeding populations. .......................................................................................33 4.3 Polymorphic DNA at the pmt , tr1 , and h6h gene loci. ...................................34 4.4 DNA polymorphism among the breeding groups. ..........................................35 4.5 Polymorphism at the h6h gene locus in breeding population III. ...................40 4.6 Chemotype by genotype expression. ..............................................................41 5. CONCLUSIONS...........................................................................................................45 APPENDICES ............................................................... Error! Bookmark not defined. 46 A. EXTRACTION METHODS ........................................................................................46 B. BUFFERS FOR EXTRACTION OF DNA AND POLYMERASE CHAIN REACTIONS. ..............................................................................................................47 C. AGAROSE GELS. .......................................................................................................49 D. NIST PAGES FOR HYOSCYAMINE AND SCOPOLAMINE ................................51 E. U.S. ARMY MARK I NERVE AGENT ANTIDOTE KIT. .......................................53 F. TR 1 CDNA SEQUENCE ALIGNMENT WITH GENOMIC DNA. ...........................54 G. H6H CDNA SEQUENCE ALIGNMENT WITH GENOMIC DNA. ...........................56 REFERENCES