Identification of pmt, tr1, and h6h Gene Polymorphism and Tropane Alkaloid Chemotypes in ...

72 Pages·2014·2.93 MB·English

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Masters Theses 1911 - February 2014
2009
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Lawrence Kramer
University of Massachusetts Amherst
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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
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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.
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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 populations were developed to compare to the
2
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 6β-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 samples from a
2
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.
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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
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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. TR1 CDNA SEQUENCE ALIGNMENT WITH GENOMIC DNA. ...........................54
G. H6H CDNA SEQUENCE ALIGNMENT WITH GENOMIC DNA. ...........................56

REFERENCES ..................................................................................................................58

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LIST OF TABLES
Table Page

1. Solanaceous plants evaluated between February and September 2006. ....... 13
2. Hyoscyamus niger Seed Information ............................................................ 14
3. Hybrids of p06 parental group by accession. ................................................ 17
4. Hybrids of p08 parental group by plant. ....................................................... 17
5. Parents and hybrids used in the experimental design. .................................. 18
6. Primers for pmt, tr1, and h6h. ....................................................................... 26
7. Duncan groupings among p08 henbane accessions of S, H, and S:H. .......... 34
8. Breeding population III genotypes. ............................................................... 41
9. Genotype aa, ab, and bb means and coefficient of linear correlation (r)
with S, H, S+H, and S:H. .............................................................................. 44

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