A HOMOLOGY BASED PCR CLONING STRATEGY TO ISOLATE A MONOTERPENE SYNTHASE GENE INVOLVED IN β-THUJAPLICIN BIOSYNTHESIS FROM CALLUS CULTURES OF CUPRESSUS DUPREZIANA A Thesis Presented to the faculty of the Department of Chemistry California State University, Sacramento Submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in Chemistry (Biochemistry) by Soraya Ghasemiyeh SUMMER 2012 ©2012 Soraya Ghasemiyeh ALL RIGHTS RESERVED ii A HOMOLOGY BASED PCR CLONING STRATEGY TO ISOLATE A MONOTERPENE SYNTHASE GENE INVOLVED IN β-THUJAPLICIN BIOSYNTHESIS FROM CALLUS CULTURES OF CUPRESSUS DUPREZIANA A Thesis by Soraya Ghasemiyeh Approved by: __________________________________, Committee Chair Dr. Tom Savage __________________________________, Second Reader Dr. Mary McCarthy-Hintz __________________________________, Third Reader Dr. Nicholas Ewing ____________________________ Date iii Student: Soraya Ghasemiyeh I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis. __________________________, Graduate Coordinator ____________ Dr. Susan Crawford Date Department of Chemistry iv Abstract of A HOMOLOGY BASED PCR CLONING STRATEGY TO ISOLATE A MONOTERPENE SYNTHASE GENE INVOLVED IN β-THUJAPLICIN BIOSYNTHESIS FROM CALLUS CULTURES OF CUPRESSUS DUPREZIANA by Soraya Ghasemiyeh β-thujaplicin, also referred to as hinokitiol, is a tropolone monoterpenoid with antimicrobial and antifungal properties. It is responsible for the decay resistance of heartwood of trees of the Cupressaceae. It is also used as an additive to toothpaste, cosmetics, and foods in Japan and has scavenging activity against reactive oxygen species and cytotoxic activity against several cancer cells lines. Here, Cupressus dupreziana callus cultures were developed as an experimental system to isolate thujaplicin biosynthetic genes. A homology-based PCR cloning strategy was employed in an attempt to isolate a monoterpene synthase gene involved in β- thujaplicin biosynthesis. _______________________, Committee Chair Dr. Tom Savage _______________________ Date v ACKNOWLEDGEMENTS There are many people I would like to thank. First and foremost I would like to thank Dr. Tom Savage, my research advisor, for all his help and guidance throughout this process. He has been a great mentor and I am lucky to have had him as my advisor. I would also like to thank my committee members, Dr. McCarthy and Dr. Ewing, for their help and insightful comments. Next I would like to thank John Disney and Ted Ferrera. Anytime I needed a chemical or equipment John helped me find it and whenever an instrument wasn’t working Ted was there to fix it. Chatting at the stockroom window was also therapeutic. Thanks to all my fellow research students for the encouraging words and friendship. A special thank you to Barbara Coulombe for helping me with my presentation as well as keeping me sane towards the end of this process. Finally I would like to thank my family. Thank you Hamid, for encouraging and supporting me to do more - always. Thank you Naseem and Eemon, I was able to focus on my studies because you are such great kids. I love you all more than I can say. And to my parents who instilled the love of learning and always made me believe I could do anything I set my mind to. vi TABLE OF CONTENTS Acknowledgements ....................................................................................................... vi List of Tables ................................................................................................................. ix List of Figures ................................................................................................................. x Chapter 1. INTRODUCTION..………………………………………………………………….1 1.1 Decay Resistance of Wood From the Cupressaceae ............................................ 1 1.2 Tropolones ............................................................................................................ 3 1.3 Thujaplicins .......................................................................................................... 5 1.4 Terpene Biosynthesis ............................................................................................ 7 1.5 Thujaplicin Biosynthesis .................................................................................... 11 2. METHODS AND MATERIALS ............................................................................. 19 2.1 Chemicals and Reagents ..................................................................................... 19 2.2 Initiation and Maintenance of Callus Cultures Providing β-thujaplicin ............. 20 2.3 Extraction and Derivitization Of β-Thujaplicin ................................................. 21 2.4 GC-MS Analysis for β-Thujaplicin and Other Monoterpenes ........................... 22 2.5 β-thujaplicin Elicitation by Methyl Jasmonte .................................................... 23 2.6 RNA Isolation ..................................................................................................... 24 2.6.1 Equipment Preparation .............................................................................. 24 2.6.2 RNA Extraction and Analysis ................................................................... 24 2.7 Amplification of Monoterpene Synthase Gene Candidates ............................... 27 2.7.1 Degenerate Primers ................................................................................... 27 2.7.2 Synthesis of 5’ RACE-ready cDNA and 3’ RACE-ready cDNA ............. 28 2.7.3 Rapid Amplification of cDNA Ends (RACE) ........................................... 29 2.7.4 Gel Purification of PCR Products .............................................................. 30 vii 2.7.5 Cloning of Amplified Sequences ............................................................... 30 2.7.6 Sequencing and Analysis ........................................................................... 32 3. RESULTS AND DISCUSSION ............................................................................... 33 3.1 Initiation and Maintenance of Callus Cultures ................................................... 33 3.2 Analysis of β-thujaplicin and Other Monoterpene Content in Callus Cultures ............................................................................................................... 35 3.3 Elicitation of β-thujaplicin by Methyl Jasmonate .............................................. 41 3.4 Isolation of RNA from C. dupreziana Callus Cultures ...................................... 42 3.5 Degenerate Primer Design .................................................................................. 48 3.6 Rapid Amplification of cDNA Ends (RACE) .................................................... 51 3.7 Conclusions ........................................................................................................ 68 References .................................................................................................................... 71 viii LIST OF TABLES Tables Page 1. Comparison of chemical components of Western Red Cedar, Western Hemlock and Douglas Fir, adapted from (2). Numbers expressed as a percentage of moisture-free weight of wood. ......................... 2 2. Degenerate primers designed using CODEHOP. N=A+T+G+C, D=G+A+T, H=A+T+C, R=A+G, W=A+T and Y=C+T. ............................. 28 3. Codon degeneracy ......................................................................................... 48 4. Degenerate primers for amplification of monoterpene symthase cDNA. N=A+T+G+C, D=G+A+T, H=A+T+C, R=A+G, W=A+T and Y=C+T. ................................................................................................... 51 5. Gene specific primers. .................................................................................... 56 6. Most homologous sequence based on BLASTx and tBLASTx searches of NCBI protein and nucleotide databases of 3’ RACE products. ......................................................................................................... 61 7. Most homologous sequence based on BLASTx and tBLASTx searches of NCBI protein and nucleotide databases of degenerate PCR products. ................................................................................................ 62 8. Sequence analysis from gel purified 3’ RACE PCR. ..................................... 66 ix LIST OF FIGURES Figure Page 1. Structure of tropolone (2-hydroxy-2,4,6,-cycloheptatrien-1-one). ................. 4 2. Some common tropolones and their structures from Cupressaceae (20). ......................................................................................... 6 3. Organization of terpene biosynthesis in plants. DMADP, dimethylallyl diphosphate. IDP, isopentyl diphosphate. ............................... 9 4. A mechanistic model of the monoterpene synthase reaction (12). ............... 10 5. Label positions of β-thujaplicin derived from [1-13C]-, [2-13C]-, and [U-13C]-glucose with assumed labeled positions of GPP on the way to β-thujaplicin shown in parenthesis. Numberings of geraniol carbons are shown with primes to distinguish them from β- thujaplicin carbons. Dots indicate 13C enriched, bold lines indicate short-range coupling and double-sided arrows indicate long-range coupling (31). ..............................................................................................