A Thesis Presented to the Faculty of the Department Of

Home , Cupressus dupreziana, Monoterpene, Resin acid, Thujaplicin

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

Chemistry

(Biochemistry)

Soraya Ghasemiyeh

SUMMER 2012

Soraya Ghasemiyeh

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

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

Abstract

A HOMOLOGY BASED PCR CLONING STRATEGY TO ISOLATE A MONOTERPENE

SYNTHASE GENE INVOLVED IN β-THUJAPLICIN BIOSYNTHESIS FROM CALLUS

CULTURES OF CUPRESSUS DUPREZIANA

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.

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

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)...... 15 6. Proposed pathway in β-thujaplicin biosynthesis based on label localization studies (31)...... 15 7. Proposed pathway in β-thujaplicin biosynthesis (32)...... 16 8. Proposed pathway for biosynthesis of β-thujaplicin (17, 34)...... 17 9. Mechanism of first-strand SMARTer 5’ cDNA synthesis...... 29 10. Mechanism of first-strand SMARTer 3’ cDNA synthesis...... 29 11. Average callus size after six weeks. Values and error bars represent means and standard errors of seven calli...... 34 12. C. dupreziana callus cutures...... 34 13. Derivatization of β-thujaplicin ...... 35

14. Total ion chromatogram of derivatized 1.0 mg/mL β-thujaplicin with 1.0 mg/mL 1,3-napthalenediol as the internal standard...... 36 15. Mass spectrum of derivatized β-thujaplicin standard...... 37 16. Total ion chromatograms of derviatized β-thujaplicin and 1,3 napthalenediol standards (top panel) and derivatized extract of C. dupreziana callus (bottom panel)...... 38 17. Mass spectrum of β-thujaplicin standard after derivatization (top panel) and derivatized β-thujaplicin from extracts of C. dupreziana (lower panel)...... 39 18. Total ion chromatogram of C. dupreziana callus culture (1) α-pinene, (2) 3-carene, (3) limonene, (4) terpinolene, (5) carvacrol and (6) β-thujaplicin. ………………………………………...41

19. UV-VIS spectrum of isolated RNA from callus B...... 44 20. Denaturing electrophoretic agarose (1.0 %) gel of RNA extracted from calli. Lane 1: wheat grass RNA control, Lane 2 : callus A, Lane 3 : callus B (From left to right)...... 45 21. Total ion chromatogram of 1.0 mg/mL β-thujaplicin with 0.1 mg/mL 2-napthol as the internal standard...... 46 22. Mass spectrum of β-thujaplicin standard...... 46 23. Total ion chromatograms of β-thujaplicin and 2-napthol standards (upper panel) and C. dupreziana callus B extract with 0.025 mg/mL 2-napthol as internal standard (lower panel)...... 47 24. Sections of the aligned monoterpene synthase sequences with

RRX8W and DDXXD motifs highlighted and the primer blocks marked above the sequences…………………………………………….....49 25. Overview of 5' and 3' first-strand cDNA synthesis and 5' and 3' RACE PCR…………………………………………………………….. .53

26. Agarose gel electrophoresis of 3’ and 5’ RACE products. Lane 1: 100 bp ladder (Invitrogen), Lane 2-4: 3’ RACE with Block C, Lane 5-7: 5’ RACE with Block O primer, Lanes 8-10: 5’ RACE with Block K primer, Lane 11: 100bp ladder (Invitrogen)…………………………………………………..54 27. Agarose gel electrophoresis of 5’ RACE products. Lane 1: Lamda III Hind digest (Invitrogen), Lane 2: Callus B 5' RACE with Block O primer; boxed band excised (~1500 base pairs), Lane 3: 100 base pair ladder (Fisher Scientific), Lane 4: Callus B 5’ RACE with Block K primer; boxed band excised (~850 base pairs)...... 55 28. Lane 1: 100 base pair ladder (Fisher Scientific), Lane 2: Callus B 3’ RACE with GSP 1, Lane 3: Callus B 3’ RACE with GSP 2 (excised bands boxed)………………………………………....57 29. Lane 1: 100 base pair ladder (Fisher Scientific) Lane 2: Fresh 3' RACE PCR product using GSP 2...... 58 30. Colony screening via lysis and electrophoresis…………………………….59 31. PCR screening of colonies with insert………………………...... 60 32. 5' RACE PCR products. Lane 1: 100 bp ladder (Fisher Scientific), Lane 2: 5’ RACE with Block K primer, Lane 3: 5’ RACE with Block O primer………..…………………………..62 33. 3' and 5’ RACE PCR products. Lane 1: 100 bp ladder (Fisher-Scientific), Lane 2: 3’ RACE with Block C primer, Lane 3: 5’ RACE with Block K primer, Lane 4: 5’ RACE with Block O primer...... 65

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Chapter 1

INTRODUCTION

Here, I discuss its role in the decay resistance of woods and how its antimicrobial activity has led to interest in understanding its biosynthetic origin to enable its production using biotechnology.

The goal of this study was to initiate and maintain callus cultures that actively produce β-thujaplicin in order to isolate the RNA. An RNA isolation protocol was developed for callus cultures and once isolated, the RNA was used in first-strand complementary DNA (cDNA) synthesis followed by 5’ and 3’ RACE so as to elucidate the DNA sequence of a monoterpene synthase gene used in β-thujaplicin production.

1.1 Decay Resistance of Wood From the Cupressaceae

Conifer trees from the family Cupressaceae (cedars and cypresses) are found globally, with the exception of Antarctica, and are well known for their resistance to decay (7). They have naturally durable softwood and traditionally the heartwood is

2 used in applications where decay resistance is necessary such as utility poles, fence posts, shingles and exterior construction. This quality spurred studies in the first half of the twentieth century demonstrating that the heartwood is rarely attacked by decay fungi (8-10). What differentiates the heartwood of the Cupressaceae from heartwood from other coniferous species is the high levels of extractives. As shown in Table 1, the wood from Western Red Cedar (a member of the Cupressaceae) has nearly twice the extractives as wood from other coniferous species, whereas levels of structural components cellulose and lignin are similar.

Table 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. Total Species Family Cellulose Hemicelluloses Lignin Extractives Western Red Cupressaceae 47.5 13.2 29.3 10.2 Cedar Western Pinaceae 48.8 14.7 28.8 5.3 Hemlock Douglas- Pinaceae 53.8 13.3 26.7 5.9 fir

Extractives, also known as secondary metabolites, are not an integral part of the cellular structure of the tree but rather are compounds soluble in neutral organic solvents or water (11). Secondary metabolites do not participate directly in growth or development of plants but have important roles in protection, plant survival and plant defense responses against insects, herbivores and microbial pathogens (12). They

3 include tannin, dyes, pitch, resins and gums that are responsible for the smell, taste and color of the wood (2).

Extractives form at the heartwood/sapwood boundary using precursors that have been transported from the phloem and sapwood. The concentration of extractives varies greatly throughout the tree with an increase of extractives observed with increasing distance from the pith (center) in heartwood and rapidly declining in sapwood. Extractives are also found in higher concentrations toward the base of the tree (2). Heartwood from younger trees contain a lesser amount of extractives than heartwood from mature trees; however, the amount in younger trees is comparable to the inner heartwood of mature trees, which is representative of the early growth period. This suggests that the extractives are stable, and as the tree matures production of extractives increases. This is consistent with the higher content of extractives found in newly formed heartwood (2).

1.2 Tropolones

Some extractives found in the Cupressaceae family contain a tropolone structure. Tropolones, are seven-membered aromatic compounds based on tropolone

(2-hydroxy-2, 4, 6-cycloheptatrien-1-one: Figure 1) that are relatively scarce in nature

(13, 14). Tropolones were not identified until 1945 when Dewar proposed this seven- membered ring structure for stipitatic acid, a compound from Penicillium stipitatum

(15). This discovery gave birth to the field of non-benzenoid aromatic compounds

(14).

Figure 1. Structure of tropolone (2-hydroxy-2,4,6,-cycloheptatrien-1-one).

Tropolones are of pharmacological interest due to their novel structures, chemical properties and biological activities (13). These special characteristics can be attributed to the 1,2 arrangement of the carbonyl and hydroxyl groups on the aromatic seven-membered ring (15). It has been suggested that these biological effects may be related to metal chelation between the carbonyl group at C-1 and the hydroxyl group of C-2 (16).

Natural tropolones are found mainly in plants, specifically in Cupressaceae and Liliaceae grass species, but a small number of bacterial and fungal tropolones exist as well (13, 17). Many different tropolones are present in the heartwood of the

Cupressaceae family, some ubiquitous and others species dependent (Figure 2).

1.3 Thujaplicins

A group of tropolone compounds found in Cupressaceae family are known as thujaplicins. The thujaplicins consist of three isomeric isopropyl tropolones known as

α-thujaplicin, β-thujaplicin (hinokitiol) and γ-thujaplicin (Figure 2). These thujaplicins are part of the volatile fraction of the heartwood extractives. Of the thujaplicins, β-thujaplicin is responsible for much of the decay resistance of the heartwood. β-thujaplicin inhibits blueing fungi more than the α-thujaplicin and γ- thujaplicin isomers or a synthetic fungicide, sodium pentacholorophenol, (8) and is also a broad-spectrum antibacterial compound, inhibiting Gram-positive and Gram- negative bacteria (7). It has been shown that β-thujaplicin can be used as a postharvest treatment to prevent decay in some fruits and is a government-approved food additive in Japan (4). β-thujaplicin also activates the hypoxia-inducible factor,

HIF-1, pathway by inhibiting HIF-specific hydroxylases in human HepG2 hematoma cells (ATCC HO-8065) and human HeLa cervical epithelium cells (ATCC CCL-2), making it a potential treatment of ischemic diseases (18). Recent work on the topical application of β-thujaplicin with 5% zinc oxide on the skin of patients with atopic dermatitis has shown inhibition, by interference of attachment, of Staphylococcus aureus, including methicillin-resistant S. aureus which is of concern in hospital settings (19). β-thujaplicin has been shown to have cytotoxic activity against various cancer cell lines including human stomach cancer KATO-III and Ehrlich’s ascites carcinoma (6).

Figure 2. Some common tropolones and their structures from Cupressaceae (20).

The antimicrobial and antiproliferative properties of β-thujaplicin have led to an increased demand and interest in new methods of its production. One potential source is to generate thujaplicins in fermentation organisms engineered with the ability to synthesize these compounds. However, the biosynthetic pathway and the corresponding genes that encode biosynthetic enzymes have yet to be elucidated.

Thus, there has been a recent focus on understanding the biochemistry and molecular genetics of thujaplicin biosynthesis.

1.4 Terpene Biosynthesis

The volatile fractions of the extractives found in Cupressaceae are terpenes.

Terpenes are the most structurally varied class of plant natural products and are derived by the fusion of isoprene units, branched 5 carbon units based on the

isopentane skeleton. Ten carbon (C10) monoterpenes contain two isoprene units,

fifteen carbon (C15) sesquiterpenes contain three isoprene units, and twenty carbon

(C20) diterpenes contain four isoprene units. Mono-, sesqui- and diterpenes are all found in wood from the Cupressaceae (21).

Terpene biosynthesis begins with the formation of the precursors isopentyl diphosphate (IDP) and its allylic isomer dimethylallyl diphosphate (DMADP). These precursors are derived from one of two pathways. The mevalonate (MEV) pathway is located in the cytosol and endoplasmic reticulum, and provides IDP and DMADP for sesquiterpene biosynthesis, whereas the 2-C-methyl erythritol-4-phosphate (MEP)

8 pathway occurs in plastids and generates IDP and DMADP for monoterpene and diterpene synthesis (Figure 3). These pathways differ in IDP precursors: acetyl-CoA is the precursor to IDP in the MEV pathway, whereas pyruvate and D-glyceraldehyde-

3-phosphate are the precursors to IDP in the MEP pathway.

Of the terpenes, monoterpenes are best known as constituents of volatile essences of flowers and the essential oils of herbs and spices. The biosynthesis of monoterpenes (Figure 4) starts with geranyl diphosphate (GDP) being formed in the plastid via the condensation of IDP and its isomer DMADP. Monoterpene synthases subsequently catalyze the cyclization of GDP to monoterpene hydrocarbons, alcohols or phosphates (Figure 4). Monoterpene synthases typically contain between 600 and

650 amino acid residues including an N-terminal transit peptide for plastid targeting

(22). Mechanistically, these enzymes ionize and isomerize GDP to form the tertiary allylic isomer linalyl diphosphate (LDP). Subsequent ionization of enzyme-bound

LDP leads to cyclization to form a six membered ring (the α-terpinyl carbocation) which can then undergo additional reactions including rearrangements, oxidations, addition of functional groups, or additional electrophilic cyclizations in the formation of many monoterpene skeletons (12). The monoterpene synthase-catalyzed reaction

(Figure 4) is the first committed step in monoterpene biosynthesis and is responsible for generating the skeletons that lead to all monoterpenes (23).

Figure 3. Organization of terpene biosynthesis in plants. DMADP, dimethylallyl diphosphate. IDP, isopentyl diphosphate.

Figure 4. A mechanistic model of the monoterpene synthase reaction (12)

1.5 Thujaplicin Biosynthesis

β-thujaplicin is a modified monoterpene with a tropolone structure (Figure 2) found only in small amounts in the heartwood of mature trees. For this reason, it is very difficult to acquire enough β-thujaplicin for analysis or applications (20). For example, each gram of sawdust from Thuja dolabrata only contains 200 µg of β- thujaplicin (24). Thus, callus cultures have been developed to provide a more robust and accessible experimental system to study tropolone accumulation. Calli are proliferating masses of undifferentiated cells that have the potential to produce the range of chemicals found in the parent plant (25). Callus cultures of Cupressus lusitanica and Thuja occidentalis have been established and studied for many years

(26), and these calli have been shown to produce β-thujaplicin.

De novo biosynthesis of β-thujaplicin and other monoterpenes in calli can be stimulated by treatment with methyl jasmonate or fungal extracts (26-28). β- thujaplicin accumulates initially upon elicitation in calli, and other monoterpenes, such as 4-terpineol and 1,6-epoxy-4(8)-en-p-menthene-2-ol, are produced later. A feedback regulation of β-thujaplicin exists in elicited culture, with its methyl ether being formed once a threshold of 40mg/L is reached suggesting that high levels β-thujaplicin may be toxic to callus cells (29).

Initial work to elucidate the biosynthetic pathway of β-thujaplicin occurred before it was recognized that β-thujaplicin is a monoterpene. Because polyketide- based pathways that involve polymerization of acetate or malonate are also responsible for a wide variety of natural product structures, early experiments were designed to differentiate between potential polyketide and isoprenoid origins of β- thujaplicin.

A precursor feeding study with calli by Yamaguchi et al. (30) incubated yeast- elicited C. lusitanica cells with [U-14C]glucose, [2-14C]mevalonate and [2-

14C]malonate as substrates (30). Significantly more incorporation of label into β- thujaplicin was observed from glucose than mevalonate, implying formation through a polyketide pathway. However, incorporation of radiolabel from [2-14C] malonate (a more direct polyketide precursor) was not observed, refuting the polyketide pathway.

It should be noted that the early experiments were performed before it was recognized that monoterpenes are formed from IDP generated by the MEP pathway rather than from mevalonate. Thus, the incorporation of label from glucose (a precursor for the

MEP pathway) rather than from malonate or mevalonate is consistent with thujaplicins being synthesized via a MEP-based isoprenoid pathway.

After the existence of the MEV pathway became well-established, an experiment was developed that involved feeding radiolabeled [2-14C] mevalonate, [10-

14C] geraniol and [U-14C] glucose to calli to confirm the isoprenoid origin of β-

13 thujaplicin and establish whether the isoprene substrate arises from the MEV or MEP pathway (31). Radiolabel from [10-14C] geraniol (which can be phosphorylated in vivo to GDP) was incorporated into β-thujaplicin more than 2 times greater than radiolabel from [U-14C] glucose and more than 200 times greater than radiolabel from

[2-14C] mevalonate, indicating that the isoprenoid GDP is a precursor to β-thujaplicin

(31).

Labeling experiments were then used to distinguish whether GDP incorporated into β-thujaplicin arises either from the MEV or MEP pathway. Isoprenoids produced from labeled [U-13C] glucose through the MEP pathway contain three carbons from the labeled glucose molecule incorporated into the final product that has a characteristic coupling spectra observable in 2D INADEQUATE NMR. GDP was shown to be a product of the MEP pathway due to the coupling patterns as well as negligible incorporation of [2-14C] mevalonate into β-thujaplicin.

Additional label localization studies using [1-13C] glucose, [2-13C] glucose and

[U-13C] glucose as precursors led to the development of a specific biosynthetic hypothesis (Figures 5 and 6). Geraniol derived from [1-13C]- and [2-13C]- glucose via the MEP pathway should be labeled at positions 1’, 5’, 9’ and 10’ and 2’, 3’, 6’ and 7’, respectively (Figure 5). β-thujaplicin derived from [1-13C]- and [2-13C]- glucose showed increased 13C NMR measurements, indicating labeled carbons, at positions 3,

5, 7 and 9 and at 1, 4, 6 and 8, respectively (Figure 5). This data indicates that

14 rearrangement of the isopropyl group did not occur during β-thujaplicin biosynthesis because the C-4 and C-8 were enriched with [2-13C]-glucose feeding and the long- range coupling between C-4 and C-9 (or 10) was present in [U-13C]-glucose feeding experiment. Therefore, C-3, C-4, C-8 C-9 and C-10 of β-thujaplicin corresponds to C-

5’, C-6’, C-7’, C-8’ and C-9’ of geraniol, respectively. The C-2 of β-thujaplicin is not labeled by [1-13C]- or [2-13C]-glucose feeding, but does exhibit long-range coupling with C-5 in the [U-13C]-glucose feeding experiment and must therefore be C-4’ from geraniol. An adjacent pair, C-2’ and C-3’ (enriched by [2-13C]-glucose) in geraniol was separated by an enriched carbon from [1-13C]-glucose feeding and the adjacent C-

3’ and C-10’ pair that have no long-range coupling with any other carbon in the [U-

13C]-glucose feeding remain adjacent as C-1 and C-7 of β-thujaplicin. Thus, the methyl group at the 10’-position in geraniol splits the C-2’ and C-3’ bond and therefore, C-1’, C-2’, C-3’ and C-10’ of geraniol correspond to C-5, C-6, C-1 and C-7 of β-thujaplicin. From the findings a carbon skeletal rearrangement from geraniol to

β-thujaplicin was suggested (Figure 6).

Figure 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).

Figure 6. Proposed pathway in β-thujaplicin biosynthesis based on label localization studies (31).

Another study suggested 2-carene or terpinyl acetate as possible intermediates in β-thujaplicin biosynthesis (Figure 7). This study involved feeding cultures with six unlabeled monoterpenes at different concentrations to investigate the relationship between the monoterpenes and β-thujaplicin biosynthesis. Addition of 2-carene or terpinyl acetate promoted β-thujaplicin accumulation by 2- 2.5 fold with respect to the control whereas the other monoterpenes were inhibitory. These results led the authors to propose two possible pathways, shown in Figure 7.

Figure 7. Proposed pathway in β-thujaplicin biosynthesis (32).

Finally, another pathway for β-thujaplicin biosynthesis has been proposed based on the timing of accumulation of related monoterpenes (Figure 8). By modifying the growth medium to prevent β-thujaplicin biosynthesis, 1,6-epoxy-4(8)- p-menthen-2-ol (33) rapidly accumulated whereas when the medium was modified to increase β-thujaplicin biosynthesis 1,6-epoxy-4(8)-p-menthen-2-ol remained at a basal level suggesting that this novel monoterpene may be an intermediate in β-thujaplicin biosynthesis (34).

Figure 8. Proposed pathway for biosynthesis of β-thujaplicin (17, 34).

Thus, to date, alternate proposals for the biosynthetic pathway to β-thujaplicin have been developed based on different types of experimental evidence. However, these pathways are not definitive and no progress has been made towards isolating genes encoding thujaplicin biosynthetic enzymes. Nonetheless, each proposed

18 pathway incorporates a monoterpene synthase-catalyzed reaction to produce the basic monoterpene skeleton, with subsequent modifications to generate thujaplicin.

Other studies on monoterpene biosynthesis have employed cloning techniques to isolate monotepene synthase genes. By isolating the monoterpene synthase genes, not only is it possible to arrive at more conclusive pathways in monoterpene biosynthesis but also enables transgenic manipulation to increase production of essential oils or phytopharmaceuticals (12, 21, 35).

Here we employ a molecular genetic approach to isolate a monoterpene synthase sequence involved in β-thujaplicin production. The first goal was to establish a tissue culture system that was actively producing β-thujaplicin to be used as a source of mRNA. The mRNA was isolated from the calli and consensus monoterpene synthase amino acid sequences were used to design degenerate primers for use in RT-PCR in order to amplify and isolate cDNA sequences encoding candidate monoterpene synthase genes.

Once a monoterpene synthase-like sequence is isolated we can then use portions of it as gene specific primers to get a full-length monoterpene synthase cDNA. This can then be cloned in to a microbial vector to express the enzyme in order to biochemically characterize and obtain correct functional identification of the gene product or products. This will enable a better understanding of molecular processes and characterization and may also enable microbial production of β- thujaplicin.

Chapter 2

METHODS AND MATERIALS

2.1 Chemicals and Reagents

Phytagel (plant cell culture powder), sucrose, Murashige and Skoog Basal Salt mixture (MS), Gamborg’s Vitamin Solution 1000X, α-napthalene acetic acid (NAA),

6-Benzylaminopurine (BAP), bromphenol blue, polyvinylpyrrolidone (PVP), ethidium bromide, β-mercaptoethanol (BME), and 3-(N-morpholino)propanesulfonic acid

(MOPS) were purchased from Sigma-Aldrich (St. Louis, MO). Molecular biology grade ethylenediaminetetraacetic acid (EDTA), Tris-HCl, aurintricarboxylic acid, sodium acetate and lithium chloride were purchased from Calbiochem (Darmstadt,

Germany). Optical grade cesium chloride was purchased from IBI Scientific (Pesota,

IA). N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA +

1%TMCS) was purchased from Thermo Scientific (Rockford, IL). 1,3-napthalenediol was supplied from Matheson, Coleman and Bell (Norwood, OH); dithiothreitol (DTT) from ACROS Organics (Geel, Belgium); and agarose, TBE buffer and formamide from Fisher Biotech (Fair Lawn, NJ). Amberlite MB3 resin was purchased from

Mallinckrodt (St.Louis, MO) and formaldehyde from Spectrum (Gardena, CA).

SMARTer™ RACE cDNA Amplification Kit was purchased from Clontech (Mountain

View, CA), the pCR®8/GW/TOPO® TA Cloning® Kit and custom primers were purchased from Invitrogen (Grand Island, NY), the 5 PRIME MasterMix from 5

PRIME (Gaithersburg, MD) and the Quantum Prep® Plasmid Miniprep Kit were purchased from BioRad (Hercules, CA). Thiabendazol, diethylpyrocarbonate (DEPC) and α-pinene were purchased from MP Biomedicals Inc. (Solon, OH). All water used in RNA isolation was treated with 0.1% v/v DEPC left overnight and then autoclaved before use.

2.2 Initiation and Maintenance of Callus Cultures Providing β−thujaplicin

To screen a variety Cupressus species for the ability to produce callus cultures capable of producing β-thujaplicin, young branches of C. dupreziana, C. lawsonia, C. macnabiana, C. nootkatensis and Thuja orientalis were obtained from the CSUS arboretum and samples of C. lusitanica were obtained from the UC Davis arboretum.

Specimens from the CSUS arboretum were identified by botany professor Michael

Baad and the sample from the UC Davis arboretum was identified by Arboretum

Superintendant Emeritus Warren Roberts. Branch samples were cut from the trees and placed on ice. Each sample was cut into 10 - 15 cm long pieces and surface-sterilized by soaking in 85% (v/v) ethanol solution for ten minutes, followed by soaking in 20%

(v/v) bleach and SDS solution for thirty minutes. The samples were then soaked in

70% ethanol solution for ten minutes and subsequently rinsed three times with sterilized deionized water. Each piece was then sliced into 1-2 mm thick slivers and placed on 0.15% (w/v) phytagel agar plates containing 0.43% (w/v) Murashige and

Skoog salts (36), 10-5M BAP, 10-2M NAA, 0.1% (w/v) 1000X Gamborg’s Vitamin

Solution, 2.0% sucrose and 2.5 x 10-4 M thiabendazole at pH 5.5. These plates were then sealed with parafilm and placed in the dark for two weeks. Once established, the calli were then transferred onto MS plates excluding thiabendazole to encourage more vigorous growth, and were replated every four to six weeks.

2.3 Extraction and Derivitization Of β-Thujaplicin

To analyze β-thujaplicin production in the established cultures, 0.5 g fresh callus was pulverized in a mortar and pestle and one mL of ethyl acetate containing

1.56 x 10-4 M 1,3-napthalenediol as an internal standard was added. The resulting paste was then placed in an airtight vial and the mortar and pestle was rinsed with ethyl acetate and added to the paste and left overnight. The extract was subsequently filtered through glass wool and the samples were dried on a vacuum concentrator on medium heat. The dried samples had 100 µL of methylene chloride added to insure complete removal of water and dried again. Finally, 150 µL of BFSTA–1% TMCS was added to the dried samples to generate the trimethylsilyl derivative. A sample of

6.09 x 10-4 M β-thujaplicin with 1.56 x 10-4 M 1,3-napthalenediol was subjected to the same procedure as a positive control. These solutions were placed in airtight vials until analysis by gas chromatography-mass spectrometry (GC-MS).

2.4 GC-MS Analysis for β-Thujaplicin and Other Monoterpenes

Gas chromatography-mass spectroscopy (GC-MS) analysis of derivatized β- thujaplicin standard and callus extracts were performed in electron impact mode with an Agilent 7890 GC equipped with an Agilent 5975 MS. Separation was carried out using a HP-5MS, (5%-Phenyl)-methylpolysiloxane, column, 0.25 mm diameter and

0.25µm film thickness. Split injections of 1 µL were used. The temperature program was: 40 oC for 1 minute, followed by a temperature ramp of 20 oC/min to 150 oC, 30 oC/min to 280 oC, finally holding at 280 oC for 7 minutes. The carrier gas was helium at a linear velocity of 1.2 mL/min, and the injector was maintained at 250 oC .

Analyses were performed in full scan mode over the range of m/z 40-550. The MS source and quadrapole were maintained at 230oC and 150oC, respectively.

Quantification of β-thujaplicin was performed by GC-MS with the addition of

1.56x10-4 M 1,3-napthalenediol as the internal standard. The areas of total ion peaks were integrated and levels of β-thujaplicin present in different calli were calculated based on comparison of peak areas to the known concentration of 1,3-napthalenediol.

To analyze other monoterpenes, calli were pulverized in a mortar and pestle then steam-distilled with deionized water. The distillate was extracted with ice-cold pentane, which was concentrated by evaporation with nitrogen gas and examined by

GC-MS using the same instrument and column as before except the temperature program was 40oC for one minute, followed by a temperature ramp of 5 oC/minute to

200 oC, holding for 5 minutes. The compounds were identified by comparison to the

NIST database.

For direct analysis of β-thujaplicin without derivatization, 2-napthol was used as an internal standard. The calli were pulverized in a mortar and pestle, and 1mL of ethyl acetate containing 1.73 x 10-4 M 2-napthol as an internal standard was added.

The resulting paste was placed in an airtight vial, and the mortar and pestle were rinsed with ethyl acetate, which was added to the paste, which was then left overnight at room temperature. The extracts were filtered through glass wool, and the samples were dried on a vacuum concentrator on medium heat. The dried samples were mixed with 100 µL of methylene chloride to insure complete removal of water and then dried again. A sample of 6.09 x 10-4 M β-thujaplicin with 6.94 M x 10-4 M 2-napthol was subjected to the same procedure as a positive control. These solutions were placed in airtight vials until analysis by GC-MS.

2.5 β-thujaplicin Elicitation by Methyl Jasmonte

A 200 mM solution of methyl jasmonate was made by adding 223.6 µL of methyl jasmonate to methanol for a final volume of 5 mL. Five µL of the 200 mM methyl jasmonate solution was then added to 5 mL of nanopure water for a final

200µM solution and filter-sterilized through a 0.2µm filter. A control solution was made by adding 5 µL of methanol to nanopure water and filter-sterilizing. Four C. dupreziana calli were halved and transferred to fresh MS plates; one half was treated

24 with 1 mL of the control solution while the other half was treated with 1 mL of the

200 µM methyl jasmonate solution. Each set of calli was incubated for a week at room temperature before extraction, derivatization and GC-MS analysis.

2.6 RNA Isolation

2.6.1 Equipment Preparation

All glassware and nonplastic equipment was washed and rinsed three times with nanopure water and oven dried at 185 oC overnight. All reusable plastic materials, such as polypropylene centrifuge tubes, were washed and rinsed with nanopure water followed by soaking in 3% H2O2 for 10 minutes and finally rinsed in

DEPC (0.1% v/v) treated water. Disposable plastics such as pipet tips and eppendorf tubes were DNase and RNase free. All surfaces were wiped down with ethanol and aseptic techniques employed.

2.6.2 RNA Extraction and Analysis

Six different protocols were used in an attempt to isolate RNA of sufficient yield and purity for cDNA synthesis. Methods tried involved the Spectrum™ Plant

Total RNA Kit (Sigma-Aldrich) according to manufacturer’s protocol, the Spectrum™

Plant Total RNA Kit (Sigma-Aldrich) modified by adding 1% w/v PVP to lysis solution as well as performing a 10% v/v cold ethanol precipitation step, a LiCl/hot phenol extraction (37), isolation using TRIzol® (38), Direct-zol™ RNA Miniprep Kit

(Zymo Research) and a modified version of a protocol by Lewinsohn et al (39). Only the Lewinsohn protocol afforded viable RNA from the callus cultures, however, high quality RNA from fresh wheat grass was isolated with the Spectrum™ Plant Total

RNA Kit (Sigma-Aldrich), which was used as a control for electrophoretic analysis of

RNA .

Using the Lewinsohn protocol, C. dupreziana callus cultures containing β- thujaplicin, based on GC-MS analysis, were frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. The powder was then added to the extraction buffer (200 mM Tris-HCl, 300 mM LiCl and 10 mM Na acetate in DEPC treated water, pH 8.5, containing 1% w/v PVP, 1mM aurintricarboxylic acid and 10 mM ditihiothreitol) in polypropylene tubes and vortexed, followed by centrifugation at

5000 x g for 20 minutes at 4 oC. The supernatant was decanted and 1/30 volume (110 mM final concentration) of 3.3M sodium acetate buffer, pH 6.1, and 10% (v/v) of cold ethanol were added. This was incubated on ice for 10 minutes to allow for polysaccharide precipitation and then centrifuged at 5000 x g for 20 minutes at 4oC.

The supernatant was collected and mixed with 1/9 volume (440 mM final concentration) of 3.3M Na acetate buffer, pH 6.1, and cold isopropanol was added to

33% (v/v) to precipitate nucleic acids. This solution was mixed and held at -20oC for at least two hours before centrifugation at 5000 x g for 20 minutes at 4oC. The supernatant was discarded and the pellet was dissolved in one mL of autoclaved nanopure water. CsCl was added to a final concentration of 0.4 g/ml. This solution

26 was then centrifuged at 5000 x g for 10 minutes at 4oC to remove any insoluble material. A discontinuous CsCl gradient was prepared by layering 500 µL of solution

A (5.7 M CsCl and 10mM Na-EDTA made with DEPC treated water with pH adjusted to 7.5 and autoclaved) at the bottom of an ultracentrifuge tube with a 1 mL of solution

B (2.8 M CsCl, 10mM Na-EDTA made with DEPC treated water, pH adjusted to 7.5 and autoclaved) layered on top of it followed by a layer of 500 µL of the sample.

Ultracentrifuge tubes were then balanced with solution C (2.4 M CsCl, 10mM Na-

EDTA made with DEPC treated water and pH adjusted to 7.5 and autoclaved). The samples were then centrifuged on a Beckman tabletop TL-100 ultracentrifuge in a

TLS-55 rotor at 106,000 x g for 23 hours at 11oC. The supernatant was removed by pipetting from top to bottom. The pellet containing the RNA was then resuspended in

40 µL of autoclaved nanopure water and centrifuged at 14000 x g for 15 minutes at

4oC (39). This supernatant was then subjected to spectrophotometric analysis and quantitation.

UV spectroscopy was carried out using an HP 8452 A Diode Array

Spectrophotometer. Prior to analysis, a quartz cuvette was soaked in chromic acid for ten minutes then rinsed ten times with autoclaved nanopure water to remove any

RNase contamination. A 50-fold dilution of the RNA was made by adding 6 µL of isolated RNA to 294 µL of nanopure water. Samples were scanned between 200-400 nm. Ratios of A260-A320/A280-A320 were calculated to determine RNA purity.

Quantification of RNA was done using the equation: A260 x 40 µg/mL x 50 (dilution factor).

Additionally, RNA integrity was analyzed by denaturing agarose gel electrophoresis. Electrophoresis equipment was washed and rinsed in nanopure water, soaked in 3% H2O2 for 10 minutes and rinsed with DEPC treated water. All solutions were made with DEPC treated water. RNA samples were separated by gel electrophoresis using a 1.2% (w/v) formaldehyde denaturing agarose gel (40) and stained with ethidium bromide.

2.7 Amplification of Monoterpene Synthase Gene Candidates

2.7.1 Degenerate Primers

Degenerate primers were designed based on homologous domains in known monoterpene synthase protein sequences. Twenty-six monoterpene synthase protein sequences from the order Pinales were selected from the NCBI Entrez protein database. Conserved amino acid motifs were identified by the alignment of these sequences using the ClustalW multiple alignment program (41). These sequences were subjected to the Block multiple alignment processor program and used in the consensus-degenerate hybrid oligonucleotide primer (CODEHOP) program (42). This program designs degenerate primers for use in polymerase chain reactions (PCR).

Three degenerate primers were chosen based on Tm, nucleotide length and clamp scores (Table 1).

Table 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.

Block C forward primer GAATGGGAGACTACCACTCCAAYHWNTGGRA Block K reverse primer TGGTTCCGAAGGTGTCGTANAYRTCRTC Block O reverse primer GATCCGGGGTGGTCCTTNADRTARAC

2.7.2 Synthesis of 5’ RACE-ready cDNA and 3’ RACE-ready cDNA

First-strand cDNA for 5’ and 3’ RACE was generated by reverse transcription of the isolated RNA using the SMARTer™ RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions. This system uses SMARTScribe RT

(reverse transcriptase), which has terminal transferase activity that adds 3-5 residues at the 3’ end of the first strand of 5’ cDNA. The SMARTer oligonucleotide contains a terminal stretch of modified bases that anneals to the extended cDNA, where the oligonucleotide can then serve as an extended template for the reverse transcriptase leading to the generation of a complete cDNA copy of the original RNA with the addition of the extra SMARTer sequence at the end (Figure 9). Because the template switching occurs only when the reverse transcriptase reaches the end of the RNA template the SMARTer sequence is usually only incorporated into full-length first strand cDNAs (43). The first-strand generation of 3’ RACE-ready cDNA utilizes a modified oligo T primer that contains the SMARTer oligonucleotide sequence which allows use of the universal primer for subsequent amplification (Figure 10).

29 SMARTer™ RACE cDNA Ampliﬁcation Kit User Manual Appendix A: Detailed Flow Chart of 5’ RACE

poly A+ RNA 5' – NBAAAAA– 3' NV TTTTT21 5'-CDS SMARTer ﬁrst-strand synthesis

SMARTer II A oligo – 3' XXXXX NBAAAAA 5' – XXXXX NV TTTTT21 – 5'

RT template switching

5'-RACE-Ready cDNA 5' – XXXXX NBAAAAA– 3' 3' – XXXXX NV TTTTT21 – 5' SMARTer™ RACE cDNA Amplification Kit User Manual 5'-RACE PCR Appendix Figure B: 9 .Detailed Mechanism Flow of first Chart-strand SMARTerof 3’ RACE 5’ cDNA synthesis. 5' – XXXXX NBAAAAA– 3' Long UP 3' – XXXXX NV TTTTT21 – 5' First round of PCR Incorporation of suppression PCR inverted repeat poly A+ RNA 5' – NBAAAAA– 3' elements: NV TTTTT26 3'-CDS by Long Universal Primer 5' – NBAAAAA21 – 3' GSP1 3' – NV TTTTT21 – 5' Standard first-strand synthesis Second= round SMART ofer PCR II A sequence Gene-specific synthesis 3'-RACE-Ready cDNA 5' – NBAAAAA– 3' NV TTTTT26 – 5'

5' – NBAAAAA30 – 3' 3'-RACE PCR Figure 10. MechanismShort of UP first-strand SMARTer 3’ cDNA synthesis. 3' – – 5'

5' – NBAAAAA– 3' Remaining rounds PCR GSP2 Ampliﬁcation of 5' fragment 2.7.3 Rapid Amplification3' – of cDNA EndsNV TTTTT (RACE)26 – 5'

5' – – 3' 3' – – 5' First round of PCR Following the generation of 5’-RACE-Ready cDNA and Gene-speciﬁc3’-RACE-Ready synthesis Double-stranded 5'-RACE fragment

NBAAAAA26 – 3' cDNA, 5’-RACE and 3’-RACE were performed using theLong UPSMARTer RACE cDNA 3' – NV TTTTT26 – 5' Amplification kit. 5’-RACE-Ready cDNA was used as the templateNext roundswith the of PCR universal Incorporation of suppression PCR inverted repeat primer as the forward primer and either the Block K or Block O degenerateelements: primers as Figure 5. Detailed mechanism of the 5’-RACE reactions. by Long Universal Primer 5' ––NBAAAAA26 3'

the reverse primer. Additionally3' – 3’-RACENV TTTTT-Ready26 cDNA– 5' was used as the template

Protocol No.with PT4096-1 the forward degenerate primer Blockwww.clontech.com C and the universal primer as the reverseClontech Laboratories, Inc. Version No. 011312 A Takara Bio Company 28

5' – NBAAAAA26 – 3' Short UP 3' – NV TTTTT26 – 5' Remaining rounds of PCR Ampliﬁcation of 3' fragment

5' – – 3' 3' – – 5'

Double-stranded 3'-RACE fragment

Figure 6. Detailed mechanism of the 3’-RACE reactions.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4096-1 A Takara Bio Company Version No. 011312 29 30 primer, according to the manufacturer’s protocol. PCR was performed on an

Eppendorf Mastercycler® Personal PCR Thermocycler. The PCR program used was:

35 cycles at 94 oC for 30 seconds, 46 oC for 30 seconds, and 72 oC for 3 minutes followed by 10 minute extension time at 72oC.

Samples of cDNA, synthesized with the SMARTerTM RACE kit, along with a

100 bp ladder (Fisher Scientific) were separated by electrophoresis in a 1.2% (w/v) agarose/0.005% (w/v) EtBr gel in 22.5 mM Tris-borate (pH8), 0.5mM EDTA buffer.

The gel was run at 85 volts for 2-3 hours.

2.7.4 Gel Purification of PCR Products

PCR products that had been separated by gel electrophoresis that were of appropriate size were excised and subjected to Nucleo Trap® extraction via manufacturer’s protocol. These extracts were subjected to gel electrophoresis to determine their concentration by comparison with the molecular weight markers’ concentrations and then sent for sequencing.

2.7.5 Cloning of Amplified Sequences

Gel-purified or unpurified PCR products were cloned into a pCR®8/GW/TOPO® vector and then transformed into One Shot® chemically competent E. coli cells according to the manufacturer’s protocol. These cells were

31 then plated on LB media plates containing 100 µg/ml spectinomycin and incubated overnight at 37oC.

Antibiotic-resistant colonies from the transformation were screened for inserts by electrophoretic analysis of plasmid size as follows. Colonies were picked using a sterile pipet tip, streaked on master LB spectinomycin plate (one per colony), and placed in separate polypropylene tubes containing 30 µl of 1X lysis buffer (10% w/v sucrose, 100 mM NaOH, 60 mM KCl, 5 mM EDTA, 0.25% (w/v) SDS). The tubes were incubated in a 37 oC water bath for 5 minutes, placed on ice for 5 minutes and then centrifuged at 14,000 x g for 10 minutes (44). Loading dye (2µL) was added to

10 µl of each supernatant and then loaded onto a 0.8% agarose gel made with 0.5%

TBE, along with 1 µl of a Hind III digest of lambda phage (Lambda Hind III) as a molecular marker. The gel was run at 100 volts for 1.5 – 2 hours.

Colonies containing plasmids with inserts, as determined by electrophoretic analysis, were then subjected to PCR screening using the appropriate degenerate primer and a vector-based primer to confirm that the insert was the PCR product, as follows. PCR-grade water (5 µl) was inoculated with a colony, and 8 µl of 5 Prime

Master Mix (5 Prime) was added, along with 0.5 µl of appropriate degenerate primer and 0.5 µl of vector-based primer, and 6 µl of PCR-grade water was added to bring the final volume to 20 µl (45). The PCR program used was: 94 oC for 5 minutes, followed by 29 cycles of 94 oC for 30 seconds, 60 oC for 30 seconds, 72 oC for 2 minutes,

32 followed by a 10 minute extension period at 72 oC. The products were then separated on a 1.2% agarose gel to determine directionality as well as insert size.

2.7.6 Sequencing and Analysis

Colonies that contain plasmids with inserts of the expected size as determined by PCR screening were then grown overnight in 3 ml of LB media containing 100

µg/ml spectinomycin for plasmid isolation. Plasmids were isolated using a BioRad miniprep kit, according to manufacturer’s protocols. Isolated plasmids were sequenced at Davis Sequencing on an ABI 3730 sequencer using a either a GW1 or

GW2 primer from the TOPO® Cloning Reaction (Invitrogen).

Chapter 3

RESULTS AND DISCUSSION

3.1 Initiation and Maintenance of Callus Cultures

The main was to isolate a nucleotide sequence with homology to a monoterpene synthase gene involved in the biosynthesis of β-thujaplicin. One approach is to isolate mRNA from tissue that is actively synthesizing β-thujaplicin to obtain cDNA, which can serve as a template for PCR amplification of monoterpene synthase cDNA using degenerate primers designed based on conserved amino acid sequences among monoterpene synthases. First tissue needed to be obtained that was actively producing β-thujaplicin from which to obtain mRNA. Because β-thujaplicin is normally found in the heartwood of mature trees from Cupressaceae, and a mature tree could not be sacrificed to obtain RNA from the heartwood, callus cultures were initiated and screened for β-thujaplicin accumulation. Callus cultures are undifferentiated cells that have the ability to produce the range of chemicals present in the parent plant and have been used to produce β-thujaplicin (26, 29, 32).

Sections of small branches from six different Cupressaceae species (Cupressus dupreziana, Cupressus lawsoniana, Cupressus lusitanica, Cupressus macnabiana,

Cupressus nootkatensis and Thuja orientalis) were sampled from local arboretums, surface sterilized and placed on solid Murashige and Skoog media to induce callus proliferation. It took three to six weeks for the calli to grow and become established, although cells from C. macnabiana and T. orientalis did not grow. Growth of the calli

34 were monitored, and those of C. dupreziana grew the largest (Figure 11). These calli were maintained by replating every 4-6 weeks (Figure 12).

Callus diameter (mm) Callus diameter 0

Figure 11. Average callus size after six weeks. Values and error bars represent means and standard errors of seven calli.

Figure 12. C. dupreziana callus cutures.

3.2 Analysis of β-thujaplicin and Other Monoterpene Content in Callus Cultures

To determine whether the established calli produce β-thujaplicin GC-MS analysis was employed. Initial GC-MS analysis of underivatized β-thujaplicin resulted in very poor peak shape. Compounds that are not volatile or lack thermal stability can be derivatized to allow for analysis via GC-MS, so derivatization with

BSTFA and 1% TMCS was used to increase the volatility and stability of β-thujaplicin to allow for better peak shape and easier detection and quantification by GC-MS.

Derivatization modifies the polar OH group on β-thujaplicin by replacing the hydrogen with a trimethylsilyl group allowing for a less polar and more volatile compound (Figure 13).

O O CH3

OH O Si CH3

BSFTA + 1% TMCS CH3

Figure 13. Derivatization of β-thujaplicin

A derivatized sample of β-thujaplicin standard with an internal standard, 1,3- napthalenediol, for quantification which was used in all extractions, was subjected to

GC-MS analysis for comparison with derivatized callus extracts (Figure 14).

Derivatized β-thujaplicin has a molecular weight of 236.3, and the mass ion can be detected in the mass spectrum (Figure 15). The mass spectrum also has a base ion peak of m/z 221.3, likely due to the loss of a methyl group from the trimethyl silyl group. This is the first reported mass spectrum of trimethylsilyl β-thujaplicin.

3000000

2500000 β-thujaplicin

2000000

1500000 Abundance Abundance 1000000 1,3-napthalenediol

500000

0 10.5 11 11.5 12 12.5 13 13.5 14 14.5 Retention Time (min)

Figure 14. Total ion chromatogram of derivatized 1.0 mg/mL β-thujaplicin with 1.0 mg/mL 1,3-napthalenediol as the internal standard.

Abundance

Scan 180 (12.026 min): 020909C.D\data.ms 221.3 340000

320000

300000

280000

260000

240000

220000

200000

180000

160000

Abundance 140000

120000

100000 206.3 80000 73.2 60000

40000 179.3 91.2 135.2 151.2 20000 51.0 115.2 236.3 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 m/z--> m/z

Figure 15. Mass spectrum of derivatized β-thujaplicin standard.

A callus from each established species was then extracted with ethyl acetate

containing the internal standard and analyzed by GC-MS after derivatization with

BSFTA-TMCS. Total ion chromatograms were obtained for derivatized extracts from

each callus to determine which species produced the most β-thujaplicin.

Quantification of β-thujaplicin was achieved by comparision of the peak areas to that

of the internal standard. Calli from two of the four species analyzed (C. dupreziana

and C. lusitanica) had detectable β-thujaplicin, based on a similar retention times and

mass spectra as the β-thujaplicin standard (Figure 16 and 17). To establish the

variability of β-thujaplicin production for each of these two species, five calli from C.

lusitanica and eight from C. dupreziana were analyzed. β-thujaplicin was detected in

3000000

2500000

2000000 β-thujaplicin

1500000 Abundance Abundance 1000000 1,3-napthalenediol

500000

0 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 Retention Time (min)

400000

350000 1,3-napthalenediol 300000

250000

200000

Abundance Abundance 150000

100000 β-thujaplicin

50000

0 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 Retention Time (min)

Figure 16. Total ion chromatograms of derviatized β-thujaplicin and 1,3 napthalenediol standards (top panel) and derivatized extract of C. dupreziana callus (bottom panel).

Abundance

Scan 180 (12.026 min): 020909C.D\data.ms 221.3 340000

320000

300000

280000

260000

240000

220000

200000

180000

160000

Abundance 140000

120000

100000 206.3 80000 73.2 60000

40000 179.3 91.2 135.2 151.2 20000 51.0 115.2 236.3 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 m/z--> m/z

Abundance

Scan 173 (11.874 min): 042409A.D\data.ms 221.2 16000

15000

14000

13000

12000

11000

10000

9000

8000

7000

6000 Abundance 73.0 5000

4000 206.2

3000

2000 55.0 91.1 151.1 179.2 135.1 1000 117.1 236.2 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 m/z--> m/z

Figure 17. Mass spectrum of β-thujaplicin standard after derivatization (top panel) and derivatized β-thujaplicin from extracts of C. dupreziana (lower panel).

40 five of the eight C. dupreziana calli tested, whereas β-thujaplicin was detected in only one C. lusitanica callus. The C. dupreziana calli in which β-thujaplicin was detected had average levels of β-thujaplicin of 0.01 mg per gram of callus similar to levels found in related C.lusitanica calli in other studies.

From these results, it was determined that C. dupreziana calli more consistently produced β-thujaplicin, as well as having the fastest calli growth.

Although other groups have used C. lusitanica cultures as an experimental system for studying β-thujaplicin production (26, 29, 46), C. dupreziana cells were chosen because of the more consistent production of β-thujaplicin.

To obtain a more complete understanding of the monoterpene composition within C. dupreziana callus cultures, non-thujaplicin monoterpenes were extracted by steam distillation of calli and analyzed by GC-MS. The installation of a new column on the GC-MS allowed for analysis of both non-thujaplicin monoterpenes and β- thujaplicin with good peak shape without derivatization. Based on the mass spectra and retention times, some of the monoterpenes detected in C. dupreziana callus cultures were : α-pinene, 3-carene, limonene, terpinolene, carvacrol and β-thujaplicin

(Figure 18). C. lusitanica callus cultures also contain α-pinene, limonene and terpinolene; however, other monoterpenes present in C. lusitanica calli that we did not detect in C. dupreziana include β-ocimene, myrcene and sabinene.

600000

6 500000

400000

300000 Abundance 200000

100000 1 2 3 5 4 0 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Retention Time (min)

Figure 18. Total ion chromatogram of C. dupreziana callus culture (1) α-pinene, (2) 3- carene, (3) limonene, (4) terpinolene, (5) carvacrol and (6) β-thujaplicin.

3.3 Elicitation of β-thujaplicin by Methyl Jasmonate

β-thujaplicin biosynthesis has been shown to be elicited by methyl jasmonate, a compound similar to β-thujaplicin that is involved in plant defense responses, or yeast elicitor (26-28, 46, 47). We tested the effects of methyl jasmonate on four C. dupreziana calli. After treatment with 200 µM methyl jasmonate for one week, calli showed a two- to three-fold increase in β-thujaplicin production compared to the control calli. The elicited calli had an average 0.030 mg β-thujaplicin per gram of

42 callus while the unelicited calli had an average of 0.0133 mg of β-thujaplicin per gram of callus. This three-fold increase in β-thujaplicin production is less than the 10-fold increase seen after the addition of fungal elicitors to C. lusitanica callus cultures and the ten-fold increase in β-thujaplicin production after the addition of 200 µM methyl jasomate to C. lusitanica suspension cultures (26, 34, 46).

Because calli treated with methyl jasmonate had increased β-thujaplicin production they were promising candidates for RNA isolation. However mold formed on the calli preventing their use as an RNA source. At the time we did not have enough calli and so after several unsuccessful attempts to isolate RNA from elicited calli we isolated RNA from unelicited calli which still produced significant levels of

β-thujaplicin but were less prone to mold development.

3.4 Isolation of RNA from C. dupreziana Callus Cultures

Having developed a tissue culture system that produces significant levels of β- thujaplicin, RNA isolation to generate a template for isolation of monoterpene synthase cDNAs was then pursued. RNA isolation from callus cultures is very difficult due to the high concentration of soluble polysaccharides and polyphenols

(39). Phenolic substances can bind and inactivate RNA, and polysaccharides can co- precipitate with RNA and interfere with subsequent isolation steps (39, 48). Six different RNA extraction methods, including two commercial plant RNA extraction kits, were evaluated for their ability to yield RNA of sufficient quantity and quality for

43 cDNA synthesis. A version of the Lewinsohn protocol (39), slightly modified by the absence of insoluble PVPP as well as being scaled down, yielded viable RNA. This method employs polyvinylpyrrolidone (PVP) to bind phenolic compounds, EDTA and thiourea to inhibit polyphenoloxidases, and aurintricarboxylic acid to inhibit RNases.

The method also includes a 10% (v/v) ethanol precipitation to remove non-nucleic acid associated polysaccharides.

The purity and concentration of RNA was evaluated by UV-VIS spectrophotometry. Purity of RNA is crucial for RT-PCR cDNA synthesis. Spectra in the range of 200-400 nm were obtained and readings at A260, A280 and A320 were taken.

By using the equation: A260-A320/A280-A320, an assessment of RNA purity can be made.

Pure RNA absorbs maximally at 260 nm whereas protein, a source of contamination, absorbs maximally at 280 nm. The absorbance at 320 nm is a measure of background absorbance, turbidity and scatter, and by subtracting this from A260 and A280 values a more accurate estimation of RNA purity can be obtained. If the ratio is greater than

~1.8 it is indicative of pure RNA. Furthermore, an approximate quantification of RNA can also be made by using the equation: concentration of RNA = A260 x 40µg

RNA/mL x dilution factor. RNA from two C. dupreziana calli, A and B, were isolated and analyzed using UV-VIS spectrophotometry. A260-A320/A280-A320 ratios of 1.77 and

1.74 were obtained, and concentrations of 194µg/mL and 828.7µg/mL were calculated for RNA isolated from calli A and B, respectively. The ratios were slightly lower than

44 desired but the UV spectra indicated the RNA was of sufficient quality for cDNA synthesis (Figure 19).

Figure 19. UV-VIS spectrum of isolated RNA from callus B.

To insure the isolated RNA was not significantly degraded, a sample of the isolated RNA was subjected to agarose gel electrophoretic analysis. Unlike DNA,

RNA is single-stranded and, thus, able to form secondary structures via intramolecular base pairing which can affect its electrophoretic mobility. Consequently, RNA was electrophoresed under denaturing conditions to disrupt RNA secondary structure.

After electrophoresis on a denaturing gel, ribosomal bands should be visible; if not, the RNA is not intact. An agarose formaldehyde gel revealed the presence of distinct, albeit faint, ribosomal bands for RNA from calli A and B, similar to that found in preparations of high quality RNA from wheat grass (Figure 20).

1 2 3

Figure 20. Denaturing electrophoretic agarose (1.0 %) gel of RNA extracted from calli. Lane 1: wheat grass RNA control, Lane 2 : callus A, Lane 3 : callus B (From left to right).

To confirm that the calli from which RNA was isolated were actively accumulating β-thujaplicin, half of each callus used for RNA isolation was extracted with ethyl acetate containing 2-napthol as an internal standard and analyzed by GC-

MS. A β-thujaplicin standard with 2-napthol was subjected to GC-MS analysis as a control (Figure 21). β-thujaplicin has a molecular weight of 164.1, and the mass ion can be detected in the mass spectrum, which is similar to a previously reported (24) β- thujaplicin mass spectrum (Figure 22). This control was used for comparision with extracted calli after RNA isolation. In both callus A and callus B, β-thujaplicin was

46 detected by GC-MS (Figure 23) with an average of 1.3 µg of β-thujaplicin per gram of callus.

300000

250000 β-thujaplicin 200000

150000

100000 Abundance Abundance 2-napthol 50000

0 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 Retention Time (min)

Figure 21. Total ion chromatogram of 1.0 mg/mL β-thujaplicin with 0.1 mg/mL 2- naptholAbundance as the internal standard.

Scan 1011 (8.485 min): B THUJASTND.D\data.ms 121.1 55000

50000

45000

40000

35000

30000 164.1 25000

20000

Abundance 15000 77.1 91.1 10000 103.1 65.1 5000 41.1 51.1 136.1 149.1 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 m/z--> m/z

Figure 22. Mass spectrum of β-thujaplicin standard.

300000

250000

200000 β-thujaplicin 150000

100000 2-napthol

50000

Abundance 0 8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 9.5

600000

500000

400000 2-napthol

300000

200000

100000 β-thujaplicin Abundance 0 8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 9.5 Retention time (min)

Figure 23. Total ion chromatograms of β-thujaplicin and 2-napthol standards (upper panel) and C. dupreziana callus B extract with 0.025 mg/mL 2-napthol as internal standard (lower panel).

3.5 Degenerate Primer Design

Because the goal was to identify a monoterpene synthase cDNA sequence and the exact sequence was unknown, degenerate primers were employed. Degenerate primers are used to amplify unknown DNA sequences when the protein sequence is known or when protein sequences from other species are known. Because the genetic code is degenerate, i.e., more than one codon can specify the same amino acid (Table

3), degenerate primers contain a pool of all possible combinations of nucleotides that code for a given amino acid sequence. Often protein sequences are conserved between different species, whereas DNA sequences are not, so degenerate primers designed based on conserved amino acid sequences can allow amplification of nucleotides encoding the conserved protein. This approach has been used in other studies to identify monoterpene synthase genes in novel organisms (21, 35, 49-53).

Table 3. Codon degeneracy Number of codons Amino Acids 1 Met, Trp 2 Phe, Tyr, His, Gln, Asn, Lys, Asp, Glu, Cys 3 Ile 4 Val, Pro, Thr, Ala, Gly 6 Leu, Ser, Arg

A search of the NCBI non-redundant protein sequence database revealed 183 monoterpene synthase sequences from many different species, including Abies grandis and Pinus taeda. Twenty-six monoterpene synthase sequences from conifer species were aligned using the multiple alignment sequencing program ClustalW to identify

49 conserved domains within these sequences. Monoterpene synthases have two well- known conserved motifs with known function (49, 54), the RRX8W motif commonly found in cyclizing monoterpene synthases and DDXXD motif critical in divalent cation-assisted substrate binding, which were both present in the alignments. In

Figure 24, portions of the aligned sequences are shown with the RRX8W and DDXXD motifs are highlighted.

Block C gi|62511233|sp|Q9M7C9.1|TPSDB_ V---INMKLTTVSHRDDNGGGVLQRRIADHHPNLWEDDFIQSLS-SPYGG 94 gi|7381253|gb|AAF61455.1|AF139 V---INMKLTTVSHRDDNGGGVLQRRIADHHPNLWEDDFIQSLS-SPYGG 94 gi|17367918|sp|O22340.1|TPSDA_ V---INMKLTTVSHRDDNGGGVLQRRIADHHPNLWEDDFIQSLS-SPYGG 94 gi|2429145|gb|AAB70907.1| V---INMKLTTVSHRDDNGGGVLQRRIADHHPNLWEDDFIQSLS-SPYGG 94 gi|15080741|gb|AAK83565.1|AF32 V---INMKLTTVSHRDDNDGGVLQRRIADHHPNLWEDDFIQSLS-SPYGG 94 gi|17367924|sp|O24475.1|TPSD3_ T-PSISMSSTTVVTDD-----GVRRRMGDFHSNLWDDDVIQSLP-TAYEE 88 gi|2411483|gb|AAB71085.1| T-PSISMSSTTVVTDD-----GVRRRMGDFHSNLWDDDVIQSLP-TAYEE 88 gi|15080739|gb|AAK83564.1| T-PSISMSSTTVVTDD-----GVRRRMGDFHSNLWDDDVIQSLP-TAYEE 83 gi|28894482|gb|AAO61225.1| TRASMSMNLRTAVSDD-----AVIRRRGDFHSNLWDDDLIQSLS-SPYGE 90 gi|34582667|gb|AAP72020.1| T-PSMSMSLTTTVSDD-----GVQRRMGDFHSNLWNDDFIQSLS-TSYGE 88 gi|28894486|gb|AAO61227.1| R---PSMSLSTVASED-----DIQRRTGGYLSNLWNDDVIQFLS-TPYGE 88 gi|62511221|sp|Q948Z0.1|TPSD6_ A-HSINMCLTSVASTD-----SVQRRVGNYHSNLWDDDFIQSLISTPYGA 84 gi|2411485|gb|AAB70707.1| A-HSINMCLTSVASTD-----SVQRRVGNYHSNLWDDDFIQSLISTPYGA 84 gi|28894488|gb|AAO61228.1| A-PSMSMSSTTSVSNED----GVPRRIAGHHSNLWDDDSIASLS-TSYEA 89 gi|28894490|gb|AAO61229.1| R-PSIRVSSTASVSNDD----GVRRRVGDYRYNHWDEDLIDSLA-TSYEA 88 gi|62511234|sp|Q9M7D0.1|TPSD9_ T-HSLRMSLSTAVSDDH----GVQRRIVEFHSNLWDDDFIQSLS-TPYGA 89 gi|7381251|gb|AAF61454.1|AF139 T-HSLRMSLSTAVSDDH----GVQRRIVEFHSNLWDDDFIQSLS-TPYGA 89 gi|77454875|gb|ABA86247.1| T-PSISMCWTATVLDD-----GVQRRIANHHSNLWDDSFIQSLS-TPYGE 87 gi|62511235|sp|Q9M7D1.1|TPSD8_ TP-SVSMSLTTAVSDDG-----LQRRIGDYHSNLWDDDFIQSLS-TPYGE 86 gi|7381249|gb|AAF61453.1|AF139 TP-SVSMSLTTAVSDDG-----LQRRIGDYHSNLWDDDFIQSLS-TPYGE 86 gi|77454877|gb|ABA86248.1| APASMSMILTAAVSDDD----RVQRRRGNYHSNLWDDDFIQSLS-TPYGE 90 gi|21322150|gb|AAK39127.2| APASTSMILTAAVSDDD----RVQRRRGNYHSNLWDDDFIQSLS-TPYGE 90 gi|2411481|gb|AAB71084.1| TP-SMSISLATAAPD-D----GVQRRIGDYHSNIWDDDFIQSLS-TPYGE 88 gi|17367921|sp|O24474.1|TPSD2_ TP-SMSISLATAAPD-D----GVQRRIGDYHSNIWDDDFIQSLS-TPYGE 88 gi|21322152|gb|AAK39128.2|AF36 TP-SMSMSLNTVVSDND----AVQRRIGDYHSNLWNDDFIQSLT-TPYGA 89 gi|35764438|dbj|BAC92722.1| R---NTMAMATTSVES------VTRRTGNHHGNLWDDDFIQSLPKLPYDA 79 : : : ** . * *::. * * .*

Block K gi|62511233|sp|Q9M7C9.1|TPSDB_ LLTVLDDMYDTFGTLDELQLFTTAFKRWDLSETKCLPEYMKAVYMDLYQC 432 gi|7381253|gb|AAF61455.1|AF139 LLTVLDDMYDTFGTLDELQLFTTAFKRWDLSETKCLPEYMKAVYMDLYQC 432 gi|17367918|sp|O22340.1|TPSDA_ LVTVLDDIYDTFGTMNELQLFTDAIKRWDLSTTRWLPEYMKGVYMDLYQC 432 gi|2429145|gb|AAB70907.1| LVTVLDDIYDTFGTMNELQLFTDAIKRWDLSTTRWLPEYMKGVYMDLYQC 432 gi|15080741|gb|AAK83565.1|AF32 LVTVLDDIYDTFGTMNELQLFTDAIKRWDLSTTRWLPEYMKGVYMDLYQC 432 gi|17367924|sp|O24475.1|TPSD3_ LITVLDDMYDTFGTVDELELFTATMKRWDPSSIDCLPEYMKGVYIAVYDT 423 gi|2411483|gb|AAB71085.1| LITVLDDMYDTFGTVDELELFTATMKRWDPSSIDCLPEYMKGVYIAVYDT 423 gi|15080739|gb|AAK83564.1| LITVLDDMYDTFGTVDELELFTATMKRWDPSSIDCLPEYMKGVYIAVYDT 418 gi|28894482|gb|AAO61225.1| IITVLDDMYDTFGTLDELELFTSAIKRWDPSATECLPEYMKGVYMIVYNT 424 gi|34582667|gb|AAP72020.1| IITILDDMYDTFGTVDELELFTAAMKRWDPSAADCLPEYMKGVYLILYDT 422 gi|28894486|gb|AAO61227.1| ILTVLDDMYDLFGTVDELKLFTAAIKRWDPSATDCLPQYMKGIYMMVYNT 422 gi|62511221|sp|Q948Z0.1|TPSD6_ LITVLDDMYDVFGTVDELELFTATIKRWDPSAMECLPEYMKGVYMMVYHT 413 gi|2411485|gb|AAB70707.1| LITVLDDMYDVFGTVDELELFTATIKRWDPSAMECLPEYMKGVYMMVYHT 413 gi|28894488|gb|AAO61228.1| MITILDDIYDTFGTMEELKLLTAAFKRWDPSSIECLPDYMKGVYMAVYDN 423 gi|28894490|gb|AAO61229.1| FVTVLDDIYDTYGTMEELELFTAAIKRWDPSVVDCLPEYMKGVYMAVYDT 416 gi|62511234|sp|Q9M7D0.1|TPSD9_ LITVLDDIYDTFGTMEELELFTAAFKRWDPSATDLLPEYMKGLYMVVYET 425 gi|7381251|gb|AAF61454.1|AF139 LITVLDDIYDTFGTMEELELFTAAFKRWDPSATDLLPEYMKGLYMVVYET 425 gi|77454875|gb|ABA86247.1| LGTVLDDIYDTFGTMDELELFTAAVKRWHPSAAEGLPEYMKGVYMMFYET 421 gi|62511235|sp|Q9M7D1.1|TPSD8_ LITVLDDIYDTFGTMDEIELFNEAVRRWNPSEKERLPEYMKEIYMALYEA 425 gi|7381249|gb|AAF61453.1|AF139 LITVLDDIYDTFGTMDEIELFNEAVRRWNPSEKERLPEYMKEIYMALYEA 425 gi|77454877|gb|ABA86248.1| LNTVLDDIYDTFGTMDEIELFTEAVRRWDPSETESLPDYMKGVYMVLYEA 429 gi|21322150|gb|AAK39127.2| LNTVLDDIYDTFGTMDEIELFTEAVRRWDPSETESLPDYMKGVYMVLYEA 429 gi|2411481|gb|AAB71084.1| LVTVLDDIYDTFGTIDELELFTSAIKRWNSSEIEHLPEYMKCVYMVVFET 422 gi|17367921|sp|O24474.1|TPSD2_ LVTVLDDIYDTFGTIDELELFTSAIKRWNSSEIEHLPEYMKCVYMVVFET 422 gi|21322152|gb|AAK39128.2|AF36 LVTVLDDVYDTFGKMDELELFTAAVKRWDLSETERLPEYMKGLYVVLFET 428 gi|35764438|dbj|BAC92722.1| LATIIDDIYDTYGSIEELKHFTEVFKRWDSSPPDYLPEYMKIAYSALYDG 402 : *::**:** :*.::*:: :. ..:**. * **:*** * .:.

Block O gi|62511233|sp|Q9M7C9.1|TPSDB_ DTRCYKADRARGEEASAISCYMKDHPGSTEEDALNHINVMISDAIRELNW 582 gi|7381253|gb|AAF61455.1|AF139 DTRCYKADRARGEEASAISCYMKDHPGSTEEDALNHINVMISDAIRELNW 582 gi|17367918|sp|O22340.1|TPSDA_ DTRCYKADRARGEEASAISCYMKDHPGSIEEDALNHINAMISDAIRELNW 582 gi|2429145|gb|AAB70907.1| DTRCYKADRARGEEASAISCYMKDHPGSIEEDALNHINAMISDAIRELNW 582 gi|15080741|gb|AAK83565.1|AF32 DTRCYKADRARGEEASAISCYMKDHPGSTEEDALNHINAMISDAIRELNW 582 gi|62511234|sp|Q9M7D0.1|TPSD9_ DTRCYKADRARGEEASCISCYMKENPGSTEEDAINHINAMVNNLIKEVNW 575 gi|7381251|gb|AAF61454.1|AF139 DTRCYKADRARGEEASCISCYMKENPGSTEEDAINHINAMVNNLIKEVNW 575 gi|77454875|gb|ABA86247.1| DIHSYQAERSRGEESSGISCYMKDNPESTEEDAVTHINAMINRLLKELNW 571 gi|28894488|gb|AAO61228.1| DTQCYKADRARGEEASAVSCYMKDHPGITEEDAVNQVNAMVDNLTKELNW 573 gi|28894490|gb|AAO61229.1| ------GQGSVVQEASSVSCYMKDNAGLTEEDAIHCINDMVNNLLKELNW 560 gi|17367924|sp|O24475.1|TPSD3_ DTRCYKADRARGEEASSISCYMKDNPGVSEEDALDHINAMISDVIKGLNW 573 gi|2411483|gb|AAB71085.1| DTRCYKADRARGEEASSISCYMKDNPGVSEEDALDHINAMISDVIKGLNW 573 gi|15080739|gb|AAK83564.1| DTRCYKADRARGEEASSISCYMKDNPGVSEEDALDHINAMISDVIKGLNW 568 gi|34582667|gb|AAP72020.1| DTRCYKADRARGEEASSISCYMKDNPGATEEDALDHINAMISDVIRGLNW 572 gi|28894482|gb|AAO61225.1| DTRCYQADRARGEEASCISCYMKDNPGTTEEDALNHLNAMISDVIKGLNW 574 gi|28894486|gb|AAO61227.1| DTRCYQADRARGEETSCISCYMKDNPGATEEDALNHLNVMISGVIKELNW 572 gi|62511221|sp|Q948Z0.1|TPSD6_ DTRCYKADRARGEEASSISCYMKDNPGLTEEDALNHINFMIRDAIRELNW 563 gi|2411485|gb|AAB70707.1| DTRCYKADRARGEEASSISCYMKDNPGLTEEDALNHINFMIRDAIRELNW 563 gi|62511235|sp|Q9M7D1.1|TPSD8_ DTRCYKADRDRGEEASSISCYMKDNPGLTEEDALNHINAMINDIIKELNW 575 gi|7381249|gb|AAF61453.1|AF139 DTRCYKADRDRGEEASSISCYMKDNPGLTEEDALNHINAMINDIIKELNW 575 gi|77454877|gb|ABA86248.1| DTRCYKADRARGEEASCISCYMKDNPGSTEEDALNHINSMINEIIKELNW 579 gi|21322150|gb|AAK39127.2| DTRCYKADRARGEEASCISCYMKDNPGSTGEDALNHINSMINEIIKELNW 579 gi|2411481|gb|AAB71084.1| DTRCYKADRDRGEEASCISCYMKDNPGSTEEDALNHINAMVNDIIKELNW 572 gi|17367921|sp|O24474.1|TPSD2_ DTRCYKADRDRGEEASCISCYMKDNPGSTEEDALNHINAMVNDIIKELNW 572 gi|21322152|gb|AAK39128.2|AF36 DTRCYXADRARGEEASCISCYMKDHPGSTEEDAVNHINAMINDIIRELNW 578 gi|35764438|dbj|BAC92722.1| DTRTFKAEANRGEVTSSIACYLKEHPESTEKDALKYLQFMLDENLKELNL 551 .: : :* ::**:*::. :**: :: *: : :*

Figure 24. Sections of the aligned monoterpene synthase sequences with RRX8W and DDXXD motifs highlighted and the primer blocks marked above the sequences.

These aligned sequences were then organized into blocks (short multiply aligned ungapped segments that correspond to highly conserved regions of protein) using the

Blockmaker component of the CODEHOP program (42). The CODEHOP program then designs degenerate primers for the blocks. Many primers were generated by the

CODEHOP program, and we focused on primers from blocks that are most conserved among monoterpene synthases. Three primers were chosen from the possible degenerate primers generated from the CODEHOP program (Figure 24). The primer from Block C contains a portion of the RRX8W motif whereas Block K contains a portion of the DDXXD motif, both of which are present in monoterpene synthases.

Block O was chosen from another downstream conserved region. These primers were then used in 5’ and 3’ RACE PCR.

Table 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.

Block C forward primer GAATGGGAGACTACCACTCCAAYHWNTGGRA Block K reverse primer TGGTTCCGAAGGTGTCGTANAYRTCRTC Block O reverse primer GATCCGGGGTGGTCCTTNADRTARAC

3.6 Rapid Amplification of cDNA Ends (RACE)

The first step in RACE involves the conversion of RNA to the first strand of cDNA by use of reverse transcriptase in a manner that allows subsequent amplification of either the 5’ or 3’ ends of the transcript (Figure 25). The first strand of cDNA that can serve as a template for isolating the 5’ end of the cDNA is known as

5’ RACE-ready cDNA, and the first stand of cDNA that can serve as a template for isolating the 3’ end of the cDNA is known as 3’ RACE-ready cDNA. In generating 5’

RACE-ready cDNA, the specific system used for this study employs a reverse transcriptase with terminal transferase activity that adds 3-5 residues to the 3’ end of the first strand. An oligonucleotide (SMARTer oligo) anneals to the extra bases allowing for full-length, first-strand cDNA. In generating 3’ RACE-ready cDNA, the first-strand cDNA is synthesized using a standard reverse transcription procedure but a special oligo(dT) primer that includes part of the SMARTer oligo sequence at its 5’ end. The SMARTer oligo contains a universal priming sequence for use in the 3’ and

5’ RACE reactions (Figure 25). Once first-strand synthesis is complete, the respective

5’ and 3’ RACE-ready cDNA is used in 5’ and 3’ RACE PCR reactions utilizing gene- specific primers and the universal primer that recognizes the SMARTer oligo sequence.

SMARTer™ RACE cDNA Amplification Kit User Manual III. Introduction & Protocol Overview The SMARTer RACE cDNA Amplification Kit provides a method for performing both 5’- and 3’-rapid amplification of cDNA ends (RACE). The SMARTer RACE cDNA Amplification Kit is an improved version of our original SMART RACE Kit, with a new, SMARTer II A oligonucleotide and SMARTScribe Reverse Transcriptase 53 included; it provides better sensitivity, less background and higher specificity. This powerful system allows you to isolate the complete 5’ sequence of your target transcript from as little as 10 ng of total RNA. The cornerstone of the SMARTer RACE cDNA Amplification Kit is SMART technology, whichSMARTer™ eliminates RACE the need cDNA Amplification Kit User Manual for problematic adaptor ligation and lets you use first-strand cDNA directly in RACE PCR, a benefit that makes RACE far less complexAppendix and much B: faster Detailed (Chenchik Flow et al., Chart1998). Additionally, of 3’ RACE the SMARTer RACE Kit exploits Clontech’s patented suppression PCR & step-out PCR to increase the sensitivity and reduce the background5’ of RACE the RACE reactions. As a result you can use either poly3’ ARACE+ or total RNA as starting material SMARTer™ RACE cDNA Amplificationfor constructingKit User Manual full-length cDNAs of even very rare transcripts. SMART technology provides a mechanism for generating Appendix A: Detailed Flow Chart Polyof A5’+ RNA RACE full-length cDNAs in reverse transcriptionSMARTer™ reactions RACE cDNA (Zhu Amplification Kit User Manual 5' polyA 3' poly A+ RNA 5' – NBAAAAA– 3' et al., 2001). This is made possible NVby TTTTT the26 joint action of the XXXXX 3'-CDS 5' AppendixOligo (dT) primer B: DetailedSMARTer FlowII A Oligonucleotide Chart of 3’ and RACE SMARTScribe Reverse Transcriptase (a variant of MMLV RT). The SMARTScribe RT, SMARTer II A First-strand synthesis Standard first-strand synthesis Oligonucleotide coupled with upon reaching the end of an RNA template, exhibits terminal First-strand + 5' – – 3' First-strand poly A RNA tailing by NBAAAAA 3’ = SMARTer II A sequence NV TTTTT21 transferase activity, adding 3–5 residues to the 3’ end of the SMARTScribe RT5'-CDS synthesis synthesis poly Afirst-strand+ RNA 5'5' – – cDNA (Figure 1). The SMARTerNBAAAAANBAAAAA– 3'– 3' oligo containsRACE a 3'-RACE-ReadySMARTer cDNA first-strand synthesis NVNV TTTTT TTTTT26 – 5' terminal stretch of modified bases that anneal26 3'-CDS to the extendedReady XXXXX 5' polyA SMARTer5' II A XXXXX cDNA tail, allowing the oligo to serve as a template for thecDNAStandard RT. first-strand synthesis oligo – 3' XXXXX Template switchingNBAAAAA 3'-RACE PCR 5' – XXXXX NV TTTTT21 – 5' SMARTScribe RT switches templates from the mRNA molecule = SMARTer II A sequence and extension by – 3' SMARTScribe RT 3'-RACE-Readyto cDNA the SMARTer5' – oligo, generating aNBAAAAA complete cDNA copy of 5’ NV TTTTT26 – 5' RACE- 5' RTthe template original switching5' – RNA with the additionalNBAAAAA SMARTer– 3' sequence at XXXXX polyA 3'-RACE PCR Ready XXXXX the end. Since the templateGSP2 switching activity of the RT occurs 3' – NV TTTTT – 5' 5'-RACE-ReadycDNA cDNA 5' – XXXXX NBAAAAA– 3' only when the enzyme reaches the end of 26the RNA template, 3' – XXXXX NV TTTTT – 5' Figure 1. Mechanism of SMARTer cDNA synthesis.21 First- the SMARTer5' – sequence is typicallyNBAAAAA only – incorporated3' into strand synthesis is primed using a modified oligo(dT) GSP2 First round of PCR full-length,3' – first-strand cDNAs. For NVmore TTTTT26 information– 5' about primer. After SMARTScribe Reverse Transcriptase (RT) Gene-specific synthesis reaches the end of the mRNA template, it adds several 5'-RACESMART PCR technology, read "Generation and use of high-qualityFirst round of PCR nontemplate residues. The SMARTer II A Oligonucleotide cDNA from small amounts of total RNA by SMART Gene-specific PCR" synthesis 5' – annealsXXXXX to the tail of the cDNA and servesNBAAAAA as an extend– 3' - NBAAAAA26 – 3' Longed UP template for SMARTScribe RT. (Chenchik et al., 1998). This process guarantees that the use NBAAAAA26 – 3' 3' – XXXXX NV TTTTT – 5' Long UP 21 of high quality RNA will result in the formationLong UP of a set of 3' – NV TTTTT26 – 5' FirstcDNAs round thatof3' PCR – have a maximum amountNV TTTTT of 5’26 sequence– 5' (Table I). Incorporation of suppression PCR inverted repeat NextNext rounds rounds of of PCR PCR elements: IncorporationIncorporation of of suppression suppression Table I: Additional 5’-RACE Sequence Obtained with SMART Technology PCR inverted repeat by Long Universal Primer elements:PCR inverted repeat 5' – NBAAAAA21 – 3' by Long Universal Primer 5' –– 3' elements: Human gene GSP1 Size of mRNA Additional NBAAAAAMatches26 Includes 3' – NV TTTTT21 – 5' by Long Universal Primer (kb) sequence5'3' ––– genomic 3' – 5'transcription NVNBAAAAA TTTTT26 26 3’ RACE Second round of PCR Gene-specific synthesis(bp)* sequences start site 5’ RACE 3' – NV TTTTT26 – 5' Transferrin receptor 5.0 +25 yes yes

5' – Smooth muscle g-actin NBAAAAA30 – 3' 1.28 +31 yes yes Short UP 5' – NBAAAAA26 – 3' 3' – Vascular smooth muscle – 5' actin 1.33 +17 yes Short UP yes 3' – NV TTTTT26 – 5' Cytoskeletal -actin 1.9 +1 yes yes Remaining rounds PCR Remaining rounds of PCR Amplification of 5' fragment 23 kDa HBP 0.67 5' – +9 NBAAAAAyes26 – 3' yes Amplification of 3' fragment p53 2.6 +4 yes Short UP yes 3' – – 5' 5' – – 3' NV TTTTT26 3' – 5' – – 3' Interferon- receptor – 5' 2.06 +143' – yes – 5' yes 14-3-3 protein 1.03 +1 n/a n/a Remaining rounds of PCR Double-stranded 5'-RACE fragment Double-stranded 3'-RACE fragment Amplification of 3' fragment Interferon-receptor 2.75 +17 yes yes

n/a = not available 5' – – 3' * Compared to GenBank cDNA sequence. 3' – – 5'

Figure 25. Overview of 5' and 3' first-strandFigure cDNA 6. Detailed mechanism synthesis of the 3’-RACE reactions. and 5' and 3' RACE Clontech Laboratories, Inc. www.clontech.com Double-stranded 3'-RACE fragmentProtocol No. PT4096-1 PCR. A Takara Bio FigureCompany 5. Detailed mechanism of the 5’-RACE reactions. Version No. 011312 5 Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4096-1 A Takara Bio Company Version No. 011312 Protocol No. PT4096-1 www.clontech.com Clontech Laboratories, Inc. 29 Version No. 011312 A Takara Bio Company 28 In our 3’ RACE reaction, the degenerateFigure 6. Detailed primer mechanism of the 3’-RACEBlock reactions. C is used as the forward primer, and the universal primer is used as the reverse primer. In the 5’ RACE

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4096-1 A Takara Bio Company Version No. 011312 reaction, the universal primer is used as the forward primer, and either degenerate 29 primers Block K or Block O are used as the reverse primers. Based on the location of the blocks within monoterpene synthase sequences, the expected products were

54 approximately 1700 base pairs for 3’ RACE with Block C, 1200 base pairs for 5’

RACE with Block K and 1700 base pairs for 5’ RACE with Block O. Initial attempts of 3’ and 5’ RACE PCR at high annealing temperature of 66 oC were unsuccessful, but amplification was observed when the annealing temperature was lowered to 48 oC

(Figure 26). Only 5’ RACE PCR using Block K or Block O was successful.

bp 1 2 3 4 5 6 7 8 9 10 11 2686 2000

1500

1200 1000 900 800 700 600 500

Figure 26. Agarose gel electrophoresis of 3’ and 5’ RACE products. Lane 1: 100 bp ladder (Invitrogen), Lane 2-4: 3’ RACE with Block C, Lane 5-7: 5’ RACE with Block O primer, Lanes 8-10: 5’ RACE with Block K primer, Lane 11: 100bp ladder (Invitrogen).

The PCR products from several reactions were combined, and the major PCR products were gel-purified (Figure 27). The purified products were then submitted for sequencing using the universal primer. Only the PCR product amplified with Block K primer yielded a sequence of approximately 400 base pairs.

bp 1 2 3 4

23130 9416 6557 4361

2322 2686 2027 2000 1500

1200

1000 900 800 700 600 500

564 Figure 27. Agarose gel electrophoresis of 5’ RACE products. Lane 1: Lamda III Hind digest (Invitrogen), Lane 2: Callus B 5' RACE with Block O primer; boxed band excised (~1500 base pairs), Lane 3: 100 base pair ladder (Fisher Scientific), Lane 4: Callus B 5’ RACE with Block K primer; boxed band excised (~850 base pairs).

Homology searches for all sequences were performed with BLASTx and tBLASTx programs (55). BLASTx translates a nucleotide sequence into all six reading frames and compares the results to a protein sequence database whereas tBLASTx translates a nucleotide sequence into all six reading frames and compares them to translations of a nucleotide sequence database. Based on BLASTx and tBLASTx homology searches of the NCBI non-redundant protein and nucleotide sequence databases, this sequence was not homologous to any previously described monoterpene synthase sequences. However, the 5’ end of the cDNA is likely to

56 contain an unconserved 5’ untranslated region and the 5’ end of the coding region is likely to encode a poorly conserved plastid targeting sequence (23).

Two non-degenerate gene specific primers (Table 5) were designed from the obtained sequence to perform 3’ RACE to obtain a full-length copy of cDNA sequence. Because non-degenerate primers were used the annealing temperature was raised from 48 oC to 66 oC. Amplification with GSP 1 was unsuccessful.

Amplification with GSP 2 yielded a ~ 1800 bp fragment and a ~ 3000 bp fragment

(Figure 28).

Table 5. Gene specific primers.

Gene Specific Primer 1 (GSP 1) AGGGAGATTTCTAGTGGCAGGC Gene Specific Primer 2 (GSP 2) TTCAAGGGGTGTGGCCACT

bp 1 2 3

2686 2000 1500 1200 1000 900 800 700 600

Figure 28. Lane 1: 100 base pair ladder (Fisher Scientific), Lane 2: Callus B 3’ RACE with GSP 1, Lane 3: Callus B 3’ RACE with GSP 2 (excised bands boxed).

These fragments were excised and purified. Attempts to directly sequence were unsuccessful, probably due to low concentration of DNA template, so another round of 3’ RACE with gene specific primer was performed using the same PCR program

(Figure 29) and the resultant PCR products were cloned into a pCR®8/GW/TOPO® vector and then transformed into One Shot® chemically competent E. coli cells. The vector contains primer binding sequences GW1 and GW2 flanking the 5’ and 3’ edges of the cloning site which allows for amplification and sequencing with minimal amount of vector-encoded DNA. The vector also contains a spectinomycin resistant gene for selection. The cells were plated and grown overnight on 100 µg/mL spectinomycin LB plates.

bp 1 2

2686 2000 1500

1200 1000 900 800 700 600 500

Figure 29. Lane 1: 100 base pair ladder (Fisher Scientific) Lane 2: Fresh 3' RACE PCR product using GSP 2.

To identify colonies containing plasmids with inserts, samples of 36 colonies were lysed, and the plasmid size was determined by electrophoresis (Figure 30). The pCR®8/GW/TOPO® vector without insert has a size of 2817 bp. When colonies are lysed and the contents run on an agarose gel, plasmids that are larger than the pCR®8/GW/TOPO® vector are assumed to contain insert.

23,130 9416 6557 4361 2322 2027

564

23,130 9416 6557 4362 2322 2027

Figure 30. Colony screening via lysis and electrophoresis.

Eight colonies that possessed plasmids with insert of reasonable size, of at least

3500 bp, were further screened by colony PCR using GSP 2 along with either GW1 or

GW2 vector primer. By PCR screening the vectors from each colony using either

GW1 or GW2 in conjunction with GSP2, the directionality of the insert can be discerned based on whether amplification occured with GW1 or GW2. Only four fragments were amplified from plasmids of the eight colonies (Figure 31).

bp A C B

2686 2000 1500 1200 1000 900 800 700 600 500

Figure 31. PCR screening of colonies with insert.

The isolated plasmids were then sequenced utilizing the appropriate vector primer. Three of the four plasmids yielded 400-1023 bp of sequence. The first two sequences were 90% identical to each other, but the third sequence was unrelated.

Homology searches agaist the NCBI nonredundant protein and nucleic acid databeases for all sequences were performed with BLASTx and tBLASTx programs, respectively

(55). Two of three sequences were homologous to a secretory plant peroxidase and the third had some homology to tyramine N-feruloyltransferase. The two peroxidase sequences from colony A and B were very closely related exhibiting 90.7% sequence identity (Table 6).

Table 6. Most homologous sequence based on BLASTx and tBLASTx searches of NCBI protein and nucleotide databases of 3’ RACE products. Colony Sequence BLASTx e value tBLASTx e value length A 1023 properoxidase [Picea 1e-78 Picea abies mRNA for 4e-78 abies] properoxidase B 719 properoxidase [Picea 3e-85 Picea abies mRNA for 4e-78 abies] properoxidase C 404 PREDICTED: tyramine 3e-23 Vitis vinifera tyramine 2e-23 N-feruloyltransferase N-feruloyltransferase

Because none of the sequences were highly homologous to monoterpene synthase sequences, another round of 5’ RACE was performed, using the same 5’

RACE-ready cDNA template as before and using universal primer as the forward primer and either degenerate primer, Block K or Block O, as reverse primers with the same PCR program as before (Figure 32). These fresh PCR products were cloned without gel purification as before. A total of 144 colonies were screened for inserts based on electrophoretic analysis of vector size. From these colonies, 36 had an insert, and these were subjected to PCR screening using vector primers to confirm insertion of the amplification product. From the PCR screening, 20 of the 36 colonies, 10 from

Block K primer and 10 from Block O primer, possessed inserts of appropriate size and were submitted for sequencing (Table 7). Again, none of these sequences were obviously homologous to previously described monoterpene synthase sequences.

bp 1 2 3

2686 2000 1500 1200 1000 900 800 700 600 500

Figure 32. 5' RACE PCR products. Lane 1: 100 bp ladder (Fisher Scientific), Lane 2: 5’ RACE with Block K primer, Lane 3: 5’ RACE with Block O primer.

Table 7. Most homologous sequence based on BLASTx and tBLASTx searches of NCBI protein and nucleotide databases of degenerate PCR products. Colony Sequence BLASTx e value tBLASTx e value length 1 940 ADI61831.1 9 e-12 AC241283.1 4 e-24 endonuclease-reverse Pinus taeda clone transcriptase [Bombyx mori] PT_7Ba0038G01, complete sequence 2 937 No similarity found n/a GU252851.1 1.8 Cercis canadensis clone 41 microsatellite sequence 3 222 No similarity found n/a Mus musculus chromosome 17 5 e-7 clone RP23-124G7, complete sequence 4 855 hypothetical protein 8.3 emb|CR382122.1| 2.1 STEHIDRAFT_161767 Kluyveromyces lactis strain [Stereum hirsutum FP- NRRL Y-1140 chromosome B 91666SS1] complete

5 962 XP_003520306.1 8 e-11 JF330774.1 1 e-13 Predicted: Capsicum annuum clone BAC uncharacterized protein CaCM278G16, complete LOC100807511 [Glycine max] sequence

6 405 No similarity found n/a AC185360.2 7 e-4 Populus trichocarpa clone

Pop1-53I10, complete sequence 7 967 YP_710029.1 2.4 FP102047.7 0.85 hypothetical protein Pig DNA sequence from clone BAPKO_0614 [Borrelia afzelii CH242-71K8 on chromosome PKo] X

8 948 GENE ID: 188283 T08D2.5 | 2.3 gb|AC124973.8| Mus 1.5 hypothetical protein musculus strain C57BL/6J [Caenorhabditis elegans] clone rp23-329l13 map 19, complete sequence

9 949 GENE ID: 6994889 5.9 GENE ID: 100646622 1.7 CMU_027360 | hypothetical LOC100646622 | solute carrier protein [Cryptosporidium muris family 35 member F5-like RN66] [Bombus terrestris]

10 839 No similarity found n/a GI:389600251 0.07 Leishmania braziliensis MHOM/BR/75/M2904 complete genome, chromosome 5 11 575 GENE ID: 11423145 1 e-38 dbj|AB029368.1| 2e-108 MTR_1g006120 | Ribosomal Juniperus chinensis protein S10 [Medicago mitochondrial gene for 18S truncatula] rRNA

12 789 No similarities found n/a GENE ID: 459311 0.026 MPHOSPH10 | M-phase phosphoprotein 10 (U3 small nucleolar ribonucleoprotein) [Pan troglodytes] 13 945 gb|ABK24819.1| 5e-8 gb|AC241346.1| 1e-08 unknown [Picea sitchensis] Pinus taeda clone PT_7Ba4137F02, complete sequence

14 1030 BAB01972.1 2 e-8 AC241299.1 7 e-37 copia-like retrotransposable Pinus taeda clone element [Arabidopsis thaliana] PT_7Ba2950E18, complete sequence

15 871 ABK24819.1 4 e-8 AC241346.1 1 e-08 unknown [Picea sitchensis] Pinus taeda clone PT_7Ba4137F02, complete sequence

16 734 XP_002515858.1 2 e-12 AC241348.1 1 e-14 conserved hypothetical protein Pinus taeda clone [Ricinus communis] PT_7Ba4180L06, complete >gb|EEF46527.1| sequence

17 751 ZP_04455918.1 2.8 AM910996.2 1.3 hypothetical protein Plasmodium GCWU000342_0195[Shuttlew knowlesi strain orthia satelles DSM14600] H chromosome 14, complete genome 18 1112 XP_002515858.1 7 e-12 AC241348.1 3 e-15 conserved hypothetical protein Pinus taeda clone [Ricinus communis] PT_7Ba4180L06, complete >gb|EEF46527.1| sequence

19 938 XP_002515858.1 4 e-12 AC241293.1 3 e-15 conserved hypothetical protein Pinus taeda clone [Ricinus communis] PT_7Ba2900E24, complete >gb|EEF46527.1| sequence

20 947 ZP_03702608.1 0.73 XM_001334017.3 0.22 NusA antitermination factor PREDICTED: Danio rerio [Flavobacteria bacterium NHS-like protein 2-like MS024-2A] (LOC794153)

To more exhaustively search for monoterpene synthase-related sequences, 3’ and 5’ RACE-Ready cDNA was resynthesized and 3’ RACE was performed with degenerate forward primer Block C and 5’ RACE was performed with degenerate primers Block K and Block O at an annealing temperature of 48 oC (Figure 33).

bp 1 2 3 4

2686 2000

1500

1200

1000 900 800 700

Figure 33. 3' and 5’ RACE PCR products. Lane 1: 100 bp ladder (Fisher-Scientific), Lane 2: 3’ RACE with Block C primer, Lane 3: 5’ RACE with Block K primer, Lane 4: 5’ RACE with Block O primer.

Unlike the initial attempt, both the 5’ RACE and the 3’ RACE reactions yielded products. Based on the position of the conserved domains within known monoterpene synthase genes, both 3’ RACE with Block C and 5’ RACE with Block O yielded fragments near the expected size of ~ 1700bp; however, 5’ RACE with Block K did not yield a fragment of the expected 1200 bp size.

To increase the probability of cloning a RACE product of the size expected for a monoterpene synthase fragment, bands of the expected size were isolated and purified from a low-melt agarose gel before cloning. Gel-purified fragments were then cloned as before. This time there were fewer colonies present, which was

66 expected, as the PCR product was more dilute due to the presence of agarose from the purification step with low melt agarose.

Nineteen colonies from the 3’ RACE with Block C primer and 20 colonies from the 5’ RACE with Block O primer were screened for the presence of inserts based on plasmid size. Twelve of the 3’ RACE colonies had an insert present, and 5 of the 5’ RACE with Block O primer had an insert present. These colonies were then subjected to PCR screening, which showed that only five 3’ RACE colonies had inserts of the appropriate size. Isolated plasmids were sent for sequencing, and four of the five colonies afforded sequence (Table 8).

Table 8. Sequence analysis from gel purified 3’ RACE PCR. Colony Sequence BLASTx e value tBLASTx e length value 1 937 XP_003520302.1 2e-5 FJ603644.1 2e-65 PREDICTED: pyruvate Taxus cuspidata 3'-N- kinase, cytosolic isozyme- debenzoyltaxol N- like [Glycine max] benzoyltransferase

2 942 BAB01972.1 2 e-8 AC241299.1 7 e-37 copia-like retrotransposable Pinus taeda clone element [Arabidopsis PT_7Ba2950E18, complete sequence thaliana]

3 936 No similarity found n/a FR796467.1 0.63 Leishmania infantum JPCM5 genome chromosome 35

4 345 No similarity found n/a dbj|AK396764.1| Sus scrofa 0.41 mRNA, clone: PST010087B10, expressed in prostate

In summary, a total of 39 amplification products were sequenced from all the

RACE reactions, of which 27 had some homology to proteins on the NCBI protein database. However, none had obvious homology to previously described monoterpene synthase.

Many studies with conifers have utilized homology-based PCR in order to successfully isolate terpene synthase genes (21, 35, 49-53). Our study, like those of other authors, involved isolating mRNA and designing dengenerate primers using conserved domains in homologous sequences. However, most of the previous studies constructed lamda cDNA libraries that were probed with sequences amplified with dengererate primers and we did not. Because a cDNA library was not constructed the sampling of colonies was random, wherereas a cDNA library allows for the use of the degenerate primers to probe for potential candidates. The absence of a cDNA library makes finding the gene of interest more difficult and less probable, even though we did try to maximize the probability of isolating a monoterpene synthase gene by gel purification of PCR products.

In the other studies, RNA was isolated from bark, shoot tissue or sapling stems

(21, 35, 49-53), whereas we used callus cultures. Callus cultures presented challenges, not only in mRNA isolation due to their high polysaccharide and polyphenol content, but also in their initiation and maintenance. Calli are prone to mold, especially during initiation, but also when replating. Each time the calli needed to be replated (every 4-

6 weeks), some succumbed to mold development. Due to mold issues, at the RNA

68 isolation stage, there were not enough calli to perform multiple RNA isolation attempts, which might have afforded higher quality mRNA.

3.7 Conclusions

A homology based PCR strategy was used to try to isolate a monoterpene synthase gene involved in the biosynthesis of β-thujaplicin from C. dupreziana callus culture. Although an obvious monoterpene synthase gene candidate was not identified, several significant goals were accomplished.

We developed a robust system for studying β-thujaplicin biosynthesis. This is the first report of C. dupreziana callus cultures being used for β-thujaplicin investigation; most investigations of β-thujaplcin with callus cultures use C. lusitanica calli. These C. dupreziana callus cultures were initiated and have been maintained for over three years and have been shown to actively produce β-thujaplicin whereas unelicited callus cultures of C. lusitanica have been reported to produce no β- thujaplicin (47).

Additionally, a method for GC-MS analysis of β-thujaplicin that is less sensitive to column conditions was developed. By derivatization with BSFTA+1%

TMSC, β-thujaplicin can be analyzed even with a suboptimum column. This is the first report of analysis of derivatized β-thujaplicin.

Finally, we also developed a method for RNA isolation from callus cultures.

Because of the high polysaccharide and polyphenol levels present in callus cultures, it

69 was necessary to incorporate a 10% ethanol precipitation to remove polysaccharides,

EDTA and thiourea to inhibit polyphenoloxidases and aurintricarboxylic acid to inhibit RNases.

In spite of these accomplishments, a cDNA with high homology to a monoterpene synthase was not isolated. From the GC-MS analysis of the monoterpene content we know that monoterpene synthases must be present, even if we were unable to isolate a monoterpene synthase sequence. The failure to isolate an apparent monoterpene synthase cDNA may be due to several factors. First, the degenerate primers we used may not have the appropriate sequence for amplifying a monoterpene synthase cDNA from Cupressus species. In this study we used three degenerate primers, one forward primer (Block C) and two reverse primers (Block K and Block O). The first successful isolation of a monoterpene synthase sequence from a gymnosperm via similarity based PCR strategy used four primers, three forward and one reverse, and cDNA from a phage library as the template (35). Only one primer was successful. Another study utilized 22 primers to attempt to amplifiy product (49).

For C. dupreziana, designing a bigger pool of degenerate primers or even using primers from other studies isolating monoterpene synthase sequence that have proven successful may result in amplification of our sequence of interest. Additionally, the other studies employed a “broad range of amplification conditions” (35), and more optimization of annealing temperatures with our primers may lead to amplification of a monoterpene synthase sequence. Alternatively, monoterpene synthases in

Cupressaceae may be substantially different from other monoterpene synthase sequences, so that some of the sequences obtained actually encode a monoterpene synthase.

Amplification may also not have been successful due to a low abundance of monoterpene synthase in mRNA preparations. Isolating mRNA from methyl jasmonate-elicited cultures may increase the abundance of monoterpene synthase transcripts in the mRNA template, increasing the probability of successful amplification of monoterpene cDNA. Further optimization of the RNA isolation protocol may also lead to a higher quality RNA template.

Finally, although less probable, amplification of an apparent monoterpene synthase cDNA may not have been successful because monoterpene synthases from

Cupressaceae may be so divergent from the other conifer monoterpene synthases that a homology-based approach is less likely to succeed. Using a more diverse collection of sequences that include non-conifer monoterpene synthases in the CODEHOP primer design process would broaden the potential range of monoterpene synthase sequences potentially amplified by the designed degenerate primers.

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