characterisation of genes related to sesquiterpene

CHARACTERISATION OF GENES RELATED

TO SESQUITERPENE COMPOUNDS IN

AGARWOOD PRODUCTION FROM

Aquilaria malaccensis

ROFIZA BINTI MANSOR

MASTER OF SCIENCE

(BIOTECHNOLOGY)

UNIVERSITI MALAYSIA PAHANG

i

SUPERVISOR’S DECLARATION

We hereby declare that we have checked this thesis and in our opinion, this thesis is

adequate in terms of scope and quality for the award of the degree of Master of Science

(Biotechnology).

_______________________________

(Supervisor’s Signature)

Full Name : DR SAIFUL NIZAM BIN TAJUDDIN

Position : ASSOCIATE PROFESSOR

Date :

_______________________________

(Co-supervisor’s Signature)

Full Name : DR. AIZI NOR MAZILA BINTI RAMLI

Position : SENIOR LECTURER

Date :

ii

STUDENT’S DECLARATION

I hereby declare that the work in this thesis is based on my original work except for

quotations and citation which have been duly acknowledged. I also declare that it has

not been previously or concurrently submitted for any other degree at University

Malaysia Pahang or any other institutions.

_______________________________

(Student’s Signature)

Full Name : ROFIZA BINTI MANSOR

ID Number: MKT13007

Date :

ii

CHARACTERISATION OF GENES RELATED TO SESQUITERPENE

COMPOUNDS IN AGARWOOD PRODUCTION FROM Aquilaria Malaccensis

ROFIZA BINTI MANSOR

Thesis submitted in fulfillment of the requirements

for the award of the degree of

Master of Science (Biotechnology)

Faculty of Industrial Science & Technology

UNIVERSITI MALAYSIA PAHANG

AUGUST 2019

ii

ACKNOWLEDGEMENT

This research project has been both a challenge and an experience to cherish for a life

time. Although a lot of hard work and sacrifice did come from my part, there are many

without whom this research would not have even lifted off the ground, let alone come to

completion. First and foremost, I would like to express my humble thanks to Allah

S.W.T for the strength, inspiration and encouragement given to me throughout the

completion of this thesis without any obstacles.

I would like to extend my deepest gratitude to my research project supervisor, Assoc.

Prof. Dr Saiful Nizam bin Tajuddin for his endless support and guidance during the

infancy of this project and throughout its implementation. I will be forever grateful for

his professionalism and willingness to listen and consider my many suggestions and

amendment to the proposed project as well as for his brilliant advice and ideas. Not

forget my co-Supervisor Dr. Aizi Nor Mazila, who teaches me in some techniques of

molecular biology and mental support for me to finish my master study.

Finally, I acknowledge my sincere indebtedness and gratitude to my husband, family,

my pillar of life, my son and my fellow friends for their encouragements, understanding,

and supports throughout the duration of this research. I cannot find the appropriate

words that could properly describe my appreciation for their devotion, support and faith

in my ability to attain my goals. Their kindness really gives me strength to finish this

research as good as I can. I hope so this research could contribute to the research and

development of agarwood scientific knowledge.

iii

ABSTRACT

Aromatic essential oil extracted from A. malaccensis possess diverse applications in

medicinal and perfume industry. High quality of agarwood is claimed due to present of

high quantity and quality of sesquiterpenes as a market compounds. Despite the

complicated process and long period in producing high quality agarwood in nature, it

can be identified by elucidation of pathway via transcriptomic approaches. Targeted

genes of sesquiterpene in A.malaccensis were identified through transcriptome data.

cDNA library of three different sample (AM Infected-White, AM Infected-Black and

AM Uninfected) were analyzed. The data was sequence with Illumina Next Generation

Sequence (NGS) platform for the purpose of gene discovery in terpenoid biosynthesis.

Data from sequencing was analysed with Blast2Go, Gene Ontology and KEGG

software. The chemical compounds present in AM samples were determined by trapped

volatile compounds in SPME and analysed with GC-FID. Gene Ontology (GO) for each

sample had been employed to classify and arrange the particular functions. A total of

370,707 unigenes were divided into three categories with 26.77% in cellular

components, 24.55% in molecular functions and 48.68% in biological process. To better

assessment GO category, every GO term was further grouped to its parent category. The

infected stem sample shown the highest number of gene regulated in all parent category.

The transcripts were analyzed and deposited into BLASTX, six transcripts sequences

were determined. That sequences were two genes of (-)-germacrene d synthase, two

genes of δ-guaiene synthase, Sesquiterpene synthase and germacrene C synthase. The

regulation levels of the 6 genes related to sesquiterpene were analyzed by observing the

log fold change value. The positive value of the log fold change shows the upregulated

and negative value shows the downregulated of the gene. Six transcripts of

sesquiterpene group that were δ-guaiene synthase; AmguaS1, AmguaS2, and AmguaS3,

(-)-Germacrene synthase AmGdS1, AmGdS2 and Sesquiterpene synthase were indicated

present in infected and uninfected samples. Genes AmguaS1, AmguaS2, and AmguaS3

shown high regulated in the infected stem of A.malaccensis. Chemical profile analyzed

by GC-FID, infected sample that shows an abundance of sesquiterpene compounds

compare to uninfected sample. The sesquiterpene marker compounds that detected with

high concentration in inoculated agarwood tree were agarospirol and Jinkol-eremol. The

most upregulated genes that is δ-guaiene synthase were identified involve in

biosynthesis process for production of marker compounds of agarwood and assumed to

be involved in plant defense mechanism by agarwood formation synthesis.

iv

ABSTRAK

Minyak pati yang beraroma yang diekstrak dari A. malaccensis mempunyai pelbagai

aplikasi dalam industri perubatan dan minyak wangi. Gaharu yang berkualiti tinggi

dinilai dengan mempunyai kualiti dan kuantiti sesquiterpene yang tinggi sebagai

sebatian pasaran. Walaupun proses rumit dan melibatkan jangka masa yang panjang

dalam menghasilkan alam kayu gaharu yang berkualiti tinggi, ia boleh dihasilkan

menerusi pendekatan transkriptom. Gen sesquiterpene A. malaccensis yang terpilih dan

telah dikenal pasti melalui data transkriptom. Data cDNA dari tiga jenis sampel yang

berlainan (AM Infected-White, AM Infected-Black dan AM Uninfected) telah di

analisis. Data tersebut telah di hantar untuk penjujukan dengan platform Illumina Next

Generation Sequence (NGS) untuk tujuan penemuan gen dalam biosintesis terpenoid.

Data dari penjujukan dianalisis dengan perisian Blast2Go, Gene Ontology dan KEGG.

Sebatian kimia yang terdapat di dalam sampel AM ditentukan oleh sebatian mudah

meruap yang diperangkap dalam SPME dan dianalisis dengan GC-FID. Gene Ontology

(GO) untuk setiap sampel telah digunakan untuk mengklasifikasikan dan menyusun

mengikut fungsi tertentu. Sejumlah 370,707 unigen dibahagikan kepada tiga kategori

dengan 26.77% dalam komponen sel, 24.55% dalam fungsi molekul dan 48.68% dalam

proses biologi. Untuk kategori GO kategori yang lebih baik, setiap istilah GO

dikelompokkan lagi kepada kategori induknya. Sampel batang yang dijangkiti

menunjukkan bilangan tertinggi gen yang dikawal dalam semua kategori induk.

Transkrip dianalisis dan dideposit ke BLASTX, enam gen transkrip telah ditentukan.

Gen itu ialah dua gen (-) - germacrene d synthase, dua gen δ-guaiene synthase,

Sesquiterpene synthase dan germacrene C synthase. Tahap penghasilan 6 gen yang

berkaitan dengan sesquiterpene dianalisis dengan memantau nilai log perubahan kali

ganda. Enam transkrip kumpulan sesquiterpene iaitu δ-guaiene synthase; AmguaS1,

AmguaS2, dan AmguaS3, (-) - Germacrene sintase AmGdS1, AmGdS2 dan

Sesquiterpene synthase ditemui dalam sampel yang dijangkiti dan sihat. Gen AmguaS1,

AmguaS2, dan AmguaS3 menunjukkan tahap penghasilan yang tinggi yang terkawal

dalam batang A.malaccensis yang dijangkiti. Profil kimia yang dianalisis oleh GC-FID,

sampel yang dijangkiti yang menunjukkan banyak sebatian sesquiterpene berbanding

dengan sampel yang tidak dijangkiti. Sebatian sesquiterpene utama yang dikesan

dengan kepekatan tinggi dalam pokok gaharu ialah agarospirol dan Jinkol-eremol. Gen

yang menunjukkan rangsangan yang tinggi ialah δ-guaiene synthase dan dikenalpasti

terlibat dalam proses biosintesis untuk pengeluaran sebatian utama gaharu dan dianggap

terlibat dalam mekanisme pertahanan tumbuhan oleh sintesis pembentukan gaharu.

v

TABLE OF CONTENT

DECLARATION

TITLE PAGE

ACKNOWLEDGEMENTS ii

ABSTRAK iii

ABSTRACT iv

TABLE OF CONTENT v

LIST OF TABLES ix

LIST OF FIGURES xi

LIST OF SYMBOLS xii

LIST OF ABBREVIATIONS xiii

CHAPTER 1 INTRODUCTION 1

1.1 Introduction 1

1.2 Problem Statement 2

1.3 Objective 4

1.4 Scope of Study 4

CHAPTER 2 LITERATURE REVIEW 5

2.1 Aquilaria spp 5

2.2 Aquilaria malaccensis 8

2.3 Formation of Agarwood 10

2.4 Method of Agarwood Induce 12

2.4.1 Physical Wounding 13

2.4.2 Chemical Inducer 14

2.4.3 Biological Inducer 15

vi

2.5 Biosynthesis Pathway of Sesquiterpene 15

2.6 Sesquiterpene Genes 19

2.7 Illumina Sequencing of Aquilaria 20

2.8 Chemical Constituent of Aquilaria 22

CHAPTER 3 METHODOLOGY 25

3.1 Plant Materials 25

3.1.1 Sample Collection and Preparation 25

3.2 Construction of Transcriptome cDNA libraries of A. malaccensis 26

3.2.1 Total RNA Extraction 26

3.2.2 Sample Analysis 28

3.2.3 Preparation of mRNA 29

3.2.4 Preparation of cDNA Library 30

3.2.4.1 RNA Fragmentation 30

3.2.4.2 cDNASynthesize 31

3.2.4.3 3'-tagged DNA Synthesize 31

3.2.4.4 Amplification of Library 31

3.2.4.5 Library Dilution 32

3.3 Sequence Analysis of transcriptome data of infected and healthy

A.malaccensis 33

3.4 Elucidate the secondary metabolic pathway for defence mechanism

of A. malaccensis 34

3.5 Comparison of chemical profile between infected and healthy

A. malaccensis 34

3.5.1 Solid Phase Microextraction (SPME) 34

3.5.2 Gas Chromatography- Flame Ion Detector (GC-FID) 35

vii

CHAPTER 4 RESULTS & DISCUSSION 36

4.1 Total RNA 36

4.1.1 Purity of Total RNA 36

4.1.2 Extracted RNA 37

4.1.3 RNA Integrity Number 38

4.2 Isolation of mRNA 39

4.3 cDNA Library Construction 40

4.4 Transcriptome Analysis (De Novo Sequencing) 44

4.4.1 Next Generation Sequencing (NGS) 44

4.4.2 Gene Ontology (GO) 45

4.4.3 Regulated gene in A. malaccensis 52

4.4.4 Assembly and Annotation 53

4.5 Screening of Sesquiterpene Synthase Gene 54

4.5.1 Sesquiterpene Synthase 54

4.6 Genes in A. malaccensis 58

4.6.1 Gene Expression Analysis (Bioinformatics) 58

4.7 Sesquiterpene Biosynthesis Pathway 64

4.8 Gas Chromatography- Flame Ionization Detector (GC-FID) 66

4.8.1 A malaccensis Chemical profile 66

4.8.2 Agarospirol & Jinkoh-Eremol Beneficial Properties 68

CHAPTER 5 CONCLUSION 69

5.1 Conclusion 69

5.1.1 Ressponsible genes in production of Sesquiterpene 69

5.1.2 Sesquiterpene Biosynthesis Pathway 70

5.1.3 Chemical constituent of Infected and Uninfected A. malaccensis 70

viii

5.2 Recommendation 71

REFERENCES 72

APPENDIX A CALCULATION FOR LIBRARY PREPARATION 83

APPENDIX B SESQUITERPENE GENE SEQUENCES 85

APPENDIX C CHROMATOGRAM OF SESQUITERPENE (GC-FID) 102

ix

LIST OF TABLES

Table 2.1 Distribution of Aquilaria species in its origin country 6

Table 2.2 Chemical compounds of well-known Aquilaria species 24

Table 3.1 Collected Sample of A. malaccensis 26

Table 3.2 Total RNA of A. malaccensis samples 28

Table 3.3 Amount of mRNA and water needed in cDNA transcriptomic 30

Table 3.4 Pre-mix for fragmentation of mRNA 30

Table 3.5 Master mix for cDNA synthesis 31

Table 3.6 Preparation PCR Failsafe Premix 32

Table 3.7 Formula used to calculate the concentration of undiluted library

stock needed in preparations of transcriptome cDNA library 32

Table 3.8 Diluted library master mix and Pre mix 33

Table 4.1 Result of concentration and purity of total RNA that extracted 37

Table 4.2 Concentration and purity of A. malaccensis mRNA 40

Table 4.3 Ct value of each sample (data from real time PCR) 42

Table 4.4 Concentration of the undiluted library for each sample 43

Table 4.5 Total of Sequences reads in A. malaccensis cDNA library 45

Table 4.6 Number of unigenes of A. malaccensis involve in biological

process 46

Table 4.7 Number of unigenes of A. malaccensis involve in molecular

functions 48

Table 4.8 Number of unigenes of A. malaccensis involve in cellular

components 50

Table 4.9 Summary of the number of unigenes regulated in response of

stimulus immune system process,enzyme regulator activity and

response to stress for each sample of A.malaccensis which

involve in the plant defense mechanism 52

Table 4.10 Total number of transcripts sequences after assemble and

annotated 53

Table 4.11 Transcripts sequences that shown similarity when assembled

with NCBI database. 55

Table 4.12 Alignment of identified transcripts with reference genes in

BLAST 56

Table 4.13 Differential expression of transcript of Sesquiterpene groups

between each samples 58

Table 4.14 The differential of transcripts identity that involve in Sample 1 vs

2 61

x

Table 4.15 The differential of transcripts identity that involve in Sample 1 vs

3 62

Table 4.16 The differential of transcripts identity that involve in sample 2 vs

3 62

Table 4.17 The differential identity of transcripts 63

Table 4.18 List of sesquiterpene compounds detected in each of agarwood

samples 67

xi

LIST OF FIGURES

Figure 2.1 Damage Aquilaria tree due to illegal logging 7

Figure 2.2 Comparison of Aquilaria sp. 8

Figure 2.3 Distribution of A. malaccensis in Malaysia 9

Figure 2.4 Leaves and Seed of A. malaccensis 10

Figure 2.5 Natural resin formation in forest and resin formation after

inoculation technique 12 13

Figure 2.6 (A) Drilling method; (B) Bark peeling method; (C) and (D)

shown the nailing method on Aquilaria tree 14

Figure 2.7 Biosynthesis pathway of sesquiterpene 17 18

Figure 2.8 Terpenoid biosynthesis by plant. Monoterpenes, sesquiterpenes,

and diterpenes are derived from the prenyl diphosphate substrates

geranyl diphosphate (GPP), geranylgeranyl diphosphate (GGPP),

farnesyl diphosphate (FPP) 18

Figure 3.1 Flow chart of process involve in this research 25

Figure 3.2 Stem tissues disruption by using liquid nitrogen 27

Figure 3.3 Process flow for sample analysis by using Bioanalyzer 28

Figure 3.4 Method flow of mRNA isolation 29

Figure 3.5 Magnetic beads was used for mRNA purification 29

Figure 3.6 Solid Phase Micro-Extraction Mechanism (SPME) 35

Figure 4.1 Total RNA extracted from A. malaccensis stem 38

Figure 4.2 Quality check for RNA integrity number for each sample. (A)

RIN for total RNA from AM infected (black), (B) RIN for total

RNA from AM infected (white) and (C) RIN for total RNA

fromAM healthy (wild) 39

Figure 4.3 The size of cDNA library with specific size based on the 100 bp

marker 41

Figure 4.4 Standard curve for real-time PCR of 10-fold dilution of cDNA

standard of library 42

Figure 4.5 Distribution of assembly unigenes involve in every terms in

biological process. 47


molecular function. 49


cellular components. 51

Figure 4.8 The elucidated pathway of sesquiterpene synthesis involve

in aromatic plant. 65

xii

LIST OF SYMBOLS

ºC Degree Celcius

α Alpha

β Beta

δ Delta

γ Gamma

µl Micro Liter

% Percentage

g Gram

xiii

LIST OF ABBREVIATION

BLAST Basic Local Alignment Sequence Tools

bp Base Pair

cDNA Complementary DNA

CDS Coding DNA Sequence

DMAPP Dimethylallyl diphosphate

DNA Deoxyribonucleic Acid

DXP 1-deoxy-dxylulose-5-phosphate

FPP Farnesylpyrosphosphate

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GC Gas Chromatography

GC-FID Gas Chromatography-Flame Ionization Detector

GC-MS Gas Chromatography-Mass Spectrometry

GGPP Geranyl Geranyl Phosphate

GO Gene Ontology

GPP Geranyl diphosphate

IPP Isopentenyl diphosphate

KEGG Kyoto Encyclopedia of Genes and Genomes

KI Kovats Index

MCS Methyl-D-erythritol-2,4-cyclodiphosphate synthase

MK Mevalonate Kinase

NCBI National Center for Biotechnology Information (NCBI)

NGS Next Generation Sequencing

PDMS Polydimethylsiloxane

pM Per Million

RIN RNA Integrity Number

RNA Ribonucleic Acid

RPL Ribosomal Protein

SPME Solid Phase Microextraction

TSS Transcription Sites

UTR Untranslated Region

72

REFERENCES

Abd Majid, J., Hazandy, A. H., Paridah, M. T., Nor Azah, M. A., Mailina, J., Saidatul

Husni, S., & Sahrim, L. (2018). Determination of Agarwood volatile compounds

from selected Aquilaria species plantation extracted by Headspace-Solid Phase

Microextraction (HS-SPME) method. IOP Conference Series: Materials Science and

Engineering, 368(1). https://doi.org/10.1088/1757-899X/368/1/012023

Aharoni, A., Giri, A. P., W.A., F., Verstappen, Bertea, C. M., Sevenier, R.,

Bouwmeestera, H. J. (1999). Gain and Loss of Fruit Flavor Compounds Produced by

Wild and Cultivated Strawberry Species. Israel Exploration Journal, 49(1), 1–42.

https://doi.org/10.1105/tpc.104.023895.native

Aharoni, A., Giri, A. P., W.A., F., Verstappen, Bertea, C. M., Sevenier, R.,Bouwmeestera,

H. J. (1999). Gain and Loss of Fruit Flavor Compounds Produced by Wild and

Cultivated Strawberry Species. Israel Exploration Journal, 49(1), 1–42.

https://doi.org/10.1105/tpc.104.023895.

Ahmaed, D. T., & Kulkarni, A. D. (2017). Sesquiterpenes and Chromones of Agarwood :

A Review. Malaysian Journal of Chemistry, 19(1), 33–58.

https://doi.org/10.1016/j.powtec.2012.04.003

Ahmaed, D. T., Mohammed, M., Masaad, A. M., & Tajuddin, S. N. (2017). Investigation

of Agarwood Compounds in Aquilaria malaccensis & Aquilaria Investigation of

Agarwood Compounds in Aquilaria malaccensis & Aquilaria Rostrata Chipwood by

Using Solid Phase Microextraction. Biomedical Journal of Scientific and Technical

Research, 1-8.https://doi.org/10.26717/BJSTR.2017.01.000499

Akter, S., Islam, M. T., Zulkefeli, M., & Khan, S. I. (2013). Agarwood Production - A

Multidisciplinary Field to be Explored in Bangladesh. International Journal of

Pharmaceutical and Life Sciences, 2(1), 22–32.

https://doi.org/10.3329/ijpls.v2i1.15132

Azren, P. D., Lee, S. Y., Emang, D., & Mohamed, R. (2018). History and perspectives of

induction technology for agarwood production from cultivated Aquilaria in Asia: a

review. Journal of Forestry Research, (April), 1–11. https://doi.org/10.1007/s11676-

018-0627-4

Azzarina, A. B., Mohamed, R., Lee, S. Y., & Nazre, M. (2016). Temporal and spatial

expression of terpene synthase genes associated with agarwood formation in

Aquilaria malaccensis Lam. New Zealand Journal of Forestry Science, 46(1).

https://doi.org/10.1186/s40490-016-0068-9

73

Back, K., & Chappell, J. (1995). Cloning and Bacterial Expression of a Sesquiterpene

Cyclase from Hyoscyamus muticus and Its Molecular Comparison to Related

Terpene Cyclases. The Journal of Biological,Chemistry 270(31), 7375-7381.

Barden, A., Anak, N. a., Mulliken, T., & Song, M. (2000). Heart of the Matter: Agarwood

use and trade and CITES implementation for Aquilaria malaccensis. Heart of the

Matter: Agarwood Use and Trade and CITES Implementation for Aquilaria

Malaccensis, 1–52.

Beniwal, B. S. (1989). Silvical Characteristics of Aquilaria Agallocha Roxb. Indian

Forester, 115(1), 17–21. Retrieved from

http://www.indianforester.co.in/index.php/indianforester/article/view/8987

Bhuiyan, N. I., Begum, J., & Bhuiyan, N. H. (2009). Analysis of essential oil of

eaglewood tree (Aquilaria agallocha Roxb.) by gas chromatography mass

spectrometry. Bangladesh Journal of Pharmacology, 4(1), 24–28.

https://doi.org/10.3329/bjp.v4i1.851

Blanchette, Ro. A., & Beek, H. H. van. (2005). US Patent for Cultivated agarwood Patent

(Patent # 6,848,211 issued February 1, 2005) - Justia Patents Search. Retrieved

December 17, 2018, from https://patents.justia.com/patent/6848211

Buehler, B., Hogrefe, H. H., Scott, G., Ravi, H., Pabón-Peña, C., O’Brien, S.,Happe, S.

(2010). Rapid quantification of DNA libraries for next-generation sequencing.

Methods, 50(4), S15–S18. https://doi.org/10.1016/j.ymeth.2010.01.004

Chakrabarty, K., Kumar, A. and Menon, V. (1994). Trade India TRAFFIC | WWF India.

Retrieved December 5, 2018, from

https://www.wwfindia.org/about_wwf/enablers/traffic/

Chen, H., Yang, Y., Xue, J., Wei, J., Zhang, Z., & Chen, H. (2011). Comparison of

compositions and antimicrobial activities of essential oils from chemically stimulated

agarwood, wild agarwood and healthy Aquilaria sinensis (Lour.) Gilg trees.

Molecules, 16(6), 4884–4896. https://doi.org/10.3390/molecules16064884

Chen, X. (2010). Quality Assessment of RNA. Biomedicalgenomics, 1–22.

Chen, X., Zhu, X., Feng, M., Zhong, Z., Zhou, X., Chen, X.,Gao, X. (2017). Relationship

between expression of chalcone synthase genes and chromones in artificial agarwood

induced by formic acid stimulation combined with Fusarium sp. A2 inoculation.

Molecules, 22(5), 1–14. https://doi.org/10.3390/molecules22050686

Chhipa, H., Chowdhary, K., & Kaushik, N. (2017). Artificial production of agarwood oil

in Aquilaria sp. by fungi: a review. Phytochemistry Reviews, 16(5), 835–860.

74

https://doi.org/10.1007/s11101-017-9492-6

Chomtong, T., Suputtitada, S., Vuttipongchaikij, S., & Apisitwanich, S. (2012). Isolation

Of Gene Transcripts During Agarwood Resin Accumulating By Subtractive

Hybridization Technique, 44(8), 1075–1085.

https://doi.org/10.3724/SP.J.1041.2012.01075

Chowdhury, M., Hussain, M. D., Chung, S. O., Kabir, E., & Rahman, A. (2016).

Agarwood manufacturing: A multidisciplinary opportunity for economy of

Bangladesh - A review. Agricultural Engineering International: CIGR Journal,

18(3), 171–178.

CITES. (2013). Agarwood-producing taxa (Decision 15.94) ., 11(March).

Dalman, M. R., Deeter, A., Nimishakavi, G., & Duan, Z. H. (2012). Fold change and p-

value cutoffs significantly alter microarray interpretations. BMC Bioinformatics, 13

Suppl 2(Suppl 2), S11. https://doi.org/10.1186/1471-2105-13-S2-S11

Dang, J., Mendez, P., Lee, S., Kim, J. W., Yoon, J. H., Kim, T. W.,Kim, I. J. (2016).

Development of a robust DNA quality and quantity assessment qPCR assay for

targeted next-generation sequencing library preparation. International Journal of

Oncology, 49(4), 1755–1765. https://doi.org/10.3892/ijo.2016.3654

Deguerry, F., Pastore, L., Wu, S., Clark, A., Chappell, J., & Schalk, M. (2006). The

diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a

limited number of sesquiterpene synthases. Archives of Biochemistry and Biophysics,

454(2), 123–136. https://doi.org/10.1016/j.abb.2006.08.006

Dudareva, N., Andersson, S., Orlova, I., Gatto, N., Reichelt, M., Rhodes, D.,Gershenzon,

J. (2005). From The Cover: The nonmevalonate pathway supports both monoterpene

and sesquiterpene formation in snapdragon flowers. Proceedings of the National

Academy of Sciences, 102(3), 933–938. https://doi.org/10.1073/pnas.0407360102

Elias, M. F., Ibrahim, H., & Mahamod, W. R. W. (2017). A Review on the Malaysian

Aquilaria species in Karas Plantation and Agarwood Production. International

Journal of Academic Research in Business and Social Sciences, 7(4), 1021–1029.

https://doi.org/10.6007/IJARBSS/v7-i4/2911

Gao, Z. H., Wei, J. H., Yang, Y., Zhang, Z., & Zhao, W. T. (2012). Selection and

validation of reference genes for studying stress-related agarwood formation of

Aquilaria sinensis. Plant Cell Reports, 31(9), 1759–1768.

https://doi.org/10.1007/s00299-012-1289-x

75

Gardner, R. G., & Hampton, R. Y. (1999). A highly conserved signal controls degradation

of 3-hydroxy-3- methylglutaryl-coenzyme a (HMG-CoA) reductase in eukaryotes.

Journal of Biological Chemistry, 274(44), 31671–31678.

https://doi.org/10.1074/jbc.274.44.31671

GenoHub. (2018). Cluster Density Optimization on Illumina Sequencing Instruments.

Retrieved December 6, 2018, from https://genohub.com/loading-concentrations-

optimal-cluster-density/

Han, X.-J., Wang, Y.-D., Chen, Y.-C., Lin, L.-Y., & Wu, Q.-K. (2013). Transcriptome

sequencing and expression analysis of terpenoid biosynthesis genes in Litseacubeba.

PLoS ONE, 8(10), e76890. https://doi.org/10.1371/journal.pone.0076890

Harvey-Brown, Y. (2018). Aquilaria sinensis, Chinese Agarwood, 8235.

Hashimoto, K., Nakahara, S., Inoue, T., Sumida, Y., Takahashi, M., & Masada, Y. (1985).

A New Chromone from Agarwood and Pyrolysis Products of Chromone Derivatives.

Chem Pharm, 33.

Head, S. R., Komori, H. K., LaMere, S. A., Whisenant, T., Nieuwerburgh, F. Van,

Salomon, D. R., & Ordoukhanian, P. (2015). Library construction for next-

generation sequencing: Overviews and challenges. Igarss 2014, 56(2), 25.

https://doi.org/10.2144/000114133.Library

Hendra, H., Moeljopawiro, S., & Nuringtyas, T. R. (2016). Antioxidant and antibacterial

activities of agarwood (Aquilaria malaccensis Lamk.) leaves. AIP Conference

Proceedings, 1755(April 2018). https://doi.org/10.1063/1.4958565

Iijima, Y. (2004). The Biochemical and Molecular Basis for the Divergent Patterns in the

Biosynthesis of Terpenes and Phenylpropenes in the Peltate Glands of Three

Cultivars of Basil. Plant Physiology, 136(3), 3724–3736.

https://doi.org/10.1104/pp.104.051318

Ishihara, M., Tsuneya, T., & Uneyama, K. (1993). Components of the Volatile

Concentrate of Agarwood. Journal of Essential Oil Research, 5(3), 283–289.

https://doi.org/10.1080/10412905.1993.9698221

Ismail, N., Rahiman, M. H. F., Taib, M. N., Ali, N. A. M., Jamil, M., & Tajuddin, S. N.

(2013). Classification of the quality of agarwood oils from Malaysia using Z-score

technique. Proceedings - 2013 IEEE 3rd International Conference on System

Engineering and Technology, ICSET 2013, 78–82.

https://doi.org/10.1109/ICSEngT.2013.6650147

76

Ismail, S. N., Maulidiani, M., Akhtar, M. T., Abas, F., Ismail, I. S., Khatib, A.,Shaari, K.

(2017). Discriminative Analysis of Different Grades of Gaharu (Aquilaria

malaccensis Lamk.) via1H-NMR-Based Metabolomics Using PLS-DA and Random

Forests Classification Models. Molecules, 22(10), 1–13.

https://doi.org/10.3390/molecules22101612

Jayachandran, K., Sekar, I., Parthiban, K. T., Amirtham, D., & Suresh, K. K. (2014).

Analysis of different grades of Agarwood (Aquilaria malaccensis Lamk.) oil through

GC-MS. Indian Journal of Natural Products and Resources, 5(1), 44–47.

Jutarut Pornpunyapat, Chetpattananondh, P., & Tongurai, C. (2011). Mathematical

modeling for extraction of essential oil from Aquilaria crassna by hydrodistillation

and quality of agarwood oil. A Journal of the Bangladesh Pharmacological Society

(BDPS, (4), 18–24. https://doi.org/10.3329/bjp.v6i1.7902

Kalra, R., & Kaushik, N. (2017). A review of chemistry, quality and analysis of infected

agarwood tree (Aquilaria sp.). Phytochemistry Reviews, 16(5), 1045–1079.

https://doi.org/10.1007/s11101-017-9518-0

KAPA protocol, & Guide, T. (2014). Technical Guide KAPA Library Quantification

Technical Guide KAPA Library Quantification. KAPA Biosystems, (August), 1–16.

https://doi.org/10.1016/S0022-1759(01)00305-2

Kumeta, Y., & Ito, M. (2010). Characterization of -Guaiene Synthases from Cultured

Cells of Aquilaria, Responsible for the Formation of the Sesquiterpenes in

Agarwood. Plant Physiology, 154(4), 1998–2007.

https://doi.org/10.1104/pp.110.161828

Kurosaki, F., Kato, T., Misawa, N., & Taura, F. (2016). Efficient Production of δ-

Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by

Mevalonate Pathway-Engineered <i>Escherichia

coli</i> Cells. Advances in Bioscience and Biotechnology, 07(11),

435–445. https://doi.org/10.4236/abb.2016.711042

Liao, Y. C., Xu, Y. H., Zhang, Z., & Wei, J. H. (2015). Molecular cloning and sequence

analysis of lipoxygenase (AsLOX1) gene in Aquilaria sinensis (Lour.) Gilg and its

expression responding to MeJA and crush wounding treatment. Plant Gene, 2, 10–

16. https://doi.org/10.1016/j.plgene.2015.02.002

Lim, wyn teck, & Noorainie, A. A. (2010). of the Agarwood (Gaharu) Trade.

Liu, Y., Chen, H., Yang, Y., Zhang, Z., Wei, J., Meng, H.,Chen, H. (2013). Whole-tree

agarwood-inducing technique: An efficient novel technique for producing high-

quality agarwood in cultivated Aquilaria sinensis trees. Molecules, 18(3), 3086–

77

3106. https://doi.org/10.3390/molecules18033086

Liu, Y., Wei, J., Gao, Z., Zhang, Z., & Lyu, J. (2017). A Review of Quality Assessment

and Grading for Agarwood. Chinese Herbal Medicines, 9(1), 22–30.

https://doi.org/10.1016/S1674-6384(17)60072-8

Lok, & Zuhaidi, A. (2016). Growth and management of Aquilaria Malaccensis for

agarwood - A new domestication perspective. International Journal of Agriculture,

Forestry and Plantation, 3, 55–60.

López-Sampson, A., & Page, T. (2018). History of Use and Trade of Agarwood.

Economic Botany, 72(1), 107–129. https://doi.org/10.1007/s12231-018-9408-4

Mancini, E., Senatore, F., Del Monte, D., De Martino, L., Grulova, D., Scognamiglio, M.,

De Feo, V. (2015). Studies on chemical composition, antimicrobial and antioxidant

activities of five Thymus vulgaris L. essential oils. Molecules, 20(7), 12016–12028.


Martin, D., Tholl, D., Gershenzon, J., & Bohlmann, J. (2002). Methyl Jasmonate Induces

Traumatic Resin Ducts, Terpenoid Resin Biosynthesis, and Terpenoid Accumulation

in Developing Xylem of Norway Spruce Stems1. Annals of Internal Medicine,

156(2). https://doi.org/10.1104/pp.011001.McGarvey

Micheal, R. (1999). The discovery of a mevalonate-independent pathway for isoprenoid

biosynthesis in bacteria, algae and higher plants. Natural Product Reports,

16(Scheme 1), 565–574. https://doi.org/10.1136/jmg.2007.049270

Mohamad ali, N. A., Chang, Y. S., Mailina, J., A, A. S., Majid, A., Husni, J. S.,Y, N. Y.

(2008). Comparison of Chemical Profiles of Selected Gaharu Oils From Peninsular

Malaysia. The Malaysian Journal of Analytical Sciences, 12(2), 338–340.

https://doi.org/10.1016/j.mencom.2013.

Mohamed, R., Jong, P. L., & Zali, M. S. (2010). Fungal diversity in wounded stems of

aquilaria malaccensis. Fungal Diversity, 43(March), 67–74.

https://doi.org/10.1007/s13225-010-0039-z

Nagajothi, M. S., Parthiban, K. T., Kanna, S. U., & Karthiba, L. (2016). Fungal Microbes

Associated with Agarwood Formation. American Journal of Plant Sciences,

07(July), 1445–1452. https://doi.org/10.1103/PhysRevB.86.094403

Nakanishi, T., Yamagata, E., Yoneda, K., Nagashima, T., Kawasaki, I., Yoshida, T., …

Miura, I. (1984). Three fragrant sesquiterpenes of agarwood. Phytochemistry, 23(9),

2066–2067. https://doi.org/10.1016/S0031-9422(00)84975-4

78

Noor, M. H. A., Mohamad, A., Othman, R., & Baharum, S. N. (2014). Morphological

evaluation on leaf characteristics of common Aquilaria species in Malaysia. R&D

Seminar 2014: Research and Development Seminar 2014, (C), 2.

Novriyanti, E., Santosa, E., Syafii, W., Turjaman, M., & Sitepu, I. R. (2010). Anti Fungal

Activity of Wood Extract of Aquilaria crassna Pierre ex Lecomte against Agarwood-

inducing Fungi, Fusarium solani. Journal of Forestry Research, 7(2), 155–165.

https://doi.org/10.20886/ijfr.2010.7.2.155-165

Okudera, Y., & Ito, M. (2009). Production of agarwood fragrant constituents in Aquilaria

calli and cell suspension cultures. Plant Biotechnology, 26(3), 307–315.

https://doi.org/10.5511/plantbiotechnology.26.307

Okugawa, H., Ueda, R., Matsumoto, K., Kawanishi, K., & Kato, A. (1996). Effect of

jinkoh-eremol and agarospirol from agarwood on the central nervous system in mice.

Plantmedicine, 62(1), 2-6. https://www.semanticscholar.org/paper/Effect-of-jinkoh-

eremol-and-agarospirol-from-on-the-Okugawa-Ueda

Okugawa, H., Ueda, R., Matsumoto, K., Kawanishi, K., & Kato, K. (2000). Effects of

sesquiterpenoids from “Oriental incenses” on acetic acid-induced writhing and D2

and 5-Ht(2A) receptors in rat brain. Phytomedicine, 7(5), 417–422.

https://doi.org/10.1016/S0944-7113(00)80063

Pawitan, Y., Michiels, S., Koscielny, S., Gusnanto, A., & Ploner, A. (2005). False

discovery rate, sensitivity and sample size for microarray studies. Bioinformatics,

21(13), 3017–3024. https://doi.org/10.1093/bioinformatics/bti448

Persoon, G. A. (2007). Agarwood: the life of a wounded tree.

Pojanagaroon, S., & Kaewrak, C. (2005). Mechanical methods to stimulate aloes wood

formation in Aquilaria crassna Pierre ex H.Lec. (Kritsana) trees. Acta Horticulturae,

676, 161–166. https://doi.org/10.17660/ActaHortic.2005.676.20

Pripdeevech, P., Khummueng, W., & Park, S.-K. (2011). Identification of Odor-active

Components of Agarwood Essential Oils from Thailand by Solid Phase

Microextraction-GC/MS and GC-O. Journal of Essential Oil Research, 23(4), 46–

53. https://doi.org/10.1080/10412905.2011.9700468

Qingye Jiang. (2013). Aquilaria The (Endangered) Spirit of Forest. Retrieved January 23,

2019, from http://www.qyjohn.net/?p=2910

Rahman, M., Nath, N. M., Sarker, S., Adnan, M., & Islam, M. (2015). Management and

Economic Aspects of Growing Aquilaria agallocha Roxb. in Bangladesh. Small-

Scale Forestry, 14(4), 459–478. https://doi.org/10.1007/s11842-015-9298-6

79

Rasool, S., & Mohamed, R. (2016). Agarwood: Science Behind the Fragrance - Google

Books. springer. Retrieved from

https://books.google.com.my/books?id=CqlPDAAAQBAJ&pg=PA39&lpg=PA39

&dq=Understanding+Agarwood+Formation+and+Its+Challenges&source=bl&ots=

lfrPNTk0_4&sig=7An7o9V7VXNKIEnaJ734z1wm57Y&hl=en&sa=X&ved=2ahU

KEwjry5-pw4rfAhULEnIKHQWyC1UQ6AEwA3oECAYQAQ#v=onepage&q=U

Saikia, P., & Khan, M. L. (2011). Agar (Aquilaria malaccensis Lam.): A promising crop

in the homegardens of Upper Assam, northeastern India. Journal of Tropical

Agriculture, 50, 8–14.

Sallaud, C., Rontein, D., Onillon, S., Jabes, F., Duffe, P., Giacalone, C.,Tissier, A. (2009).

A Novel Pathway for Sesquiterpene Biosynthesis from Z,Z-Farnesyl Pyrophosphate

in the Wild Tomato Solanum habrochaites. The Plant Cell Online, 21(1), 301–317.

https://doi.org/10.1105/tpc.107.057885

Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M.,Ragg,

T. (2006). The RIN: An RNA integrity number for assigning integrity values to RNA

measurements. BMC Molecular Biology, 7, 1–14. https://doi.org/10.1186/1471-

2199-7-3

Selvan, T., D, N., & Kaushik, pawan K. (2014). Agarwood production for intensive

income generation. Review of Research, 3(5).

Sen, S., Talukdar, N. C., & Khan, M. (2015). A simple metabolite profiling approach

reveals critical biomolecular linkages in fragrant agarwood oil production from

Aquilaria malaccensis - A traditional agro-based industry in North East India.

Current Science, 108(1), 63–71.

Sharon-Asa, L., Shalit, M., Frydman, A., Bar, E., Holland, D., Or, E.,Eyal, Y. (2003).

Citrus fruit flavor and aroma biosynthesis: Isolation, functional characterization, and

developmental regulation of Cstps1, a key gene in the production of the sesquiterpene

aroma compound valencene. Plant Journal, 36(5), 664–674.

https://doi.org/10.1046/j.1365-313X.2003.01910.x

Siah, C. H., Namasivayam, P., & Mohamed, R. (2016). Transcriptome reveals senescing

callus tissue of Aquilaria malaccensis, an endangered tropical tree, triggers similar

response as wounding with respect to terpenoid biosynthesis. Tree Genetics and

Genomes, 12(2). https://doi.org/10.1007/s11295-016-0993-z

Sidik, A. B. (2008). Comparison Of Gaharu (Aquilaria Malaccensis) Essential Oil

Composition Between Each Country, (May).

80

Steele, C. L., Crock, J., Bohlmann, J., & Croteau, R. (1998). Sesquiterpene Synthases

from Grand Fir (Abies grandis). The Journal of Biological Chemistry, 273(4), 2078–

2089. https://doi.org/10.1074/jbc.273.4.2078

Suhendra, D. I. and A. (2013). Uji Inokulasi Fusarium Sp Untuk Produksi Gaharu Pada

Budidaya A.Beccariana. Jurnal Sains Dan Teknologi Indonesia2, Vol.14(No. 3),

182–188.

Sumarna, Y. (2008). The Effects of Seed Maturity Condition and Media Type on Growth

of Agarwood Seedlings of KarasTrees (Aquilaria malaccensis Lamk.), (c), 129–135.

Süntar, I., Akkol, E. K., Nahar, L., & Sarker, S. D. (2012). Wound healing and antioxidant

properties: do they coexist in plants? Free Radicals and Antioxidants, 2(2), 1–7.

https://doi.org/10.5530/AX.2012.2.2.1

Swee, L., & Chua, L. (2018). Agarwood (aquilaria malaccensis) in malaysia, Case Study

3, (January 2008).

Tajuddin, S. N., Muhamad, N. S., & Yusoff, M. A. Y. and M. M. (2008). Characterization

of the chemical constituents of agarwood oils from Malaysia by comprehensive two-

dimensional gas chromatography–time-of-flight mass spectrometry. The Malaysian

Journal of Analytical Sciences, 12(2), 338–340.

https://doi.org/10.1016/j.mencom.2013.

Talucder, M. S. A., Haque, M. M., & Saha, D. (2016). Development Of Agar ( Aquilaria

Malaccensis ) Cultivation , Propagation Technique And Its Potentiality As

Agroforestry Component In Bangladesh : A Review, 3(2), 149–157.

Trapp, S. C., & Croteau, R. B. (2017). Genomic Organization of Plant Terpene Synthases

and Molecular Evolutionary Implications. Voprosy Filosofii, (7), 140–146.

Venkataramanan, M. N., Borthakur, R., & Singh, H. D. (1985). Occurrence of

endotrophic mycorrhizal fungus in agarwood plant Aquillaria agallocha (Roxb.).

Current Science, India, 54(18), 928.

https://eurekamag.com/research/001/420/001420423.php

Wang, S., Yu, Z., Wang, C., Wu, C., Guo, P., & Wei, J. (2018). Chemical constituents

and pharmacological activity of agarwood and aquilaria plants. Molecules, 23(2).


Wetwitayaklung, P., Thavanapong, N., & Charoenteeraboon, J. (2009). Chemical

Constituents and Antimicrobial Activity of Essential Oil and Extracts of Heartwood

of Aquilaria crassna Obtained from Water Distillation and Supercritical Fluid Carbon

Dioxide Extraction. Silpakorn U Science & Technology Journal, 3(1), 25–33.

81

https://doi.org/10.14456/sustj.2009.3

Wong, M., Siah, C., Faridah, Q., & Mohamed, R. (2013). Characterization of wound

responsive genes in Aquilaria malaccensis. Journal of Plant Biochemistry and

Biotechnology, 22(2), 168–175. https://doi.org/10.1007/s13562-012-0144-z

Wong, Y. F., Chin, S. T., Perlmutter, P., & Marriott, P. J. (2015). Evaluation of

comprehensive two-dimensional gas chromatography with accurate mass time-of-

flight mass spectrometry for the metabolic profiling of plant-fungus interaction in

Aquilaria malaccensis. Journal of Chromatography A, 1387, 104–115.

https://doi.org/10.1016/j.chroma.2015.01.096

Xu, Y., Zhang, Z., Wang, M., Wei, J., Chen, H., Gao, Z.,Li, W. (2013). Identification of

genes related to agarwood formation: Transcriptome analysis of healthy and

wounded tissues of Aquilaria sinensis. BMC Genomics, 14(1).

https://doi.org/10.1186/1471-2164-14-227

Yagura, T., Shibayama, N., Ito, M., Kiuchi, F., & Honda, G. (2005). Three novel diepoxy

tetrahydrochromones from agarwood artificially produced by intentional wounding.

Tetrahedron Letters, 46(25), 4395–4398.

https://doi.org/10.1016/j.tetlet.2005.04.072

Yang, J. S., & Chen, Y. W. (1983). [Studies on the constituents of Aquilaria sinensis

(Lour.) Gilg. I. Isolation and structure elucidation of two new sesquiterpenes,

baimuxinic acid and baimuxinal]. Yao Xue Xue Bao = Acta Pharmaceutica Sinica,

18(3), 191–198. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6615594

Ye, W., Wu, H., He, X., Wang, L., Zhang, W., Li, H.,Gao, X. (2016). Transcriptome

sequencing of chemically induced aquilaria sinensis to identify genes related to

agarwood formation. PLoS ONE, 11(5), 1–16.

https://doi.org/10.1371/journal.pone.0155505

Yokomori, R., Shimai, K., Nishitsuji, K., Suzuki, Y., Kusakabe, T. G., & Nakai, K.

(2016). Genome-wide identification and characterization of transcription start sites

and promoters in the tunicate Ciona intestinalis. Genome Research, 26(1), 140–150.

https://doi.org/10.1101/gr.184648.114

Yoneda, K., Yamagata, E., Nakanishi, T., Nagashima, T., Kawasaki, I., Yoshida,

T.,Miura, I. (1984). Sesquiterpenoids in two different kinds of agarwood.

Phytochemistry, 23(9), 2068–2069. https://doi.org/10.1016/S0031-9422(00)84976-6

Ngeow.Y.F (2014). Real-time PCR detection of male-specific coliphages Real-time PCR

detection of male-specific coliphages, (January 2007), 2–6.

82

Zaremski, C., Andary, C., & Ducousso, M. (2017). NGS analysis of fungal OTUs in

Aquilaria sp . from French Guyana , Cambodia , Laos and Thailand, (August).

Zhang, X. L., Liu, Y. Y., Wei, J. H., Yang, Y., Zhang, Z., Huang, J. Q., Liu, Y. J. (2012).

Production of high-quality agarwood in Aquilaria sinensis trees via whole-tree

agarwood-induction technology. Chinese Chemical Letters, 23(6), 727–730.

https://doi.org/10.1016/j.cclet.2012.04.019

Zhang, Z., Wei, J., Han, X., Liang, L., Yang, Y., Meng, H.,Gao, Z. (2014). The

sesquiterpene biosynthesis and vessel-occlusion formation in stems of aquilaria

sinensis (lour.) gilg trees induced by wounding treatments without variation of

microbial communities. International Journal of Molecular Sciences, 15(12),

23589–23603. https://doi.org/10.3390/ijms151223589

t of RNA. Biomedicalgenomics, 1–22.

characterisation of genes related to sesquiterpene

Documents