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UNIVERSITI PUTRA MALAYSIA
CDNA CLONING AND EXPRESSION ANALYSIS OF TOCOPHEROL CYCLASE GENE FROM VARIOUS OIL PLAM TISSUES
(Elaeis guineensis and Elaeis oleifera)
LAI MUN SEONG
FP 2011 32
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CDNA CLONING AND EXPRESSION ANALYSIS OF TOCOPHEROL
CYCLASE GENE FROM VARIOUS OIL PLAM TISSUES
(Elaeis guineensis and Elaeis oleifera)
By
LAI MUN SEONG
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Master Science
December 2010
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of
the requirement for the degree of Master of Science
CDNA CLONING AND EXPRESSION ANALYSIS OF TOCOPHEROL
CYCLASE GENE FROM VARIOUS OIL PALM TISSUES
(Elaeis guineensis and Elaeis oleifera)
By
LAI MUN SEONG
December 2010
Chair: Associate Professor Datin. Siti Nor Akmar Abdullah, PhD
Faculty: Faculty of Agriculture
Tocopherol cyclase (TC) which catalyses the formation of γ-tocopherol from reduced
(quinol form) 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ) is one of the key
enzymes in tocopherol biosynthetic pathway. The aim of this study is isolate the
cDNA clone encoding TC and to study its expression profile in different oil palm
tissues and different developmental stages in the mesocarp. A partial cDNA 0.5 kb in
length was amplified using gene-specific primer from an oil palm expressed sequence
tags sequence and found to have high sequence identity with other plant species TC
such as Oryza sativa (81%) and Helianthus annus (76%) at the amino acid level.
Rapid amplification of cDNA ends (RACE) and end to end PCR successfully
amplified the cDNA for the full mature protein of TC from two oil palm species (E.
guineensis and E. oleifera) of 1. 233 kb in length encoding 411 amino acids. The
amino acid identity between the E. guineensis and E. oleifera sequence was 99.6%.
Both sequences have three long stretches of hydrophobic residues and 30 carboxyl-
terminal amino acid unique for plants (not found in cynobacteria TC). However, the
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reported conserved 5 amino acid carboxyl domain KPPGL in plant TC has 1 variant
amino acid in both oil palm sequences (RPPGL). Quantitative gene expression
analysis performed using Real-time PCR showed expression of E. guineensis TC in
mesocarp was moderately high at 7 w.a.a., dropped at 10 w.a.a then increased steadily
from 10 w.a.a and reached a maximum level at 17 w.a.a and the expression level
dropped significantly at 19 w.a.a. The overall expression levels in leaves were lower
than mesocarp with young leaves having a higher expression level than mature leaves.
Real-time gene copy number analysis indicated only one copy of TC exists in the oil
palm genome. The isolated mature protein sequence of oil palm TC can be used in
functional study to understand the vitamin E biosynthetic process in oil palm and
plants in general. It can also be used in genetic engineering efforts to modify vitamin
E content and composition in plants.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
PENGKLONAN CDNA DAN ANALISIS PENGEKSPRESAN GEN
TOKOFEROL SIKLASE DARIPADA PELBAGAI JENIS TISU KELAPA
SAWIT
(Elaeis guineensis dan Elaeis oleifera)
Oleh
LAI MUN SEONG
JUN 2010
Pengerusi: Profesor Madya Datin Dr Siti Nor Akmar Abdullah
Fakulti: Fakulti Pertanian
Tokoferol siklase (TC) yang memangkinkan tindak balas penghasilan “γ-tocopherol”
daripada “2,3-dimethyl-6-phytyl-1,4-benzoquinone” (DMPBQ) adalah salah satu
enzim yang penting dalam tapak biosintesis tokoferol. Matlamat kajian ini adalah
untuk memencilkan cDNA yang megkodkan tokoferol siklase and menganalisis corak
pengekspresan TC dalam pelbagai peringkat tisu mesokarpa. Jujukan cDNA bersaiz
0.5 kb telah diamplifikasi dengan mengunakan pencetus spesifik yang direka daripada
tag mendedahkan urutan dan didapati mempunyai peratusan persamaan jujukan yang
tinggi di peringkat asid amino dengan tumbuhan lain seperti Oryza sativa (81%) dan
Helianthus annus (76%). Amplifikasi cepat hujung cDNA (RACE) dan PCR hujung
ke hujung berjaya mengamplifikasikan cDNA untuk protein matang daripada dua
spesies kelapa sawit (Elaeis guinenesis dan Elaeis oleifera) dengan kepanjangan
1,233 kb dan mengkodkan 411 asid amino. Peratus kesamaan jujukan asid amino TC
antara E. guineensis dengan E. oleifera adalah 99.6%. Kedua-dua jujukan
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mengandungi tiga turutan panjang residu hidrofobik dan 30 asid amino di bahagian
hujung karboksil yang unik pada tumbuhan (tidak dijumpai dalam TC cyanobacteria).
Namun, terdapat satu variasi asid amino yang dijumpai dalam jujukan TC bagi kedua-
dua spesis kelapa sawit (RPPGL) berbanding dengan 5 asid amino pada hujung
karboksil domain (KPPGL) yang dilaporkan terpulihara dalam TC tumbuhan yang
lain. Analisis ekspresi gen secara kuantitatif dilaksanakan dengan menggunakan
tindak-balas rantai polimerase “masa sebenar” menujukkan ekspresi TC E. guineensis
adalah sederhana tinggi dalam tisu mesokarpa 7 minggu selepas pendebungan (w.a.a),
menurun pada minggu ke-10 w.a.a dan beransur naik sehingga mencapai ekspresi
yang maksima pada minggu ke-17 w.a.a dan menurun dengan signifikan pada minggu
ke 19 w.a.a. Secara keseluruhan, ekspresi TC dalam tisu daun adalah rendah
berbanding dengan tisu mesokarpa; dan daun muda mempunyai ekspresi yang lebih
tinggi berbanding dengan daun matang. Analisis salinan gen menggunakan “masa
sebenar” menunjukkan hanya terdapat satu salinan TC wujud dalam genom kelapa
sawit. Jujukan protein matang yang dipencilkan daripada kelapa sawit boleh
digunakan dalam kajian kefungsian untuk memahami proses biosintesis vitamin E
dalam kelapa sawit dan tumbuhan lain secara umum. Ia juga boleh digunakan dalam
kejuruteraan genetik untuk memanipulasi kandungan dan komposisi vitamin E.
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ACKNOWLEDGEMENTS
First of all I would like to express my most sincere gratitude to Assoc. Prof.
Datin Dr. Siti Nor Akmar Abdullah, Department of Agriculture Technology,
Universiti Putra Malaysia, who is the chairman of my supervisory committee. She has
provided me with many helpful suggestions, important advice and constant
encouragement during the course of this work. Her broad knowledge and logical way
of thinking have been of great value for me. Her understanding, encouragement and
personal guidance have provided a good basis for the present thesis.
I wish to express my warm and sincere thanks to the member of my
supervisory committee, Assoc. Prof. Dr Ho Chai Ling from Department of Cell and
Molecular Biology and Assoc. Prof. Dr. Maheran Abdul Aziz from Department of
Agriculture Technology. They have provided many valuable suggestions and gave
constructive advice throughout this course.
I owe my sincere gratitude to Assoc. Prof. Halimi Mohd Saud, Head of
Department of Agriculture Technology and Assoc. Prof. Mihdzar Abdul Kadir, who
gave me the opportunity to carry out my molecular work in their agro-biotechnology
laboratory. I am also indebted to Puan Norfitriah, Siti, Puan Tina, Puan Wana and Ila
for their valuable assistance in the laboratory.
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I would also like to express my gratefully appreciation to the support of some
very special individuals, Chia Sok Hua, Eum Sang Mi, Alireza Khosravi, Chin Wai
Hoe, Ashida Othman, Siti Mariyam Ijab, and Hasnul Hakim. They helped me
immensely by giving me encouragement and friendship, providing numerous ideas
and useful discussion and devoting their precious time to made many valuable
suggestions.
Last but not least, my special gratitude is extended to my parents and for their
constant guidance, support and sacrifices since my childhood. Without their support
and encouragement, this study is impossible to be completed.
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I certify that a Examination Committee has met on 23th
Dec 2010 to
conduct the final examination of Lai Mun Seong on his master thesis entitled “cDNA
Cloning and Expression Analysis of Tocopherol Cyclase Gene from Various Oil Palm
Tissues (Elaeis guineensis and Elaeis oleifera)” in accordance with the Universities
and University Colleges Act 1971 and the Constitution of the Universiti Putra
Malaysia [P.U(A) 106]. The Committee recommends that the student be awarded the
relevant degree.
Members of the Thesis Examination Committee were as follows:
Chairperson, PhD
Title: Assoc. Prof. Dr. Mihdzar Abdul Kadir
Name of Faculty: Agriculture
Universiti Putra Malaysia
(Chairman)
Examiner 1, PhD
Title: Assoc. Prof. Dr. Halimi Mohd Saud
Name of Faculty: Agriculture
Universiti Putra Malaysia
(Internal Examiner)
Examiner 2, PhD
Title: Dr. Parameswari Namasivayam
Name of Faculty: Biotechnology and Biomolecular Science
Universiti Putra Malaysia
(Internal Examiner)
External Examiner, PhD
Title: Assoc. Prof. Dr. Zamri Zainal
Name of Department/ Faculty: Science and Technology
Name of Organization (University/ Institute) Universiti Kebangsaan Malaysia
Country: Malaysia
(External Examiner)
_________________________
BUJANG KIM HUAT, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Datin Siti Nor Akmar Abdullah, PhD
Assoc. Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Chairman)
Ho Chai Ling, PhD
Assoc. Professor
Faculty of Biotechnology and Biomolecular Science
Universiti Putra Malaysia
(Member)
Maheran Abdul Aziz, PhD
Assoc. Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
____________________________
HASANAH MOHD GHAZALI, PhD Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously and is not
concurrently, submitted for any other degree at Universiti Putra Malaysia or at any
other institution.
__________________
LAI MUN SEONG
Date: 23 December 2010
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TABLE OF CONTENT
Page
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENTS vi
APPROVAL viii
DECLARATION x
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVAITIONS xviii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW
2.1 Oil Palm 4
2.1.1 Physiology of Oil Palm 4
2.1.2 Oil Palm Production 6
2.1.3 Triacylglycerol in Palm Oil 7
2.1.4 Minor Component in Palm Oil 8
2.2 Vitamin E 11
2.2.1 Introduction to Vitamin E 11
2.2.2 Biosynthesis of Tocopherol 14
2.2.3 Beneficial Properties of Tocopherol
2.2.4 Tocopherol Cyclase
18
21
2.3 Introduction to Real-Time Chemistries 25
2.3.1 Absolute Quantification 27
2.3.2 Relative Quantification 29
3 MATERIALS AND METHODS
3.1 Sample Collection 31
3.2 Isolation of Total RNA 32
3.2.1 RNA Extraction Method 32
3.2.2 RNA Quantification 33
3.3 First-Strand cDNA Synthesis 33
3.4 Isolation of House Keeping Gene 34
3.4.1 Primer Design Based on the Oil Palm Glyceraldehyde 3-
Phosphate dehydrogenase Gene (GAPDH)
34
3.4.2 RT-PCR Analysis of GAPDH Gene in Different Oil
Palm Tissues
35
3.4.3 Cloning of the PCR Products 35
3.4.4 Plasmid Isolation 36
3.4.5 Screening for the Recombinant Plasmid 37
3.5 Isolation of Tocopherol Cyclase Gene 38
3.5.1 Amplification of Tocopherol Cyclase Gene from
Different Oil Palm Tissues
38
3.5.2 Gel Extraction of PCR Products 38
3.6 SMARTTM
RACE cDNA Amplification 39
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3.6.1 Design of Gene Specific Primers for 5’ RACE and
3’RACE Amplification
39
3.6.2 First-Strand cDNA Synthesis 40
3.6.3 Isolation of the 5’ and 3’ cDNA ends of Tocopherol
Cyclase from Elaeis guineensis
40
3.6.4 Isolation of the 5’ and 3’ cDNA ends of Tocopherol
Cyclase from Elaeis oleifera
41
3.6.5 Long Distance PCR 42
3.6.6 Sequence Analysis of the Full-Coding Region of
Tocopherol Cyclase Gene from Elaeis guineensis and
Elaeis oleifera
43
3.7 Molecular Characterization of Tocopherol Cyclase 44
3.7.1 Determining the Gene Copy Number of Tocopherol
Cyclase using Real-Time PCR
44
3.7.2 Gene Expression Analysis via Absolute Quantification
Approach
51
4 RESULTS
4.1 Sample Collection and Total RNA Isolation 55
4.2 Partial cDNA Clone Isolation 61
4.3 Isolation of cDNA clone Encoding the Full-Mature Protein of
Tocopherol Cyclase from Elaeis guineensis and Elaeis oleifera
63
4.4 End to End PCR 73
4.5 Comparison of Tocopherol Cyclase from Two Oil Palm Species 78
4.6 Characteristic of the cDNA Encoding the Tocopherol Cyclase 84
4.7 Molecular Characterization of Tocopherol Cyclase 98
4.7.1 Gene Copy Number Determination of Tocopherol
Cyclase in Elaeis guineensis using SYBR Green
Approach
98
4.7.2 Gene Expression Analysis of Tocopherol Cyclase with
Absolute Quantification Real-Time PCR
102
5 DISCUSSION
5.1 Analysis of the cDNA Clone Encoding the Full-Mature Protein
of Tocopherol Cyclase
110
5.2 Gene Copy Number of Tocopherol Cyclase in Oil Palm
5.3 Transcripts Accumulation and Expression of Tocopherol
Cyclase
113
114
5.4 Future Studies 117
6 CONCLUSION 119
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BIBLIOGRAPHY
121
APPENDIX A 134
APPENDIX B 136
APPENDIX C 137
APPENDIX D 138
APPENDIX E 140
BIODATA OF STUDENT 144