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