UNIVERSITI PUTRA MALAYSIA

DIFFERENTIAL GENE EXPRESSION OF OIL PALM (ELAEIS GUINEENSIS JACQ.) FRUITS DURING LATE RIPENING STAGES AND MOLECULAR CHARACTERISATION OF METALLOTHIONEIN-LIKE TRANSCRIPTS

AHMED BAKHIT SALIM AL-SHANFARI

FP 2012 17

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DIFFERENTIAL GENE EXPRESSION OF OIL PALM ( ELAEIS GUINEENSIS JACQ.) FRUITS DURING LATE RIPENING STAGES AND MOLECULAR CHARACTERISATION OF METALLOTHIONEIN-LIKE TRANSCRIPTS

By

AHMED BAKHIT SALIM AL-SHANFARI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2012

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DEDICATIONS

This thesis is dedicated to my wonderful parents, who have raised me to be the person I am today. To my wife, sisters, and children, you have been with me every step of the way, through good times and bad. Thank you for all the unconditional love, guidance, and support that you have always given me, helping me to succeed and instilling in me the confidence that I am capable of doing anything I put my mind to. Thank you for everything. I love you! Finally, this thesis is dedicated to all those who believe in the richness of learning.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Doctor of Philosophy

DIFFERENTIAL GENE EXPRESSION OF OIL PALM ( ELAEIS GUINEENSIS

JACQ.) FRUITS DURING LATE RIPENING STAGES AND MOLECULAR CHARACTERISATION OF METALLOTHIONEIN-LIKE TRANSCRIPTS

By

AHMED BAKHIT SALIM AL-SHANFARI

September 2012

Chairman: Associate Professor Datin Siti Nor Akmar binti Abdullah, PhD

Faculty: Agriculture

Oil palm (Elaeis guineensis Jacques) is a major oil crop of economic importance in the

world. Different biological and physiological mechanisms occur during oil palm fruit

ripening. These mechanisms are regulated by the genes expressed during fruit ripening

processes. Therefore, determining the structure and function of the expressed genes

involved in these mechanisms will help to improve the important characters for

enhancing palm fruit oil yield and quality. A forward suppression subtractive

hybridization (SSH) library was constructed to identify genes involved in fruit ripening

in oil palm. Oil deposition, as an oil palm fruit ripening indicator, will start at

approximately 12 weeks after anthesis (w.a.a.) with an active period of oil synthesis at

15-16 w.a.a. until fruit maturity at about 20 w.a.a. in the mesocarp. Hence, the

suppression was performed with cDNA extracted from fruits at 12 w.a.a. as “driver” and

fruits at 17 w.a.a. as “tester”. A single-pass sequencing from the 5′-end of 2,112

randomly selected cDNA clones resulted in 2,019 high-quality expressed sequence tags

(ESTs). Clustering of the 2,019 EST sequences showed 20 unigenes consisting of 9

contigs and 11 singletons. Among the edited EST sequences, 1,109 (14 unigenes) had

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significant matches with protein sequences in the public databases. The matched ESTs

were further classified into six putative cellular functions including unclassified proteins

(38.0%), cell rescue and defense (33.8%), proteins with binding functions (27.7%),

biogenesis of cellular component (0.3%), metabolism (0.1%), and cell cycle (0.1%). At

17 w.a.a., the 20 unigenes were expressed at higher abundance compared to 12 w.a.a.

Two of the abundant transcripts encoding type 2 metallothionein-like proteins

designated as MET2a and MET2b were selected for further study due to their high

abundance (16.05%) in the SSH library and their involvement in biological processes in

fruit development and maturation. The second part of the present study involved the

isolation of the full-length cDNA encoding MET2a and MET2b from the ripening oil

palm fruit mesocarp at 17 w.a.a. and examing their expression patterns compared to the

other two previously reported type-3 MT members (MT3-A and MT3-B) in various oil

palm organs including different vegetative and reproductive tissues. The full-length

cDNA sequence of MET2a and MET2b were 571 and 553 bp designated as EgMT2a and

EgMT2b, respectively. Their sequences were homologous (67–77%) with several type-2

MTs in plants. All four genes were differentially expressed in the ripening oil palm

mesocarp tissues but undetectable in the vegetative tissues examined. All the MT genes

examined were significantly up-regulated in the mature developmental stages of oil palm

fruit mesocarp except for EgMT2b which was expressed only at 17 w.a.a. but at a much

lower level. The type 2 MT-like proteins are more in relation with late fruit ripening

stage than the type 3 MT-like proteins consistent with their reported functions in the

homeostasis or detoxification. The findings in the present study will contribute to a

better understanding of the molecular mechanisms involved in fruit ripening in oil palm.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PEMBEZAAN PENGEKSPRESAN GEN SEMASA TEMPOH AKHIR

KEMATANGAN BUAH KELAPA SAWIT ( Elaeis guineensis Jacq.) DAN PENCIRIAN MOLEKULAR TRANSKRIP-TRANSKRIP BAK-

METALLOTIONIEN YANG TELAH DIKENALPASTI

Oleh

AHMED BAKHIT SALIM AL-SHANFARI

September 2012

Pengerusi: Professor Madya Datin Siti Nor Akmar binti Abdullah, PhD

Fakulti: Pertanian

Kelapa sawit (Elaeis guineensis Jacques) adalah tanaman minyak utama sebagai

penyumbang ekonomi dunia yang penting. Pembezaan mekanisma biologi dan fisiologi

berlaku ketika pemasakan buah sawit. Mekanisma-mekanisma ini dikawalatur oleh gen

terekspres ketika proses pemasakan buah. Oleh itu, penentuan struktur dan fungsi gen

terekspres yang terlibat dalam mekanisma ini akan membantu meningkatkan ciri-ciri

berkepentingan untuk meningkatkan hasil dan kualiti minyak kelapa sawit. Satu

perpustakaan subtraktif hibridisasi (SSH) supresi kehadapan telah dibina untuk

mengenalpasti gen-gen yang terlibat dengan kemasakan buah dalam kelapa sawit (Elaeis

guineensis Jacq). Pengumpulan minyak sebagai petunjuk pemasakan buah sawit akan

bermula kira-kira 12 minggu selepas antesis (m.s.a) dengan tempoh aktif ketika 15-16

m.s.a. sehingga buah masak pada 20 m.s.a. dalam mesokarp. Oleh itu, teknik supresi ini

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telah dijalankan dengan menggunakan cDNA yang telah diekstrak daripada buah sawit

pada 12 minggu selepas antesis (m.s.a) sebagai “pemacu” dan daripada 17 m.s.a sebagai

“kajian”. Penjujukan sekali penghujung-5’ yang dijalankan keatas 2,112 klon-klon

cDNA yang dipilih secara rawak telah menghasilkan 2,019 penanda jujukan terekspres

(ESTs) berkualiti tinggi dengan purata panjang bacaan sebanyak 383 bp. Pengklusturan

2,019 jujukan EST menggunakan StackPACK menunjukkan 20 unigen yang terdiri dari

9 kontig dan 11 tunggalan. Di antara jujukan EST yang telah disunting, 1,109 (14

unigen) mempunyai padanan yang signifikan dengan jujukan protein di pangkalan data

umum. EST yang mempunyai padanan seterusnya diklasifikasikan kepada enam fungsi

selular putatif termasuk protein yang tidak diklasifikasikan (38.0%), penyelamat dan

pertahanan sel (33.8%), protein dengan fungsi pengikat (27.7%), biogenesis komponen

selular (0.3%), metabolisma (0.1%) dan kitaran sel (0.1%). Pada m.s.a ke 17, 20 unigen

telah dikespreskan pada paras tertinggi berbanding pada m.s.a ke 12.

Dua transkrip paras tertinggi yang mengkodkan protein bak-metalotionin jenis ke-2

dilabelkan sebagai MET2a dan MET2b telah dipilih untuk kajian lanjut disebabkan

ketinggian paras (16.05%) di dalam perpustakaan SSH dan penglibatannya di dalam

proses biologi ketika pengembangan dan pematangan buah. Bahagian kedua kajian ini

melibatkan pemencilan klon-klon cDNA lengkap MET2a dan MET2b daripada

mesokarp buah kelapa sawit yang masak pada 17 m.s.a dan menguji corak

pengekspresan yang dibandingkan dengan jenis ke-3 (MT3-A dan MT3-B) seperti yang

dilaporkan terdahulu di dalam pelbagai organ-organ kelapa sawit termasuk tisu vegetatif

dan reproduktif yang berbeza. Jujukan-jujukan cDNA MET2a and MET2b yang lengkap

bersaiz 571 dan 553 bp yang dilabelkan sebagai EgMT2a dan EgMT2b mengikut

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turutan. Kedua-dua jujukan tersebut mempunyai homologi (67-77%) dengan beberapa

MT jenis ke-2 dalam tumbuh-tumbuhan. Keempat-empat gen tersebut telah

diekspreskan secara berbeza pada tisu mesokarp buah kelapa sawit yang masak tetapi

ianya tidak dapat dikesan pada tisu-tisu vegetative yang telah diuji. Kesemua gen MT

yang telah diuji menunjukkan peningkatan ketara dalam regulasi semasa peringkat

matang perkembangan mesokarpa buah kelapa sawit kecuali EgMT2b yang hanya

diekspreskan pada 17 m.s.a pada paras yang rendah. Protein bak-MT jenis ke-2

mempunyai hubungkait yang tinggi dengan proses kemasakan buah pada tempoh akhir

berbanding dengan protein bak-MT jenis ke-3. Ini adalah konsisten dengan fungsi yang

telah dilaporkan dalam homeostasis atau detoksifikasi. Hasil kajian ini boleh

menyumbang kepada pemahaman yang lebih meluas tentang mekanisma molekular yang

terlibat dalam kemasakan buah kelapa sawit.

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ACKNOWLEDGEMENTS

The most important acknowledge is to our Lord Most Merciful Most Wise by whose

mercy I was able to complete this study. May Allah’s peace and blessing be upon our

Beloved Prophet Muhammad who was a mercy unto us from Allah s.w.t., whose

character and nobility none has seen before or after Him (Pbuh).

I would like to gratefully acknowledge the enthusiastic supervision of Associate

Professor Datin Dr. Siti Nor Akmar binti Abdullah, the chairman of the supervisory

committee, and for her technical support and the preparation of this thesis. The members

of the supervisory committee Assoc. Prof. Dr. Halimi Mohd Saud, and Assoc. Prof. Dr.

Suhaimi Napis for their guidance, understanding and invaluable advices throughout the

duration of this study.

I am sincerely grateful to Islamic Development Bank (IDB) as my sponsor that they

accept me as one of their scholars, for funding me throughout my study period covering

all expense based on the scholarship office program rules. Thank you for the financial

support to pursue my PhD degree. I also want to thank the Ministry of Agriculture in

Oman, special grateful to His Excellency A’Shaikh Salim bin Abdullah Al-Khalily the

Minister of Agriculture, to recent Oman's Minister of Agriculture & Fisheries Dr. Fuad

Jaffer Al-Sajwani, to Dr. Ishaq Al-Ruqaishi Under-Secretary of the Minister of

Agriculture & Fisheries, to Dr. Ahmed Al-Bakri the Director General of the Agriculture

and Animal Wealth Research Directorate, and Mr. Saoud Al-Harthi the Director General

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of Agriculture Directorate in Dhofar Governorate for their supports and encouragements

to pursue my study.

To my colleagues and friends in the Faculty of Agriculture – UPM especially the

Agriculture Technology Laboratory, thank you so much for the help and contributions

towards the completion of this thesis. The lab is much merrier with your presence and

laughter.

Several people have played a decisive role in saving me from any difficulties during my

study period. These persons are: my family members, my friends at the university

campus and accommodation. Without your willingness and suggestions this PhD thesis

would not have been written.

Last but not least, I wish to express my sincere thanks to all those who have one way or

another helped me in making this study a success.

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I certify that a Thesis Examination Committee has met on 3 September 2012 to conduct the final examination of Ahmed Bakhit Salim Al-Shanfari on his thesis entitled "Differential Gene Expression of Oil Palm (Elaeis guineensis Jacq.) Fruits during Late Ripening Stages and Molecular Characterisation of Metallothionein-Like Transcripts" in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the PhD degree. Members of the Thesis Examination Committee were as follows: Mohd Rafii Yusop, PhD Associate Professor Institute of Tropical Agriculture Universiti Putra Malaysia (Chairman) Ho Chai Ling, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Mihdzar Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Ton Bisseling, PhD Professor Laboratory of Molecular Bilogy Wageningen University Holland (External Examiner) ____________________________ SEOW HENG FONG, 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 Doctor of Philosophy. The members of the Supervisory Committee were as follows: Siti Nor Akmar Abdullah, PhD Associate Professor Institute of Tropical Agriculture Universiti Putra Malaysia (Chairman) Halimi Mohd Saud, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Suhaimi Napis, PhD Associate Professor Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Member) _________________________ BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION

I declare that the thesis is 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. _____________________________________ AHMED BAKHIT SALIM AL-SHANFARI Date: 3 September 2012

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TABLE OF CONTENTS Page ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL x DECLARATION xii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATION xix CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 2.1 The Oil Palm - Elaeis guineensis Jacq. 4

2.1.1 Origin and History of Cultivation 4 2.1.2 Taxonomy 4 2.1.3 Growth and Propagation 6 2.1.4 Fruit Ripening Stages 7

2.2 Palm Oil 9 2.2.1 Oil Palm as the Most Productive Oil Crop 9 2.2.2 The Uses and the Nutritional Value of the 10 Palm Oil 2.2.3 Oil Production in Oil Palm Fruits 12 2.2.4 The Physiological and Biological Changes 13

during Oil Palm Fruit Ripening 2.3 Gene Expression 14

2.3.1 Isolation of Differentially Expressed Genes 15 2.3.2 Expressed Sequence Tag (EST) 21 2.3.3 Gene Expression Profiles 23 2.3.4 Isolation of Full-length cDNA Clones 26

2.4 Genes Expressed in Ripening Fruits 27

2.4.1 Genes Expressed in Ripening Oil Palm Fruits 29 2.5 Plant Metallothionein-like Genes 30

2.5.1 Metallothionein-like Genes Isolated from Oil 32 Palm

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3 DIFFERENTIAL GENE EXPRESSION IDENTIFIED 34

BY SUPPRESSION SUBTRACTIVE HYBRIDIZATION DURING LATE RIPENING OF FRUIT IN OIL PALM (Elaeis guineensis Jacq.) 3.1 Introduction 34

3.2 Materials and Methods 36

3.2.1 Plant Materials 36 3.2.2 Total RNA Extraction 36 3.2.3 Estimation of RNA Integrity and Purity 38 3.2.4 Messenger RNA (mRNA) Isolation 39 3.2.5 Construction of Subtracted cDNA Library 40 3.2.6 Analysis of Subtraction Efficiency 50 3.2.7 Cloning of Subtracted cDNA 52 3.2.8 Analyzing Transformants 53 3.2.9 Gel Extraction 55 3.2.10 Template Preparation and DNA Sequencing 56 3.2.11 Generation and Analysis of Expressed 57 Sequence Tags (ESTs) 3.2.12 Homology Comparison via Database Searches 58 and Gene Annotation 3.2.13 Analysis of Differential Gene Expression 59

3.3 Results 60

3.3.1 Total RNA Extraction 60 3.3.2 Messenger RNA (mRNA) Isolation 62 3.3.3 Characteristics and Efficiency of cDNA 62 Subtraction 3.3.4 Characterization of the Subtracted cDNA 66 Library 3.3.5 Sequencing and Assembly 71 3.3.6 Blast Searches and Sequence Similarities 75 3.3.7 ESTs with Significant Homology Match 75 (Class I) 3.3.8 ESTs with No Significant Homology Match 78 and No Hits Match (Class II) 3.3.9 EST Redundancy 78 3.3.10 Classification of EST According to Predicted 79 Function 3.3.11 Abundance of Unigene Transcripts during the 81 Late Stages of Fruit Ripening 3.3.12 Expression Patterns of Unigenes 83

3.4 Discussion 87 3.5 Conclusion 92

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4 ISOLATION AND EXPRESSION ANALYSIS 93

OF GENES ENCODING METALLOTHIONEINS IN OIL PALM ( Elaeis guineensis Jacq.) 4.1 Introduction 93

4.2 Materials and Methods 94

4.2.1 Plant Materials 94 4.2.2 Total RNA Extraction 95 4.2.3 Isolation of Full-length cDNAs 95 4.2.4 Analysis of RACE amplified PCR product 97 4.2.5 MT Genes Expression Analysis 100 4.2.6 Multiple Sequence Alignment and Phylogenetic 104 Analysis

4.3 Results 104 4.3.1 Isolation of the Two Selected MT-Like Genes 104 Full-length cDNA Clones 4.3.2 Characteristics of Oil Palm Type 2 113 Metallothionein-Like Genes and their Encoded Proteins 4.3.3 MTs Genes Expression 119

4.4 Discussion 125 4.5 Conclusion 130

5 SUMMARY, GENERAL CONCLUSION AND 132

RECOMMENDATIONS FOR FUTURE RESEARCH

BIBLIOGRAPHY 136 APPENDICES 154 BIODATA OF STUDENT 163 LIST OF PUBLICATIONS 164