generation of expressed sequence tags (ests) … of...primer-based approach not only enables...
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GENERATION OF EXPRESSED SEQUENCE TAGS (ESTs) DATABASE AND ISOLATION OF ALCOHOL DEHYDROGENASE GENE FROM
YOUNG LEAF SAMPLES OF Metroxylon sagu
Wee Ching Ching
Master of Science 2011
Pusat Khidmat MaklumatAkadem& UNRrF, RST17 4'4"LAYSiA SA RAW; K
GENERATION OF EXPRESSED SEQUENCE TAGS (ESTS) DATABASE AND ISOLATION OF ALCOHOL DEHYDROGENASE
GENE FROM YOUNG LEAF SAMPLES OF Metroxylon sagu P. KHIDMAT MAKLUMAT AKADEMIK
MAß
1000246359
WEE CHING CHING
A thesis submitted in fulfillment of the requirement for the Degree of
Master of Science (Biotechnology)
Faculty of Resources Sciences and Technology UNIVERSITI MALAYSIA SARAWAK
2011
ACKNOWLEDGEMENTS
First of all, I would like to thank my supervisor, Dr Hairul Azman Roslan for your guidance
throughout the project. Thanks also for giving me the opportunity to learn and explore even
more in the field of biotechnology.
Second, I would like to thank my friends: Shayrul Bariyah, Nur Qistina, Tan Sia Hong and
Lee Jong Jen for your assistance, encouragement and sharing useful knowledge to help me
troubleshoot my problem encountered.
Next, I would like to thank lab assistant, Mr Aziz for your help in fording chemicals and
equipments needed for me to carry out my research smoothly.
,I also would like to take this opportunity to thank my parents for their encouragement,
understanding and also supporting in every aspects of my life.
Last but not least, I want to thank Ministry of Science, Technology and Innovation for
awarding me scholarship and also thanks for the Sciencefund Grant, E 14088-02-01-09-
SF0008 for fund support in buying things required to carry out the project. Thanks again to all
of you!
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ABSTRACT (Alcoholic
fermentation in plants with reduction of acetaldehyde by alcohol dehydrogenase to
produce energy is one of the metabolic pathways that enables plant to withstand stress. Sago
palm is important to the state of Sarawak as one of the most important crops that brings huge
revenue. It has the ability to withstand stress by growing well in waterlogged area. Shoots of
sago palm in the early development stages has a high alcohol dehydrogenase gene expression.
In this study, the gene expression in selected sago palm tissue was studied via the generation
of expressed sequence tags of young leaves and isolation of full length Adh cDNA from sago
palm leaves using a combination of RACE and DNA walking method) From the EST,
sequence assembly yielded 50 contigs and 347 singletons with a total of 397 tentative unique
genes. The redundancy of the library is 29%. Most of the transcripts were involved in the
primary metabolites and transcripts related to stress were also detected. A partial Adh
transcript (1385 bp in length) which is termed msAdh class III had also been detected in the
library with 95% similarity with Epipremnum aureum class III Adh. In silico analysis of the
polypeptide of msAdh class III indicated that it contains all the conserved amino acids at both
substrate-interacting and coenzyme-interacting segments. Both Arg-l 15 and Asp-57 that play
an main role in binding of glutathione for formaldehyde dehydrogenase activity were also
detected in msADH class III. Meanwhile, a complete Adh cDNA, termed msAdhl, was
successfully isolated using the RACE and DNA walking techniques. DNA walking using
primer-based approach not only enables isolation of Adh gene successfully but also help in
identifying the promoter sequence and the intron sequence of msAdhl gene. In this method,
around 1.2 kb msAdhl genomic DNA sequence was obtained. Sequence analysis of this
sequence detects promoter sequences which contains TATA box, CAT box motif and also
AGGA box that located at -144 to -136, -395 to -391 and 275 to 272 from the initiation of
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translation sites, respectively. The intron detected was the first intron site of sago palm
msAdhl gene with a total length of 477 bp. Therefore, full length msAdhl cDNA has
nucleotide sequence of 1464 bp and encodes for 380 amino acid sequences. This cDNA has
91% and 85% homology with Elaeis guineensis and Washingtonia robusta respectively.
Several Adh-specific motifs such as the two zinc-binding domains located at Cys-48/His-
70/Cys-178 and Cys-100/Cys-103/Cys-106/Cys-114, Asp-227 that bind to the adenosine
ribose of the coenzyme and the amino acid Phe 93 and Leu 116 that bind to alcohol substrate
were also found in the ADH protein sequences.
IV
Penjanaan databes penanda jujukan terekspres dan pemencilan gen alkohol dehidrogenase dari sampel daun muda Metroxylon sagu
ABSTRAK
Penghasilan tenaga hasil daripada fermentasi beralkohol tumbuhan melalui cara penurunan
asetaldehida oleh alkohol dehidrogenase merupakan salah satu jalur metabolisme yang
membolehkan tumbuhan tahan daripada tekanan. Pokok sagu merupakan hasil tanaman yang
penting di Sarawak kerana ia mendatangkan pendapatan yang lumayan. Pokok ini memiliki
kemampuan tahan tekanan dan dapat tumbuh dengan subur di kawasan perairan. Tunas pokok
sagu mempunyai tahap-tahap perkembangan awal seiringan dengan peningkatan dalam
ekspresi gen Adh. Dalam kajian ini, expresi gen daripada tisu pokok sagu yang terpilih telah
dikaji melalui penjanaan penanda jujukan terekspres daun muda dan rantai Adh cDNA telah
dipencilkan melalui gabungan kaedah RACE dan penitian DNA. Daripada ESTs, penindihan
jujukan menghasilkan 50 kontig dan 347 singleton dengan jumlah 397 gen unik jangkaan
(TUGs). Redundansi daripada perpustakaan adalah 29%. Kebanyakkan transkrip adalah
terlibat dalam metabolit utama dan transkrip yang berkaitan dengan tekanan juga dikesan.
Transkrip Adh separa (sepanjang 1385 bp), juga dikenali sebagai msAdh kelas III, telah
dikesan di perpustakaan dengan 95% persamaan dengan Epipremnum aureum kelas III Adh.
Analisis in silico polipeptida kelas III msAdh menunjukkan bahawa ia mengandungi semua
asid amino dilestarikan di kedua-dua daerah interaksi-substrat dan interaksi-co-enzim. Kedua-
dua Arg-115 and Asp-57 yang memainkan peranan utama dalam pengikatan gluthione untuk
kegiatan formaldehid dehydrogenas juga dikesan di msADH class III. Sementara itu, cDNA
Adh lengkap, dinamakan msAdhl, telah berjaya dipencilkan dengan menggunakan teknik
RACE dan penitian DNA. Penitian DNA dengan menggunakan pendekatan berasaskan primer
bukan saja berjaya memencilkan gen Adh tetapi juga membolehkan jujukan proba dan jujukan
v
intron pertama msAdhl gen dikenalpasti. Dalam kaedah ini, jujukan genom msAdhl DNA
sepanjang 1.2 kb telah diperolehi. Analisis jujukan ini mengesan jujukan proba yang
mengandungi petak TATA, petak motif CAT dan juga petak AGGA yang terletak di posisi -
144 ke -136, -395 ke -391 dan -275 to -272 masing-masing dari permulaan tempat translasi.
Intron yang dikesan adalah intron pertama dalam gen msAdhl pokok sagu dengan panjang
477 bp. Oleh itu, panjang penuh msAdhl cDNA memiliki jujukan nukleotida 1464 bp dan
380 jujukan asid amino. cDNA ini mempunyai 91% dan 85% homologi masing-masing
dengan Elaeis guineensis dan Washingtonia robusta. Beberapa Adh-motif tertentu seperti dua
domain pengikatan zink yang terletak di Cys-48/His-70/Cys-178 dan Cys-100/Cys-103/Cys-
106/Cys-114, Asp-227 yang mengikat kepada adenosin ribosa dari koenzim dan asid amino
Phe 93 dan Leu 116 yang mengikat substrat alkohol juga boleh dijumpai dalam jujukan
protein ADH.
ýI
Pusat Khidmat Maklumat Akademik U1vIVERSITi MALAYSIA SARAWAK
TABLE OF CONTENTS
Contents
Acknowledgement
Abstract
Table of contents
List of Figures
List of Tables
List of Abbreviations
Chapter 1: INTRODUCTION
1.1 Background
1.2 Objective of this study
Chapter 2: LITERATURE REVIEW
2.1 Background to sago palm
2.1.1 Names and taxonomy
2.1.2 Botanical description of sago palm
2.1.3 Ecology and distribution
2.1.4 Functional uses
2.2 Alcohol dehydrogenase
2.2.1 Classification
2.2.2 Alcohol dehydrogenases in plants
2.2.3 Discovery of ADH and its evolution
2.2.4 Structure of alcohol dehydrogenases
2.2.5 Mechanism of alcohol dehydrogenase
2.2.6 Alcohol dehydrogenase expression in plants
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2.3 Methods for gene isolation 17
2.3.1 Rapid Amplification of Complementary Deoxyribonucleic 17
Acid (cDNA) Ends (RACE)
2.3.2 DNA walking 18
2.4 Characteristic of promoter 20
2.4.1 Characterization of Adh promoter in plants species 22
2.5 Expressed Sequence Tags (ESTs) 24
2.5.1 Definition of expressed sequence tags 24
2.5.2 cDNA library construction 25
2.6 Gene expression study 26
2.6.1 Control of gene expression in eukaryotes 26
2.6.2 Bacterial expression systems 27
Chapter 3: GENERATION OF EXPRESSED SEQUENCE TAGS (ESTs) FROM 29
ME TR OX YL ON SAGU
3.1 Introduction 29
3.1.1 Bioinformatics 31
3.2 Materials and methods 32
3.2.1 Plants materials 32
3.2.2 RNA isolation 33
3.2.3 Preparation of DNA-free total RNA
3.2.4 RNA analysis
3.2.5 First-strand cDNA synthesis
3.2.6 Second strand cDNA synthesis
3.2.7 cDNA library constructed by tailing method
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3.2.8 cDNA library constructed using subtractive hybridization 36
3.2.9 cDNA library construction by using cDNA library 38
construction kit (Stratagene)
3.2.9.1 Phagemid or plasmid isolation and DNA sequencing 43
3.2.10 EST processing, contig assembly and analysis 44
3.3 Results 45
3.3.1 Integrity of RNA 45
3.3.2 RNA purification 45
3.3.3 RNA purity and yield 46
3.3.4 Characteristic of the constructed cDNA library 47
3.3.5 ESTs clustering and assembly 51
3.3.6 Sequence analysis of ESTs 52
3.3.7 Detection of alcohol dehydrogenase class III transcript from 61
EST database
3.4 Discussions 67
3.4.1 Extraction of RNA from sago palm 67
3.4.2 EST library 68
3.4.3 Detection of Adh class III from EST database 74
3.5 Conclusions 76
Chapter 4: ISOLATION OF ALCOHOL DEHYDROGENASE (ADH) cDNA 77
FROM METROXYLON SAGU
4.1 Introduction 77
4.2 Materials and methods 79
4.2.1 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 79
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4.2.2 3'RACE
4.2.3 5'RACE
4.2.4 Second RACE
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4.2.5 Full-length Adh cDNA amplification 82
4.2.6 Purification of PCR product 82
4.2.7 Ligation into plasmid 82
4.2.8 Calcium chloride (CaC12) bacteria competent cells preparation 83
4.2.9 Transformation
4.2.10 Colony PCR
4.2.11 Miniprep
4.2.12 Sequencing and sequence analysis
4.3 Results
4.3.1 RT-PCR analysis
4.3.2 Adh cDNA isolation using conserved region
4.3.3 3'RACE
4.3.4 5'RACE
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4.3.5 PCR using 5RACE2 primer 90
4.3.6 Verification of full length Adh cDNA 93
4.3.7 Analysis of the predicted protein sequence of sago palm 93
msAdhl
4.3.8 Molecular evolution of sago palm msADH 1 94
4.4 Discussion 99
4.5 Conclusions 102
X
Chapter 5: ISOLATION OF PROMOTER SEQUENCES OF ALCOHOL 103
DEHYDROGENASE GENE FROM METROXYLON SAGU THROUGH DNA
WALKING
5.1 Introduction 103
5.2 Materials and methods 105
5.2.1 Genomic DNA extraction 105
5.2.2 DNA purification 105
5.2.3 DNA analysis 106
5.2.4 DNA walking 106
5.2.5 Sequencing and sequence analysis
5.3 Results
5.3.1 Integrity of DNA
5.3.2 Removal of RNA molecules
5.3.3 DNA purification
5.3.4 DNA walking
5.3.5 DNA nucleotide sequence analysis
5.3.5.1 Kpn400 and OKpn400
5.3.5.2 YSacl200 and OSacl200
5.4 Discussion
5.5 Conclusions
Chapter 6: PROTEIN EXPRESSION ANALYSIS
6.1 Introduction
6.2 Materials and methods
6.2.1 Cloning of msAdhl into pET vector
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6.2.2 Transformation into expression host, BL21(DE3)
6.2.3 Induction and optmize expression of target
6.2.4 Cell lysis, protein extraction and quantification
6.2.5 Total protein detection through denaturing PAGE
6.2.6 ADH staining
6.2.7 ADH enzyme assay and protein quantification
6.3 Results
6.3.1 Restriction enzyme digestion
6.3.2 Sequencing result
6.3.3 Total protein detection through SDS-PAGE
6.3.4 ADH staining and enzyme activity assay
6.3.5 Protein quantification
6.4 Discussion
6.5 Conclusions
CHAPTER 7: CONCLUSION
REFERENCES
APPENDIX A
APPENDIX B
APPENDIX C
APPENDIX D
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LIST OF FIGURES
Figure Pages
Figure 2.1: The geographic distribution of Metroxylon sagu. 7
Figure 2.2: Applications of sago palm. 8
Figure 2.3: Diagram of alcoholic fermentation pathway. 9
Figure 3.1: (a) Sago palm; (b) Young leaves; (c) Mature leaves 32
Figure 3.2: Assembly of the drip column 41
Figure 3.3: Agarose gel (1.0%) electrophoresis of RNA isolated from sago 45
palm.
Figure 3.4: Agarose gel (1.0%) electrophoresis of RNA before and after 46
treated.
Figure 3.5: Blue-white color selection and primary titering of the cDNA 47
library constructed using commercial kit.
Figure 3.6: Transformation of different dilution factor of phagemid into SOLR 48
strain.
Figure 3.7: Agarose gel (1.5%) electrophoresis of colony PCR products from 49
libraries constructed manually.
Figure 3.8: Agarose gel (1.5%) electrophoresis of colony PCR products from 50
library constructed using commercial kit.
Figure 3.9: Agarose gel (1.0%) electrophoresis of plasmid. 50
Figure 3.10: The ten most frequently matched plants according to the BLASTX 53
EST search results (Blast2Go software).
Figure 3.11: Pie chart representation of GO-annotation classification of 58
Metroxylon sagu ESTs by putative biological processes (level 3).
X111
Figure 3.12: Pie chart representation of GO-annotation classification of 59
Metroxylon sagu ESTs by putative molecular function (level 3).
Figure 3.13: Pie chart representation of GO-annotation classification of 60
Metroxylon sagu ESTs by putative cellular component (level 4).
Figure 3.14: Residues at functionally important positions in substrate binding 61
and coenzyme binding for the pea and sago palm class P (bottom)
and class III (top) alcohol dehydrogenase.
Figure 3.15: Alignment of the deduced amino acid sequences between 62
msADHI and msADH class III of young leaves of Metroxylon
sagu.
Figure 3.16: Phylogenetic tree of msADH class III of sago palm and other 63
species class III ADH. It is constructed by using clustal method of
Lasergene Megalign (DNASTAR, Inc., Madison, WI) based on
amino acid similarities of the sequences.
Figure 3.17: Nucleotide msAdh class III sequence of sago palm leaf cDNA and 64
deduced amino acid sequence. Underlined regions represent
putative polyadenylation sites (AATGAA).
Figure 3.18: Alignment of the deduced amino acid sequences of msADH class 65
III between Metroxylon sagu and selected plant species.
Figure 4.1: Position of the primers in accordance with Adh cDNA. 80
Figure 4.2: Agarose gel (1.5%) electrophoresis of RT-PCR products with ef- 85
1a primers.
Figure 4.3: Agarose gel (1.5%) electrophoresis of the RT-PCR (gradient, as in 86
figure 4.4) products of young leaves cDNA using morADH-f and
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morADH-r.
Figure 4.4: Agarose gel (1.5%) electrophoresis of the gradient PCR products 86
of young leaves cDNA using morADH-f and morADH-r.
Figure 4.5: A 1.5 % agarose gel electrophoresis showing the PCR products of 87
young leaves cDNA using morADH8-f and adaptor(dt) 17 primers.
Figure 4.6: Figures showed the 3'RACE-PCR products of mature leaves and 88
young leaves on a 1.5% agarose gel.
Figure 4.7: Gradient PCR products of young leaves cDNA on a 1.5 % agarose 89
gel of the using GSP3 and adaptor(dt) 17 primers.
Figure 4.8: 5'RACE products of young leaves cDNA on a 1.5 % agarose gel 89
using GSP3 and adaptor(dt) 17 primers.
Figure 4.9: PCR product using primer 5RACE2 and GSP3 on 1.5% agarose 90
gel.
Figure 4.10: Alignment and mapping between 5' and 3' RACE products 92
showed complete msAdhl cDNA of sago palm.
Figure 4.11: PCR product of full length msAdhl cDNA. 93
Figure 4.12: Nucleotide msAdhl sequence of sago palm leaf cDNA and 95
deduced amino acid sequence.
Figure 4.13: Alignment of the deduced amino acid sequences of msADHI 96
between Metroxylon sago and selected plant species.
Figure 4.14: Phylogenetic tree of msADHI of sago palm and other species 97
ADH.
Figure 5.1: DNA isolation from (A) young leaves and (B) mature leaves. 108
Figure 5.2: Comparison of DNA before and after treatment by RNase A. 109
x\
Figure 5.3: Agarose gel electrophoresis result of DNA walking PCR of mature 110
leaves.
Figure 5.4: Agarose gel electrophoresis result of DNA walking PCR of young 111
leaves. (A) First round PCR products.
Figure 5.5: Alignment between msAdh gDNA (after intron removed) with 113
cDNA to indicate the similarity between the gDNA and cDNA.
Figure 5.6: Analysis of the upstream sequences of msAdhl gene. 114
Figure 6.1: Agarose gel (1.0%) electrophoresis of Xbal restriction digests of 124
pET-41 a(+) without insert and with msAdh insert (pET-
41 a(+)/Adh).
Figure 6.2: Nucleotide sequencing result using GSP3 primer indicating the 126
pET-41 a(+) vector sequences before the msAdhl cDNA startsite.
Figure 6.3: Nucleotide sequencing result using mor8F primer indicating the 127
pET-41 a(+) vector sequences after the msAdhl eDNA.
Figure 6.4: SDS-PAGE electrophoresis. Protein expression at 30°C in E. coli 127
BL21(DE3) transformed with pET-41 a(+)/Adh.
Figure 6.5: SDS-PAGE electrophoresis. Protein expression at 30°C in E. coli 128
BL21(DE3) transformed with pET-41 a(+)/Adh and pET-41 a(+)
only.
Figure 6.6: Standard curve of protein using bovine serum albumin (BSA). 129
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LIST OF TABLES
Table Pages
Table 2.1: Classes of alcohol dehydrogenases. 11
Table 2.2: Principal classes of alcohol dehydrogenases in plants. 12
Table 3.1: Spectrophotometer reading of RNA extracted from mature leaves and 47
young leaves of sago palm.
Table 3.2: Comparison of ESTs for cDNA library constructed manually and using 51
commercial kit.
Table 3.3: Summary of total EST sequencing. 52
Table 3.4: Distribution of ESTs according to the TopBLASTX search results. 52
Table 3.5: Identity of clusters containing greater than 3 ESTs. 54
Table 3.6: Number of ESTs of different form of heat shock proteins. 55
Table 3.7: Number of ESTs of different types of protein kinase. 56
Table 3.8: Comparison between msADH3 class III of sago palm with other plant 63
class III ADH deduced amino acid sequences.
Table 3.9: Percentage similarity and divergence of msAdh3 and class III Adh 66
nucleic acid sequences between sago palm and other species using the
multiple-sequence alignment program LaserGene by DNASTAR Inc.
(Madison, WI, USA).
Table 4.1: Comparison between msADH 1 with other species ADH deduced amino 94
acid sequences.
Table 4.2: Pair-wise comparison of the percentage similarity and divergence for 98
msADHI amino-acid sequences between sago palm and other species
using Lasergene Megalign by DNASTAR Inc. (Madison, WI, USA).
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Table 5.1: Nucleotide sequence of the overhanging primers (OHP); adaptor primer 106
(AP); nested primer (NP) and gene specific primer (GSP4) that was
designed from the Adh cDNA isolated in the chapter 4.
Table 5.2: Spectrophotometer reading of DNA extracted from mature leaves and 109
young leaves of sago palm.
Table 6.1: Spectrophotometer readings for standard BSA at 595 nm wavelength. 129
Table 6.2: Spectrophotometer readings for protein samples at 595 nm wavelength 130
and protein concentration (mg/ml).
xviii
LIST OF ABBREVIATIONS
EST Expressed Sequence Tag
ADH alcohol dehydrogenase
LDH lactate dehydrogenase
ms Metroxylon sagu
GAPDH glyceraldehyde-3 -phosphate dehydrogenase
PAT phosphinothricin acetyltransferase
Hsp heat shock protein
HSF heat shock factor
MAP mitogen-activated protein
GO gene ontology
ROS Reactive oxygen species
NAD+ Nicotinamide adenine dinucleotide
BLAST Basic Local Alignment Search Tool
NCBI National Center for Biotechnology Information
bp base pair
cDNA complementary DNA
DNA deoxyribonucleic acid
DNase deoxyribonuclease
RNA Ribonucleic acid
RNase ribonucleases
RT-PCR Reverse Transcription Polymerase Chain Reaction
DDRT- PCR Differential Display Reverse Transcription PCR
RACE Rapid amplification of cDNA ends
X1\
DEPC diethyl pyrocarbonate
GSP gene specific primer
UTR untranslated region
LiCI litium chloride
CTAB cetyl trimethyl ammonium bromide
PVP 40 polyvinylpyrrolidone
EDTA disodium ethylenediaminetetra-acetate 2H20
LB Luria- Bertani
LA Luria-Agar
IPTG isopropyl-B-D-thiogalactopyrano side
X-gal 5-bromo-4-chloro-3-indolyl- ß-D-galactoside
Amp ampicillin
Kan kanamycin
M-MuLV-RT Moloney Murine Leukimia Virus Reverse Transcriptase
OD optical density
pfu plaque forming units
ORF open reading frame
PCR polymerase chain reaction
rpm revolutions per minute
RT reverse transcriptase
TAE tris-acetate
TdT terminal deoxynucleotidyl transferase
T. E tris EDTA
RE restriction enzyme
x\
ml
µl
TUG
hr
milliliter
microliter
tentative unique gene
hour
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CHAPTER 1
INTRODUCTION
1.1 Background
Sago palm (Metroxylon sagu Rottb. ) is a monocotyledonous plant belonging to the order
Arecales, family Palmae, and subfamily Calamoideae. It is also well known as one of the
agricultural crops that bring economical income to the state of Sarawak. According to Tie and
Lim (1991), the present area planted with sago palms in Sarawak is around 19,720 hectares.
Sarawak is the largest sago growing areas and the world's biggest exporter of sago by
exporting 44,700 tonnes of sago start in 2007 to Penisular Malaysia, Japan, Taiwan,
Singapore and other countries (http: www. doa. sarawak. gov. my/statistik07_6_3 4. pdf).
Sago palm is one of the agricultural crops that is able to withstand stress by growing
well in harsh swampy area. Different environmental stresses are encountered by plants during
their growth and development. In order to survive well, plants respond by changing in their
cell structures, biochemistry and gene expression. Study to identify and characterize both
alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) enzyme of sago palm roots
and leaves that grew in dry and waterlogged areas had been conducted by Roslan and
Sundaraj in 2007. The study (Roslan & Sundaraj, 2007) revealed that there was an increase in
the ADH expression in the waterlogged roots compared to non-waterlogged roots.
Interestingly, the expression level of ADH in young leaves of non-waterlogged sago palm is
the highest compared to the waterlogged roots. This may be due to the various physiological
and biochemical changes in the maturation of young leaf and because of that it has brought up
i
the interest to study the gene expression profile and also the isolation of the Adh cDNA from
shoots of this crop.
Gene expression study was carried out by the construction of an expressed sequence
tag (EST) database. ESTs are generated by sequencing of cDNA library. It is the fastest way
to identify transcribed genes and at the same time to discover novel genes. To date, there is no
ESTs reported on sago palm. Here, the study reports the preliminary ESTs database of young
leaves from sago palm. The set of ESTs obtained will be useful for further analysis.
Apart from that, isolation of full length sequence of msAdhl cDNA from sago palm
leaves was carried out using a combination of RACE method and genome walking. Rapid
amplification of complementary deoxyribonucleic acid (cDNA) ends (RACE) is one of the
rapid ways to isolate transcript of interest for investigation in gene expression and function
and protein structure. As a result, full length sequence of msAdhl cDNA had been
successfully isolated from both young and mature leaves of sago palm. In addition, the
promoter sequence of msAdhl was also isolated using a genome walking method.
To determine the function of ADH protein, it is heterologously expressed in bacterial
system. This system was used because it is cost-saving, high productivity and not time
consuming. In addition, purification is easier to be done in prokaryotic system compare to
eukaryotic system. Initial trial on the ADH protein expression was performed in the pET-
41 a(+) system from Novagen using BL21(DE3) (Invitrogen) as host cells but it was not
successful.