UNIVERSITI PUTRA MALAYSIA

MOLECULAR CHARACTERIZATION OF AVNA LIBRARY FROM THE FLESH OF DURIAN (DURIO ZIBETHINUS MURR.) CLONE D24

HUYNH KY.

FBSB 2005 19

MOLECULAR CHARACTERIZATION OF cDNA LIBRARY FROM THE FLESH OF DURIAN (Durio zibethinus Murr.) CLONE D24

BY

HUYNH KY

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

APRIL 2005

Dedicated to my parents

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in Fulfilment of the requirement for the degree of Master of Science

MOLECULAR CHARACTERIZATION OF cDNA LIBRARY FROM THE FLESH OF DURIAN (Durio zibethinus Murr.) CLONE D24

BY

HUYNH KY

April 2005

Chairman :

Faculty

Associate Professor Suhaimi bin Napis, Ph.D.

Biotechnology and Biomolecular Sciences

The genus Durio is originated from Malaysia. Out of 198 registered clones,

D24 is known as the best variety not only in Malaysia but also in the world.

However, molecular genetic study on durian is still limited. This study was

one of the initial molecular work which could contribute to the database of

clone D24 durian. Firstly, we have compared three conventional methods for

total RNA isolation from different types of durian tissues. The results showed

that the CTAB procedure produced the best yield and high quality of total

RNA from 4-month old durian flesh. The yield ranged from 75.8 pg to 272.5

pg per gram of frozen durian tissue with an average purity ratio A26dA280 of

1.78. The RNA from 4-month old durian flesh of clone D24 was successfully

used for the construction of a cDNA library.

The titer of the primary cDNA library was 5.1 x106 pfulml. The percentage of

recombinant plaques was 99% which indicated a sufficient cDNA library

quality for full-length cDNA screening. A total of 30 randomly collected clones

were generated from the cDNA library. Based on the comparison of these

sequences with the GENEBANK, the sequences were classified into 7

groups according to their putative functions i.e. general metabolism

(1 0 sequences); DNA and protein synthesis (4 sequences); glycosylation and

transport proteins (1 sequence); respiration chain and photosynthesis

(1 sequence); regulation mechanism (6 sequences); immunology

(1 sequence); and novel genes (7 sequences). The initial molecular biology

information of durian will also support further basic molecular work for genetic

engineering and crop improvement application in the future.

Consequently, two interesting clones, putative FKBP12 and quinone

reductase, were subjected to further characterization. These genes were

found to be present in the genome as single copy genes and expressed not

only in the 4-month old durian flesh but also in other tissues such as the

leaves and young flower buds. However, these genes were not found to be

expressed in ripening flesh tissues. The former, FKBP12 has a main function

of dissecting higher plants ca2+-dependent signal pathway. The latter,

quinone reductase plays an important role in xenobiotic detoxification.

Abstrak thesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENClRlAN MOLEKUL PERPUSTAKAAN IS1 DURIAN KLON D24

Oleh

HUYNH KY

April 2005

Pengerusi

Fakulti

Profesor Madya Suhaimi bin Napis, Ph.D.

Bioteknologi dan Sains Biomolekul

Genus Durio (Durio zibethinus Murr.) adalah berasal dari Malaysia. Daripada

198 klon durian yang telah didaftarkan, klon D24 merupakan klon yang

terbaik di Malaysia malahan di seluruh dunia. Walaubagaimana pun, analisis

genetik molekular ke atas durian masih terhad. Kajian yang telah dijalankan

ini merupakan langkah awal dalam kajian molekular ke arah

menyumbangkan data molekular bagi durian klon 024. Pada peringkat

pertama kajian, perbandingan terhadap tiga kaedah pengekstrakan RNA

telah dikaji dengan menggunakan pelbagai jenis sampel tisu durian.

Keputusan menunjukkan kaedah CTAB memberikan hasil RNA yang paling

memuaskan dari segi kualiti dan kuantiti berbanding dengan dua kaedah

yang lain. Hasil RNA yang diperoleh adalah dalam lingkungan 75.80 ug

hingga 272.5 ug daripada setiap gram sampel tisu durian yang digunakan.

Hasil tersebut memberikan purata nisbah ketulenan A2601A280 sebanyak 1.78.

Perpustakaan cDNA telah berjaya dibina dengan menggunakan sampel RNA

daripada isi durian yang berusia empat bulan.

Titer bagi perpustakaan cDNA primer adalah berjumlah 5 . 1 ~ 1 0 ~ pfulml.

Peratusan plak rekombinan yang terhasil adalah 99%, menunjukkan kualiti

pupustakaen cDNA yang mencukupi untuk penyaringan. Sebanyak 30 klon

telah dipencilkan daripada perpustakaan cDNA ini. Berdasarkan

perbandingan homologi antara klon dan GENEBANK, jujukan-jujukan

tersebut boleh dikelaskan kepada tujuh kumpulan berdasarkan kepada

anggapan fungsi iaitu metabolisme umum (10); DNA dan sintesis protein (4);

glikosilasi dan protein pengangkutan (1); respirasi dan fotosintesis (1);

mekanisme regulasi (6); imunologi (1) dan gen novel (7). Adalah diharapkan

maklumat biologi molekul durian ini pada masa depan dapat menyokong

kajian molekul asas lain untuk kerja-kerja kejuruteraan genetik dan

peningkatan hasil durian.

Dua klon yang menarik, iaitu gen FKBP12 dan gen bagi quinone reductase

telah didalami dalam kajian yang seterusnya. Gen-gen ini adalah didapati

sebagai gen tunggal didalam genom dan diekspresi bukan sahaja didalam isi

durian berusia empat bulan, malahan beberapa tisu durian lain seperti daun

dan putik bunga muda. Walau bagaimanapun, gen-gen ini tidak dieskpresi

oleh isi durian yang masak. FKBP12 berfungsi dalam laluan isyarat

penggantungan ion kalsium bagi tumbuhan peringkat tinggi. Bagi quinone

reductase pula, ia memainkan peranan yang penting dalam detoksifikasi

xenobiotik.

vii

ACKNOWLEDGEMENTS

Deepest in my heart, I would like to thank Assoc. Prof. Dr. Suhaimi Napis, the

chairman of my supervisory committee, for his patience and trust in me, and

his excellent guidance, encouragement and support throughout my study.

The inevaluble help of Prof. Dr. Ghizan Saleh and Assoc. Prof. Dr. Norihan

Mohd. Saleh, members of the supervisory committee, are gratefully

acknowledged for the discussion during my project.

I am grateful to Cantho University, Vietnam for providing the financial support

during my study. I would also like to acknowledge IRPA grant and internal

funds (UPM and Department of Agriculture) for supporting this research

project.

My sincere gratitude to the laboratory members of Genetic Laboratory at

faculty of Biotechnology and Biomolecular Sciences, UPM: Mr. Ong, Choong,

Panca, Ummi, Niza, Alfred, Joey, Kam Lock, Sock Hwa, Tzer Ying for

supporting and providing a friendly environment to me while conducting my

research. Warm thoughts go to my Vietnamese friends in UPM for their

caring and the sense of belongingness in a big family during my residence in

Malaysia. I wish to thank Encik Jabi Bin Tananak and his staff at Tenom

Agricultural Research Station, Encik Ramlee Bin Md. Afandi and his staff at

Department of Agriculture Serdang, and Encik Yahaya and his staff at Scion

Production Farm, University Agriculture Park, Universiti Putra Malaysia, for

their valuable assistance in collecting samples.

I certify that an Examination Committee met on lgth April 2005 to conduct the final examination of Huynh Ky on his Master of Science thesis entitled "Molecular Characterization of cDNA Library from Flesh of Durian (Durio zibethinus Murr.) Clone D24" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Raha Abdul Rahim, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Maheran Abd. Aziz, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

Siti Nor Akmar Abdullah, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

Mohamad Osman, PhD Associate Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (External Examiner)

~ro fessor l~e~h&/bean School of Gra . ate Studies Universiti Putra Malaysia

This thesis 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 are as follows:

Suhaimi Bin Napis, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Norihan mohd. Saleh, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

Ghizan Saleh, Ph.D Professor. Faculty of Agriculture Universiti Putra Malaysia (Member)

AlNl IDERIS, Ph.D. ProfessorIDean School of Graduate Studies Universiti Putra Malaysia

Date: 1 5 JUL 2005

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

... ... ... ... ... ... ... ... ... ..- ... ... ....

HUYNH KY

Date: 04-1 0 ~ / 9 - 0 0 5

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LlST OF TABLES LlST OF FIGURES LlST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS

CHAPTER

INTRODUCTION

LITERATURE REVIEW 2.1 Durian

2.1 .I Taxonomic History 2.1.2 Morphology 2.1.3 Nutritive Composition and Uses of the Fruit 2.1.4 Other Uses 2.1.5 Clonal Selection and Hybridization 2.1.6 Clonal Identification 2.1.7 Economy and Fruit Production Molecular Markers in Plant breeding 2.2.1 Protein Markers 2.2.2 DNA Markers cDNA Library 2.3. I Applications 2.3.2 Limitations

METHODOLOGY 3.1 Plant Materials 3.2 Isolation of Genomic DNA, Total RNA and mRNA

3.2.1 Extraction of Genomic DNA (Lipp et a/., 1999) 3.2.2 Evaluation of Three Conventional Methods

for Plant Total RNA Isolation 3.2.3 Isolation of mRNA Construction of cDNA Library 3.3.1 First Strand Synthesis 3.3.2 Second Strand Synthesis 3.3.3 Blunting the cDNA Termini 3.3.4 Ligation the EcoR I Adaptors 3.3.5 Phosphorylating the EcoR I End 3.3.6 Digestion with Xho I 3.3.7 Size Fraction of cDNA Fragments

I I ... 111

v vii viii ix xiii xiv xvi

xii

3.3.8 DNA Ethidium Plate Assay 44 3.3.9 Ligating cDNA into Uni Zap XR 44 3.3.1 0 Packaging with Gigapack II Extract 44 3.3.1 1 Preparation of E. coli Cells for Plating 45 3.3.12 Phage Plating and Titering 45 3.3.13 Random Selection of Plaque from cDNA Library 46 3.3. 14 In vivo Excision Phage 46 3.3.15 Plasmid Preparation 47 3.3.16 Amplification of the Inserted Fragment into the vector 48

3.4 Sequencing Analysis 49 3.5 Amplification of cDNA Library 50 3.6 Southern Blot Analysis 5 1 3.7 Northern Blot Analysis 52 3.8 Probe Preparation 54 3.9 Hybridization, Washing and Detection 54

RESULTS AND DISCUSSION Genomic DNA Isolation, Total RNA Isolation and mRNA Purification 4.1 .I Genomic DNA Isolation 56 4.1.2 Total RNA Isolation 58 4.1.3 Purification of mRNA 67 cDNA Library Construction from 4-month Old Durian Flesh 70 Plaque Screening of cDNA Library Using 72 PCR-Based Selection General Survey of Randomly Collected Clones from 4-Month74 Old Durian Flesh cDNA Library Molecular Characterization of two Interesting Clones 80 4.5.1 Clone cDlO 80 4.5.2 Clone cD l 89

CONCLUSIONS AND SUGGESTIONS

REFERENCES APPENDICES BIODATA OF THE AUTHOR

LIST OF TABLES

Table

I The nutritive constituents of durian fruits

Durian production in Penisular Malaysia

Page

7

15

3 The interaction of the yield (pglg) between three RNA 60 extraction methods and different type of durian tissues

4 The interaction of the A260/A280 ratio between RNA extraction 61 methods and different types of durian tissues

The interaction of the A260/A230 ratio between RNA extraction 62 methods and different types of durian tissues

Effect of three different method on the purity, concentration 63 and yield of RNA from durian tissues

Effect of durian sample on purity, concentration and yield of 64 RNA from SDS method, CTAB method and GTTC methods

The 4-month old durian flesh cDNA library clones with 75 significant sequence similarity at amino acid level to the sequences in GENEBANK

xiv

LIST OF FIGURES

Figure

cDNA synthesis flow chat

Southern blot and Northern blot via gravitational transfer

Page

43

53

Gel electrophoresis (1% (wlv)) of 4-month old durian flesh 57 DNA isolated using CTAB method, a) Undigested durian genomic: 1 and 2, Genomic DNA; M, 1 Kb ladder (Biolabs, UK) DNA; b) Durian genomic DNA digested with BamH Ill ( I ) , EcoR 1 (2), EcoR V (3), Ssp 1 (4), respectively; M, 2-Log DNA ladder (Biolabs, UK).

Formaldehyde denaturing agarose gels 1% (wlv) were used 59 to separate total RNA from five tissues types from different extraction procedures. (a) Durian young bud; (b) Ripening flesh; (c) Leaves; (d) I -month old durian flesh; (e) 4-month old durian flesh (e); lane 1, SDS method; lane 2, CTAB method; lane 3, GTTC method

Messenger RNA was isolated from total RNA of 4-month old 69 durian flesh using pMAC Kit. 1. 500 ng RNNml; 2. 250 ng RNAImL; 3. 100 ng RNNmL; 4. 50 ng RNAImL; 25 ng RNA/mL, and mRNA = 90 ng1mLx 600 mL

The size distribution of the synthesized cDNA separated on 71 agarose gel 1.2 % (wlv). The size fraction from 500 bp to 3 kb was used to construct cDNA library. M, LambdalPst I ladder (Fermentas)

PCR amplification of cDNA inserts separated on 1% (wlv) 73 agarose gel. M. LambdalPst I ladder (Fermentas); 1, Negative control; 2-1 3 cDNA inserts amplified by PCR.

The comparison of clone cD1O putative translated protein 82 sequence with two other similar proteins from V. faba and A. thaliana and to indicate their similarities

Northern blot with 20pg of total RNA from five different types 83 of durian tissues, total RNA (20pg) were separated on 1.2% (wIv) formaldehyde denaturing agarose gel. (a) lane 1-1 - month old flesh; 2- 4-month old flesh; 3- ripening flesh; 4- leave; 5- young bud; (b) total RNA was hybrdized with the cD1O cDNA probe and exposed to X-ray film for 14 hours.

Analysis of the copy number of the FKBP 12 gene. (a) 85 Genomic DNA from durian was digested with EcoR I (I), Hind 111 (2), BamH 1 (3), Taq 1 (4), LambdalPst I marker (M). (b) After electrophoresis, DNA was transfered onto membrane and hybridized with cDlO cDNA probe.

Mechanism of FKBP12 gene express in human.

The comparison of clone cD1, putative quinone reductase gene with two other similar proteins from A. thaliana and R. rubrum to indicate their similarities

Northern blot with 20pg of total RNA from five different types of durian tissues, total RNA (20 pg) were separated on 1.2% (wlv) formaldehyde denaturing agarose gel. (a) lane 1-1 month old flesh; 2- 4-month old flesh; 3- ripening flesh; 4- leave; 5- young bud; (b) total RNA was hybrdized with the cD1 cDNA probe and exposed to X-ray film for 14 hours.

Analysis of the copy number of the putative quinone reductase gene. (a) Genomic DNA (10 pg) from durian was digested with Hind Ill (I) , EcoR 1 (2), EcoR V (3), Ssp 1 (4), Bgl 1 (5), and Taq 1 (6) , 2-Log DNA marker (M)). The blots were separated on 1.2 % (wlv) agarose gel. (b) After electrophoresis, DNA was transfered onto membrane and hybridized with cD1 cDNA probe.

xvi

LIST OF ABBREVIATIONS

AFLP

AP-PCR

ATP

BLAST

BSA

Ca

CAPS

cDNA

cpDNA

CTAB

DAF

DEPC

DMSO

DNA

dNTP

DTT

EB

EDTA

EST

EtBr

F buffer

GTTC

HCI

Amplified fragment length polymorphism

Abitrarily primer polymerase chain reaction

Adenosine triphosphate

Basic local alignment search tool

Bovine serum albumine

Calcium

Cleaved amplified polymorphic sequences

Complementary deoxyribonucleic acid

Chloroplast deoxyribonucleic acid

Hexadecyl-trimethyl ammonium bromide

DNA amplification fingerprinting

Diethyl pyrocarbonate

Dimethyl sulfoxide

Deoxyribonucleic acid

Deoxynucleoside triphosphate

Dithiothreitol

Elution buffer

Ethylene-diamine-tetra acetic acid

Expressed sequence tag

Ethidium bromide

Formaldehyde buffer

Guanidine thiocynate

Hydrochloric acid

xvii

l PTG

ITS

LB

LB-Broth

LiCl

MgS04

MOPS

mRNA

NaOC

NaOH

NCBl

NZY

PABP

PC1

PCR

PVP

RAF

RAPD

rDNA

RFLP

RNA

RNase

RT

SDS

SM

lsopropyl p-D-thiogalactopyranoside

Internal sequence region

Luria bertaini

Luria bertaini broth

Lithium chloride

Magnesium sulphate

3-[N-morpholino] propanesulfonic acid

Messenger ribonucleic acid

Sodium acetate

Sodium hydroxide

National Center for Biotechnology Information

Z-amine - Yeast extract

Polyadenylate-binding protein

Phenol: chloroform:isoamyl alcohol

Polymerase chain reaction

Polyvinyl polypyrrolidone

Randomly amplified DNA fingerprinting

Randomly amplified polymorphic DNA

Ribosome deoxyribonucleic acid

Restriction fragment length polymorphism

Ribonucleic acid

Ribonuclease

Reverse transcriptase

Sodium dodecyl sulfate

Sodium chloride-Magnesium sulfate buffer

xviii

SNP

SSC

SSR

TAE

TE

Single nucleotide polymorphism

Sodium saline citrate

Microsatellite

Tris- acetate- EDTA buffer

Tris-EDTA buffer

Ultraviolet

XIX

M

ng

ODx

%

pmol

pfu1mL

LIST OF SYMBOLS AND UNITS

Alpha

Base pair

Beta

Centimeter

Degree celsius

Enzyme unit

gamma

Gram

Gravity (relative centrifuge force)

Kilo-base pair

Kilodalton

Kilogram

Microgram

Microliter

Milligram

Milliliter

Millimolar

Molar

Nanogram

Optical density at wavelength x nanometer

Percent

Picomole

Plaque forming units per milliliter

v

Vol

vlv

wlv

Round per minute

United States Dollar

Voltage

Volume

Volume per volume

Weight per volume

CHAPTER 1

INTRODUCTION

Durio zibethinus Murr., one of the most well known tropical fruits in the world,

is native to South East Asia with its center of diversity in Borneo. It is

considered as highly priced fruit in culturally, and economically aspects in

South-East Asia and sometimes referred to as 'The King of Fruits' (Zappala

et at., 2002). However, westerners often quickly retreat or decline any

invitation to try the unique taste experience of the King of Fruits. On the other

hand, Alfred Russel Wallace said [flo eat Durian is a new sensation, worth a

visit to the East to experiencenand @he more you eat of it, the less you feel

inclined to stop0 They also compared the role of Durian in SouthEast Asian

societies as to Champagne in the West (Piper, 1989).

In Malaysia, a total of 179 clones had been registered, and Clone D24 in

particular has been a popular commercial cultivar (Chan, 2000). Some other

clones (D2, D98, etc) are much sought after and sold at a higher price in the

market. The value of durian exports alone accounted for over 40% of total

fruit exports in 1989 (Ali, 1993). However, little research has been carried out

in the past on durian.

Recently, the improvement of fruit production and quality is the main strategy

of the national agro-industry, of which eight fruits have been given the priority

for research and production including durian for the fresh market. Application

of molecular approach to develop durian variety was emphasized in the

National Agriculture Policy (NAP3) (Rahman, 2000). For this reason,

construction of a cDNA library from durian flesh will play an important role in

the future durian breeding and improvement.

A cDNA library contains DNA fragment that is derived from cellular mRNA by

reverse transcriptase. The advantage of a cDNA library is that genes may be

studied as they exist in the organism with control elements intact. A cDNA

clone would have no introns or control elements, but if supplied with bacterial

promoter, it can still be expressed. Based on the database from a cDNA

library, new genes can be discovered and studied such as genes controlling

odor, color or with medicinal application. Moreover, crop improvement and

the molecular marker system may be applied with the valuable information

from a cDNA library.

The objectives of this study were:

To evaluate effective methods for the isolation of total RNA from

different types of durian tissue;

To construct a cDNA library;

To generate and analyze some clones from the cDNA library; and

To isolate and characterize some intersting genes in durian.

CHAPTER 2

LITERATURE REVIEW

2.1 Durian

2.1 .I Taxonomic History

In the early time, the family Bombacaceae includes three sub-tribes,

Adansonieae, Matisieae, and Durioneae. The divisions were classified based

on differences in leaf morphology. The genus Durio belongs to the Durioneae

which is characterized by simple entire penninervate leaves (Brown, 1997).

The Bombacaceae actually shares more anatomical similarities with the

Malvaceae (especially the genus Hibiscus) than they do with the

Sterculiaceae. The difference in chromosome number is one of the evidence

that can be used to distinguish Durioneae from the other Bombacaceous

tribes. Chromosome counts of durian (2n=28) is lower than those of

Bombacaceous species (2n=72) from other tribes but closer to those found in

related Sterculiaceae, Tiliaceae and Malvaceae (Baum and Oginuma, 1994).

Recently, phylogenetic relationship within Durioneae was studied based on

sequences of chloroplast (ndhF) and nuclear ribosomal (ITS). The data

suggested that Durio and its relatives appear to be more closely related to

the tribe Helicters and Reevesia (Sterculiaceae) than to Bombacaceae

(Alverson et a/., 1999; Nyffeler and Baum, 2000).

The genus of Durio consists of 28 species. However, there are only six

species with edible fruits, of which include D. oxleyanus Griffith,

D. graveolens Becc., D. dulcis Becc., D. grandiflorus Becc., D. kutejensis

Becc. and D. zibethinus Murr. (Kanzaki et a/., 1998). Among them,

Durio zibethinus Murr. is one of the most interesting tropical fruits found in

South East Asia. The species Durio zibethinus was attributed by Murray in

1774 (Brown, 1997).

2.1.2 Morphology

Durian tree, a tall handsome tree reaching up to 37 m in height, is usually

straight, rough, with peeling trunk up to 1.2 m in diameter. The bark is grey or

reddish-brown (Brown, 1997). The evergreen, alternate leaves are densely

covered with golden hair on the underside. The leaves are 6-22 cm long and

2.5-8 cm wide, depending on the variety (Yaacob and Subhadrabandhu,

1995). The fruits are egg-shaped or oblong to nearly round. The shape may

be short or long depending on the variety and ranges from 15 to 30 cm long

from 12.5 to 50 cm wide, and up to 8 kg in weight (Morton, 1987). The yellow

or yellowish skin is covered with many pointed spines about 1-2 cm long, and

very hard, almost woody, protecting the flesh inside when the fruit falls from a

great height. A mature tree, between 15-20 years old, can produce 100 or

more fruits per year while a young tree bears about 50 fruits, depending on

the variety and location (Piper, 1989). The aroma develops as the fruit ripens

and reaches its peak 2-3 days after the fruit drops. At this particular time, the

durian's taste is very delicious or the flavor is at its best.