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.
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