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SCREENING FOR FLOWER-SPECIFIC eDNA SEQUENCES IN SAGO PALM (Metroxylon sagu) VIA
A DIFFERENTIAL DISPLAY TECHNIQUE
Bong Shiaw Kong
Master of Science (Molecular Biology)
2004
Pusat Khidmat MakJumat Akademik UNlVERSITI MALAYSIA SARAWAK
SCREENING FOR FLOWER-SPECIFIC eDNA SEQUENCES IN SAGO PALM (Metroxylon sagu) VIA
A DIFFERENTIAL DISPLAY TECHNIQUE PKHIOMAT MAKLUMAT AKAOEMIK
111111111 rlI~ii 111111111 1000246514
Bong Shiaw Kong
BSc (Hons) in Biotechnology
A thesis submitted in full fulfillment of the requirement for the degree of Masters of Science
Faculty ofResource Science and Technology UNIVERSITY MALAYSIA SARA W AK
2004
DECLARATION
No portion of the work referred to in this thesis has been submitted in support of an application for anoth~r degree or qualification to this or any other university or institution of
higher learning
Bong Shiaw Kong 730302-13-5109
ii
ACKNOWLEDGEMENT
I would like to express my gratitude and sincere appreciation to my supervisor Professor Dr Mohd Azib Salleh for his guidance and inspiration throughout this work and also his help in the preparation of this thesis
My apprecation also goes to Mr Amin Manggi in the Faculty of Resource Science and Technology University Malaysia Sarawak for his technical assistance To Dr Sim Soon Liang and Dr Hairul Azman thank you for your advice and support I would also like to thank the Ministry of Science Technology and Environment (MOSTE) for giving me the Postgraduate Fellowship to support my studies
To my wife Jane my parents brother and sisters thank you so much for your understanding
and support
III
Abstract
( Differential display is rapid and economical method compared to traditional
differential screening of cDNA libraries or construction of subtracted cDNA libraries for the
identification of differentially expressed genes This technique is used in the present study to
identify genes that are specifically expressed in flower tissues of sago)
Sago palm maturation period is a major obstacle in the development of this plant as a
commercial crop Previously it has been found that the flowering of sago palm is a direct
indicator for the maximum level of starch content in the trunk This study focuses on the
isolation of specific genes that can be expressed only in the flower tissue Characterization of
these genes can lead to the discovery of the regulatory gene for maturation in sago palm A
nonradioactive differential display technique which takes advantage of chemiluminescent
technology has been adopted for this study This adaptation has proven to be successful
compared to other nonradioactive techniques The results were more convincing and
reproduceable A random primer was used to amplify the cDNA generated from mRNA of
different tissues and the differentially expressed cDNA bands were displayed in the
lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from
the cDNA fingerprints The bands were then excised from the cDNA fingerprints and
reamplified using the same random primer These differentially expressed bands were then
analyzed by blot analysis to determine their specificity They showed positive results in all
tI
blotting experiment including cDNA blotting and Northern blotting The blotting experiments
also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned
into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and
stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high
IV
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Pusat Khidmat MakJumat Akademik UNlVERSITI MALAYSIA SARAWAK
SCREENING FOR FLOWER-SPECIFIC eDNA SEQUENCES IN SAGO PALM (Metroxylon sagu) VIA
A DIFFERENTIAL DISPLAY TECHNIQUE PKHIOMAT MAKLUMAT AKAOEMIK
111111111 rlI~ii 111111111 1000246514
Bong Shiaw Kong
BSc (Hons) in Biotechnology
A thesis submitted in full fulfillment of the requirement for the degree of Masters of Science
Faculty ofResource Science and Technology UNIVERSITY MALAYSIA SARA W AK
2004
DECLARATION
No portion of the work referred to in this thesis has been submitted in support of an application for anoth~r degree or qualification to this or any other university or institution of
higher learning
Bong Shiaw Kong 730302-13-5109
ii
ACKNOWLEDGEMENT
I would like to express my gratitude and sincere appreciation to my supervisor Professor Dr Mohd Azib Salleh for his guidance and inspiration throughout this work and also his help in the preparation of this thesis
My apprecation also goes to Mr Amin Manggi in the Faculty of Resource Science and Technology University Malaysia Sarawak for his technical assistance To Dr Sim Soon Liang and Dr Hairul Azman thank you for your advice and support I would also like to thank the Ministry of Science Technology and Environment (MOSTE) for giving me the Postgraduate Fellowship to support my studies
To my wife Jane my parents brother and sisters thank you so much for your understanding
and support
III
Abstract
( Differential display is rapid and economical method compared to traditional
differential screening of cDNA libraries or construction of subtracted cDNA libraries for the
identification of differentially expressed genes This technique is used in the present study to
identify genes that are specifically expressed in flower tissues of sago)
Sago palm maturation period is a major obstacle in the development of this plant as a
commercial crop Previously it has been found that the flowering of sago palm is a direct
indicator for the maximum level of starch content in the trunk This study focuses on the
isolation of specific genes that can be expressed only in the flower tissue Characterization of
these genes can lead to the discovery of the regulatory gene for maturation in sago palm A
nonradioactive differential display technique which takes advantage of chemiluminescent
technology has been adopted for this study This adaptation has proven to be successful
compared to other nonradioactive techniques The results were more convincing and
reproduceable A random primer was used to amplify the cDNA generated from mRNA of
different tissues and the differentially expressed cDNA bands were displayed in the
lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from
the cDNA fingerprints The bands were then excised from the cDNA fingerprints and
reamplified using the same random primer These differentially expressed bands were then
analyzed by blot analysis to determine their specificity They showed positive results in all
tI
blotting experiment including cDNA blotting and Northern blotting The blotting experiments
also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned
into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and
stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high
IV
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
DECLARATION
No portion of the work referred to in this thesis has been submitted in support of an application for anoth~r degree or qualification to this or any other university or institution of
higher learning
Bong Shiaw Kong 730302-13-5109
ii
ACKNOWLEDGEMENT
I would like to express my gratitude and sincere appreciation to my supervisor Professor Dr Mohd Azib Salleh for his guidance and inspiration throughout this work and also his help in the preparation of this thesis
My apprecation also goes to Mr Amin Manggi in the Faculty of Resource Science and Technology University Malaysia Sarawak for his technical assistance To Dr Sim Soon Liang and Dr Hairul Azman thank you for your advice and support I would also like to thank the Ministry of Science Technology and Environment (MOSTE) for giving me the Postgraduate Fellowship to support my studies
To my wife Jane my parents brother and sisters thank you so much for your understanding
and support
III
Abstract
( Differential display is rapid and economical method compared to traditional
differential screening of cDNA libraries or construction of subtracted cDNA libraries for the
identification of differentially expressed genes This technique is used in the present study to
identify genes that are specifically expressed in flower tissues of sago)
Sago palm maturation period is a major obstacle in the development of this plant as a
commercial crop Previously it has been found that the flowering of sago palm is a direct
indicator for the maximum level of starch content in the trunk This study focuses on the
isolation of specific genes that can be expressed only in the flower tissue Characterization of
these genes can lead to the discovery of the regulatory gene for maturation in sago palm A
nonradioactive differential display technique which takes advantage of chemiluminescent
technology has been adopted for this study This adaptation has proven to be successful
compared to other nonradioactive techniques The results were more convincing and
reproduceable A random primer was used to amplify the cDNA generated from mRNA of
different tissues and the differentially expressed cDNA bands were displayed in the
lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from
the cDNA fingerprints The bands were then excised from the cDNA fingerprints and
reamplified using the same random primer These differentially expressed bands were then
analyzed by blot analysis to determine their specificity They showed positive results in all
tI
blotting experiment including cDNA blotting and Northern blotting The blotting experiments
also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned
into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and
stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high
IV
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
ACKNOWLEDGEMENT
I would like to express my gratitude and sincere appreciation to my supervisor Professor Dr Mohd Azib Salleh for his guidance and inspiration throughout this work and also his help in the preparation of this thesis
My apprecation also goes to Mr Amin Manggi in the Faculty of Resource Science and Technology University Malaysia Sarawak for his technical assistance To Dr Sim Soon Liang and Dr Hairul Azman thank you for your advice and support I would also like to thank the Ministry of Science Technology and Environment (MOSTE) for giving me the Postgraduate Fellowship to support my studies
To my wife Jane my parents brother and sisters thank you so much for your understanding
and support
III
Abstract
( Differential display is rapid and economical method compared to traditional
differential screening of cDNA libraries or construction of subtracted cDNA libraries for the
identification of differentially expressed genes This technique is used in the present study to
identify genes that are specifically expressed in flower tissues of sago)
Sago palm maturation period is a major obstacle in the development of this plant as a
commercial crop Previously it has been found that the flowering of sago palm is a direct
indicator for the maximum level of starch content in the trunk This study focuses on the
isolation of specific genes that can be expressed only in the flower tissue Characterization of
these genes can lead to the discovery of the regulatory gene for maturation in sago palm A
nonradioactive differential display technique which takes advantage of chemiluminescent
technology has been adopted for this study This adaptation has proven to be successful
compared to other nonradioactive techniques The results were more convincing and
reproduceable A random primer was used to amplify the cDNA generated from mRNA of
different tissues and the differentially expressed cDNA bands were displayed in the
lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from
the cDNA fingerprints The bands were then excised from the cDNA fingerprints and
reamplified using the same random primer These differentially expressed bands were then
analyzed by blot analysis to determine their specificity They showed positive results in all
tI
blotting experiment including cDNA blotting and Northern blotting The blotting experiments
also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned
into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and
stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high
IV
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Abstract
( Differential display is rapid and economical method compared to traditional
differential screening of cDNA libraries or construction of subtracted cDNA libraries for the
identification of differentially expressed genes This technique is used in the present study to
identify genes that are specifically expressed in flower tissues of sago)
Sago palm maturation period is a major obstacle in the development of this plant as a
commercial crop Previously it has been found that the flowering of sago palm is a direct
indicator for the maximum level of starch content in the trunk This study focuses on the
isolation of specific genes that can be expressed only in the flower tissue Characterization of
these genes can lead to the discovery of the regulatory gene for maturation in sago palm A
nonradioactive differential display technique which takes advantage of chemiluminescent
technology has been adopted for this study This adaptation has proven to be successful
compared to other nonradioactive techniques The results were more convincing and
reproduceable A random primer was used to amplify the cDNA generated from mRNA of
different tissues and the differentially expressed cDNA bands were displayed in the
lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from
the cDNA fingerprints The bands were then excised from the cDNA fingerprints and
reamplified using the same random primer These differentially expressed bands were then
analyzed by blot analysis to determine their specificity They showed positive results in all
tI
blotting experiment including cDNA blotting and Northern blotting The blotting experiments
also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned
into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and
stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high
IV
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Abstrak
I-
Teknik differential display merupakan satu teknik yang cepat ekonomik dalam
mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam
kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok
sagu
Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali
yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal
pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada
tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan
gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini
boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan
sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif
telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia
berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu
primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah
digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu
berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint
cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~
untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi
secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur
terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh
diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam
vi
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang
seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi
dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus
amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT
adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan
sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi
dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull
Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan
nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi
kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk
dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan
enzim spesifik yang terti bat dalam process pennatangan atau pembungaan
vii
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENT
CONTENTS PAGE
TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii
10 LITERATURE REVIEW _
11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8
141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and
RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and
RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy
----------------------------- 14 146 Ways to overcome some problems associated with
mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening
~ methods----- 19
1S Aims of this project--------------------------------------20
20 MATERIALS AND METHODS
21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22
viii
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel
electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30
251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33
26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy
-------- 34 27 Verification of differentially expressed bands 35
28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39
29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent
cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44
210 DNA sequencing ---------------------------------- 44
30 REsULTS AND DISCUSSION
31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48
32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy
----------------------~------------------------------------51
34 Reamplification of the selected bands --------------------57
ix
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66
36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71
40 GENERAL DISCUSSIONS
41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-
specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88
BmLIOGRAPHY------------------------~------------89
APPENDIX 1
APPENDIX II
x
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
LIST OF TABLES
Table No Title Page
Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display
Table 2 RAP-peR conditions 32
Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47
Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47
Table 5 Spectrophotometric readings of mRNA preparation 50
Table 6 The quality and quantity of the mRNA yielded 50
Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72
xi
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
LIST OF FIGURES
Figure No Title
Fig 1 The RAP-PCR technique
Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display
Fig 3 A flowering sago palm showing third order branching
Fig 4 Total RNA from leaf tissues
Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands
Fig 6 Differential display of all the tissues using C 1 primer
Fig7a Differential display of all tissues using C2 primer
Fig7b Differential display of all tissues using C5 primer
Fig 8 Differential display of all the tissues using C3 primer
Fig 9 The reamplified S 1 and S2 bands
Fig 10 Dot blot of the differentially expressed bands
Fig 11 cDNA blotting using the same probe as in the dot blot
Fig 12 RNA hybridization
Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23
Fig 14 Nucleotide sequence of the cDNA derived from SI band
Fig IS Nucleotide sequence of the S2 band cDNA
Xll
c -
Page
10
15
23
49
54
55
58
59
60
62
65
67
68
73
75
76
I
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Abbreviations
~g
JLl
2-BE
AMP
APS
bp
BPB
ddH20
DDRT
DEPC
DIG-dUTP
DNA
dNTP
EDTA
EtBr
IPTG
LB
LiCI
M
MgCh
microgram
microliter
Butoxyethanol
Ampicillin
Ammonium persurphate
Base pair
Bromo phenol blue
double-distilled water
Differential display reverse transcription
Diethyl pyrocarbonate
Digoxig~nin-ll-dUTP
Deoxyribonucleic acid
Deoxynucleotide
Ethylene diamine tetra acetate
Ethidiwn bromide
isopropylthio-j3-D-galactoside
Luria-Bertani me~ium
Lithiwn chloride
molar
Magnesium chloride
xiii
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
MMLV-RT
OMT
PAGE
peR
pSI
PVPP
RAP
RNA
RNase
rpm
SA-PMP
SDS
TAE
TBE
TE
TENfED
V
vv
W
X-gal
Moloney murine leukimia virus-reverse transcriptase
O-methyltransferase
Polyacrylamide gel electrophoresis
Polymerase chain reaction
Pound per square inch
polyvinyl-polypyroridon
Random arbitrary primer
Ribonucleic acid
Ribonuclease
rev01ution per minute
Streptavidin-paramagnetic particle
Sodium dodecyl sulfate
Tris-acetatelEDTA electrophoresis buffer
Tris-boriclEDTA electrophoresis buffer
Trist-EDTA
Tetramethy ethyl enediamine
Volt
volume over volume
Watt
5-bromo-4-chlorlt-3-indolyl-p-D-galactoside
xiv
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
10 LITERATURE REVIEW
11 Introduction
Metroxylon Sagu commonly known as sagu or mulung locally is one of the
earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)
Bellwood (1985) noted that the sago palm was one of the most important cultivated
plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice
etc Among the earliest record of sago plantation as a domestic crop was that described
in a Chinese geography text published in the late 13 th century (Takaya 1985) According
to Takaya the sago palm was grown extensively in the areas stretching from southern
Mindanao to Borneo Northern Suiawesi and the Maluk-u islands
In Sarawak sago palm has been grown for at least 400 years and is concentrated
mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the
Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan
division A detailed study on the distribution of sago palm in Sarawak has been carried
out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by
smallholders and predominantly by the Melanau community as their main cash crop
(Anonymous 1986) It was the principal source of revenue during the Brooke reign in
bull the 19th century but at present it only contributes about 4 of the state revenue for
agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about
20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat
Division The total annual production of sago S$ch from the state is about 55000
tonnes
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
The total acreage of sago plantations in South East Asia is about 375 million ha
with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and
Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)
Chemically sago starch is quite similar to that of com potato tapioca and wheat
starches Sago starch can be used to make biscuits bread cakes and as thickeners for
chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food
made from sago flour Sago starch has also be utilized extensively in the manufacture of
high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy
textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as
feed in the local pig industry The rrajor conswners of sago starch are from the Far East
such as China Japan and Korea These countries import sago starch for specialize food
and up-market food outlet There has been an increasing demand for sago starch recently
by the Japanese as it has specific properties for the manufacture of up-market products
which other starches lack (personal communication Mr J Takara [2001])
Despite recent advance in farming techniques and starch processing methods the
importance 9f sago palm as a cash crop has been decreasing This was due to several
factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce
commercial plantation In addition the economic return of sago is low compared to other
crops such as pepper cocoa and oil palm However the most significant factor is the
long and non-uniform maturation period which makes harvesting difficult t~ manage if
the crop is grown in large-scale plantations
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Nevertheless sago palm is said to rival the root crops as a major starch producing
crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach
maximum level is however a major disadvantage for sago palm compared to for
example 4-6 months for sweet potato Biotechnological techniques including molecular
genetics and tissue culture sago starch utilization and modification and the treatment of
waste and waste water from sago processing plants have been the topic of discussion
among researchers and major sago growers in the state Worldwide the Tsukuba Sago
Fund has been encouraging and supporting research and industrial application of sago
starch However there has been very little effort to investigate the underlying genetic and
biochemical mechanisms that control starch biosynthesis in sago
In Sarawak Sago is mainly grown under a semi-wild condition with minimal
maintenance There seems to be no definite planting spacing or pattern In older plots
this problem of spacing is further complicated by the fact that sago grows in clumps and
new suckers can creep along the ground before growing upwards at some distance from
the mother palm The ability to produce suckers also tends to vary Furthermore sago
palms have such a long maturing period that many growth st~ges can be encountered in a
single garden Thus the best planting material for sago palm will be young sucker that
were readily available in most older plots Seed gennination is not a popular choice
among small holders as it requires higher mairitenance and a well-planed cultivation
scheme The vast majority of the small holders in Sarawak still maintain the semi-wild
method of sago cultivation In the early 1990s the Sarawak Department of Agriculture
has established the Land Custody and Development Authority (LCDA) an agency tasked
3
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
with carrying out intensive research and development programmes for sago This has
resulted in the setting up of the first large-scale sago palm plantation in the Mukah
district
12 Starch storage in sago and its yield at different stages of growth
The biochemical pathways for starch biosynthesis in plants have been well
studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon
synthesized in the photosynthetic pathway starch is produced in the leaves of the sago
palm and then stored in the trunk However the starch content depends on the starch
density in the pith and the trunk size The starch density in the pith probably depends on
the irradiation captured by the leaves The trunk size increases quadratically with girth
The trunk height is mainly governed by light a trunk growing in the shade will try to
reach fun sunshine and thus use the limited amount of photosynthates produced in the
crown first for trunk elongation Short stout trunks of the saine palm type are thus
expected to contain more starch than tall slender trunks (Flach 1991)
Starch flour yield ofnonnal stands of sago palm varies The variation is a result of
several factors namely type of soil rain falls and most importantly stages of growth
(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while
Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and
range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be
produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the
Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and
4
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK
Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch
flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-
about 189kg from one matured trunk in commercially grown sago ill all these studies
time of harvesting has become the factor Sim and Ahamd (1978) had conducted an
experiment based on the stage of growth in sago palm The findings of their work showed
that at the early flowering stage (average age of about 11 years) the tree could give the
maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication
of starch yield at different geographical locations and under different environmental
condition According to Sim et al (1978) in Sarawak it is a general belief that felling of
sago palm is best carried out after flowering but before the fruiting stage
Johnson and
Raymond (1965) claimed that the- maximum starch content occurs at the stage after
flowering Flach (1972) however reported that the sago trunks are best harvested during
the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in
their assessment agreed to this Sim and Ahmad also suggested that starch stored would
have been used for the fonnation of seed after the flowering process shy
These findings proved that the flowering stage is a vital indicator for us to identify
the maturation of the sago palm as this is the only physiological factor that can be
examined by plant breeders and plant cultivators Therefore the study of the flowering
process would provide us with a possible clue to what control the starch accumulation
and physiological development process
5
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
13 The flowering process in sago
The transition to flowering can be a remarkable change in the life of a plant In
many species such as in most perennials reproductive development occurs in certain
regions of the plant but vegetative growth of the plant continues The transition occurs
in shoot meristems which are reprogrammed to make inflorescence or floral organs
rather than vegetative organs on receiving appropriate environmental or development
signals From a developmental perspective therefore the floral transition is as much
about reprogramming the shoot meristems as it is about the actual production of
inflorescence flowers However it is not known whether the anatomical changes are the
cause or the results of changes in growth status of the meristem The floral transition
marks the beginning of reproductive development and in many plants such as sago
palm which bas a single bunch of flowers it also signifies the end of indeterminate
growth There are two distinct transition processes that can be distinguished genetically
Different types of inflorescence are formed in detenninate and indetenninate species
(Weberling 1989) In determinate species the inflorescence meristem forms terminal
flowers that end any fwther inflorescence growth In indetenninate species flowers are
fonned on lateral branches or inflorescence and not from terminal buds
The development of flowers is required for the alteration of the sporophytic to the
gametophytic generation the production or gametes for fertilization and seed
development These reproductive processes require the production of specialized organs
for the development of the gametophytes and to ensure fertilization The evocation
morphogenesis and function of these specialized organs is regulated through complex
6
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
mechanisms that have both genetic and environmental components (Greyson 1994) The
environmental component for example is the requirement that some plants have for a
specific photoperiod in order to initiate the flowering process But in the~case of sago
palm the environmental condition might only be the water content in the soil which has
yet to be studied and fully understood Other affecting factor might be their own
physiological changes which can include the starch content Hence the genetic
component of flowering is evident from the numerous mutations that identify genes
affecting flower morphology or function (Westhoff et al 1998) Although the majority
of the mutations are inherited as simple recessive traits and many of the mutations have
been thoroughly described morphologically and genetically (Howell 1998) the function
of the gene and the mechunism through which altered development occurs are not known
The determination of the molecular basis of such flotal mutation has been impeded by the
lack of a simple method for the isolation of the affected gene on the basis of phenotype
and mapping alone The morphogen~sis of flowers is associated with differential
expression of genes (Jordan 1993) The differentially expressed genes between
reproductive and vegetative organs are the basis of a strategy for the molecular analysis
of the genetic component of flowering The major difficulty in isolating genes involved
in the flowering process is that little is known of the identity of the proteins they encode
the tissues in which they are expressed or the time at which they are active during plant
development Methods of gene isolation which are closely based on knowledge of
genetics are therefore the most likely to be successful Thus the study of flowering
process should start with isolation of the regulatory gene of this physiological process
7
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
14 The isolation of flower specific gene
Prior to finding the regulatory gene and the biochemical pathway flower-specific
gene(s) must first be identified Tissue-specific gene(s) can be studied using many
approaches As they are differentially expressed the methods for identifying them can
be based on two different approaches - firstly differential screening of cDNA libraries
(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries
(Sargent and David 1983) These approaches have been successfully applied in other
plants but they are rather laborious and time-conswning and require large amounts of
RNA Differential screening detects only abundant mRNAs while subtractive
hybridization is more sensitive but even more difficult to set up Finatly a major
limitation of both procedures is that only one pair of RNA between them can be
perfonned at any given time
A new method known as RNA tmgerprinting through random peR amplification
is a good alternative for studying tissue specific gene expression or any regulatory gene
expression The method is rapid and fingerprints of any tissue-specific RNA can be
easily produced This method offers numerous advantages over other methods mentioned
above including its simplicity and its ability to compare the fluctuations in gene
expression between multiple samples simultaneously using only nanograms amounts of
RNA In addition it can also yield information on the overall patterns of gene expression
between different cell types or between different physiological conditions of the same
cell type (McClelland et ai 1995)
8
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
141 RAP-peR and differential display of mRNA
RNA finger printing or differential display was first introduced by Liang and
Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns
and subsequently for the isolation and cloning of gene sequences with desired expression
characteristics The technique relies upon the use of RNA arbitrary primers or any
random primer and the polymerase chain reaction (peR) and it is similar to the more
established techniques such as randomly amplified polymorphic DNA (RAPD) analysis
of genomic DNA conceptually Informative patterns or fingerprints of the reamplified
products can be produced even when no previous information is available concerning
primer binding sites or expected products The fmgerprints provide the basis for
selecting and ultimately isolating differentially expressed genes and have even been
suggested as a means for identifying and classifying different RNA sources (Liang et al
1993)
142 RAP-peR technique
Figure 1 depicts the overall concept of the RAP-peR technique During first
strand synthesis a single 18-base arbitrary primer anneals and extends from sites
contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from
conventional differential display of mRNA where an oligodeoxythymidine primer
oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand
synthesis proceeds in a similar manner during a single round of low-stringency peR
peR amplification at high stringency proceeds by virtue of having incorporated the
arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent
9
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10
RAP-peR
------------- - -AAA ~CC TCCA
_ pt olf04r
First-strand s~ntnesis
~ RNA -------------- AAA
eDNA ---- CCATCCA
W7
ACGTACC~ eDA CCT GC
Second-strand synthesis ~-
ACCT ACC ------------- GCT GCA
peR amplification my
Figure 1 The RAP-peR technique (Buchner 1994)
10