Isolation and Characterization of Partial Gene Sequence ... and Characterization of Partial... · 3.2.3 Nucleic Acids Extraction 21 3.2.3.1 Total RNA Isolation 21 3.2.3.2 Genomic

Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase)

from Kelampayan (Neolamarckia cadamba)

Lee Vivian

QK 495 US L477 2f)13 Bachelor of Science with Honours

(Resource Biotechnology) 2013

- ----~-----__-

Pusat Khidmat MakJumat Akademik lINJVIRSm MALAYSIA SARAWAK

PKHIDMAT MAKLUMAT AKADEMIK

1llIllIlIlfi~illllllllll 1000246611

Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)

Lee Vivian

A Thesis Submitted in Partial Fulfillment of The Requirement ofThe Degree of Bachelor of Science with Honours (Resource Biotechnology)

Supervisor Dr Ho Wei Seng Co-supervisor Dr Pang Shek Ling

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2013

ACKNOWLEDGEMENT

First and foremost I would like to express my profoundest and warmest gratitude to my

project supervisor Dr Ho Wei Seng for the continuous support of my study and research

for his enthusiasm patience motivation and immense knowledge along with his

invaluable advices and guidance His guidance helped me in all the time of this research

and writing of this thesis whilst allowing me the room to work in my own way

My sincere thanks also go to Dr Pang Shek Ling my co-supervisor for her time

and effort devoted in providing warm encouragement and insightful comments while

leading me on this research project Under her guidance I have successfully overcome

many difficulties and learned a lot

I would also like to acknowledge with much appreciation my fellow lab mates in

Forest Genomics and Infonnatics Lab (GiL) for the stimulating discussions and their

constant support and help throughout this research

Last but not least I would like to extend huge heartiest thanks to my family and

friends who provide a carefree environment and cheering me up through the good times

and bad with their love care and moral support

DECLARATION

I hereby declare that this thesis is of my original work except for quotations and citations

all of which have been duly acknowledged I also declare that it has not been previously

or concurrently submitted for any other degree at UNIMAS or any other institutions

Lee Vivian

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K

T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II

TABLE OF CONTENTS III

LIST OF ABBREVIATIONS VI

LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT 1

10 INTRODUCTION 2

20 LITERATURE REVIEW 5

21 Neolamarckia cadamba 5

22 Wood Formation in Forest Trees 6

221 Overview of the Development of Woody Stem 6

222 Chemical Composition of Woody Cell Walls 7

23 UDP-sugars 8

231 Biosynthesis and Biochemical Role ofUDP-sugars 8

24 UDP-sugar Pyrophosphorylase (USPase) II

241 Metabolic Role ofUSPase 11

242 USPase Protein 12

30 MATERIALS AND METHODS 16

31 Materials 16

311 Plant Materials 16

32 Methods 16

III

321 Primer Design 16

322 Collection of Plant Material 20

3221 Developing Xylem Tissues 20

3222 Leaf Tissues 20

323 Nucleic Acids Extraction 21

3231 Total RNA Isolation 21

3232 Genomic DNA Extraction and Purification 23

324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24

3241 Assessment of Total RNA Integrity 24

3242 Assessment of gDNA Integrity 25

325 Nucleic Acids Quantification 26

326 Reverse Transcription 27

327 Polymerase Chain Reaction (PCR) 28

3271 PCR of cDNA 28

3272 PCR ofgDNA 31

328 PCR Product Purification via Gel Extraction 33

329 DNA Sequencing and Sequence Data Analysis 35

40 RESULTS 36

41 Nucleic Acids Integrity and Quality 36

411 Total RNA from Developing Xylem Tissues 36

412 Genomic DNA from Leaf Tissues 37

IV

42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39

43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40


45 Purified PCR Products 42

46 DNA Sequencing and Data Analysis 43

50 DISCUSSIONS

51 Isolation ofTotal RNA from Developing Xylem Tissues 44


53 Reverse Transcription-PCR (RT-PCR) 47

54 PCRofgDNA 48



60 CONCLUSIONS AND RECOMMENDATIONS 51

REFERENCES 52

v

LIST OF ABBREVIATIONS

A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere

Basic Alignment Search Tool

Basic Alignment Search Tool for nuc1eotides

Base pair

Complementary deoxyribonucleic acid

Cytosine guanine

Cetyltrimethylarnmonium bromide

Centimeter

Double-distilled water

Dietylpyrocarbonate

Deoxyribonucleic acid

Deoxyribonucleic acid-ase

Deoxyribonucleotide triphosphate

European Molecular Biology Laboratory-European Bioinformatics Institute

gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar

National Centre for Biotechnology Information

Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV

Polymerase Chain Reaction

Pyrophosphate

Ribonucleic acid

Ribonucleic acid-ase

Revolution per minute

Ribosomal RNA

Reverse Transcription-Polymerase Chain Reaction

Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre

UDP-sugar pyrophosphorylase

U ridine-5 -triphosphate

Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES

Page Table 31 Composition of reaction mixture for first-strand

cDNA synthesis 28

Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29

Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30

Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31

Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33

Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37

Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38

Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43

VIII

LIST OF FIGURES

Figure 21 The role of products of the enzymatic reaction ofUSPase

Page

10

Figure 22 Biochemical reaction catalyzed by UDP-sugar

pyrophosphorylase (USPase) 11

Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13

Figure 24 Cryastallized structure ofUSPase from Leishmania 14

Figure 31 Partial result of mUltiple alignment between nucleotide

sequences ofArabidopsis thaliana Glycine max (soybean)

and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17

Figure 32 Output information of primer search using Primer Premier 60 19

Figure 41 Gel electrophoresis of total RNA isolated from developing

Figure 42 Gel electrophoresis of genomic DNA extracted from leaf

Figure 43 Gel electrophoresis of amplicons from gradient PCR


xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36

tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38

using cDNA as template 39

using gDNA as template 40

Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41

Figure 46 Purified DNA from purification ofPCR products with

Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42

Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43

IX

Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar

pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)

Lee Vivian

Resource Biotechnology Faculty of Resource Science and Technology


ABSTRACT

Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species

Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology

ABSTRAK

Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang

penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA

dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill

Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan

1

10 INTRODUCTION

Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree

that is being cultivated widely in recent years It belongs to the family of Rubiaceae and

nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm

(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia

Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012

Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy

sustainable harvesting of forest trees and to cater the need for commercial productions

Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel

2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber

providing the source for plywood and used for lightweight construction works Besides

Kelampayan wood is also a source of pulp for paper production (Joker 2000)

Apart from these commercial productions Kelampayan possess a wide range of

pharmacological properties The therapeutic properties are mostly found in its bark and

leaves The leaves have been used as folk remedies to pacify a wide range of such

illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers

(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at

2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf

material of Kelampayan is active against some tumors another common medicinal belief

is that the leaves of this species are antidiabetic agents and studies have been conducted

to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the

bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic

2

constipating and antiemetic properties and is given to treat the fever and inflammation of

eyes (Dubey et al 2011)

Despite the many usable values of this tree species knowledge on the structural

and regulatory genes that govern wood fonnation of Kelampayan has not been

established to a comprehensive extent as compared with other higher plant species such

as Populus and Eucalyptus trees Thus knowing that most of the production values of

Kelampayan arise from the usage of its wood it is essential to study specific genes that

contribute significance to the plant development especially in wood fonnation

UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-

monosaccharide-I-phosphate uridyltransferase is believed to be an important wood

formation gene in woody trees It is one of the key enzymes in plant carbohydrate

metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy

triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The

product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in

turn acts as a precursor for the fonnation of plant metabolites and more importantly

structural components of the cell wall which may play a significant role in wood

formation in Kelampayan

However at present the only woody plant of which its USPase gene

characterized is Populus trichocarpa Although this gene has been studied in a variety of

agricultural plant species which mostly are herbaceous it is not known whether this gene

in Kelampayan displays significant sequence and functional homologies with other

characterized USPases Although this is advantageous in a way that the sequences of the

3

t

characterized plant genomes are available one limiting factor of using these herbaceous

plants as model species is the fact that many genes expressed during wood formation in

woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)

Furthermore since there is only one woody tree species with its USPase characterized its

role in wood formation requires further validation

To address these problems this study aims to isolate and amplify the partial

cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it

by performing in-silico analyses with reference to currently available data in the public

domain The study was done at both levels of gDNA and eDNA as comparison between

them could suggest the location of transcribed region in the genome

4

Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~

20 LITERATURE REVIEW

21 Neolamarckia cadamba

Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as

Kelampayan in the Malay language It is cultivated worldwide in tropical regions with

geographical distribution covering India Pakistan Sri Lanka Thailand Indochina

eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and

Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary

rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain

year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)

(Joker 2000)

Due to its special properties Kelampayan has been propagated for a wide variety

of uses Of ecological role this tree species is suitable for reforestation because it is fast-

growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line

planting (Joker 2000) In term of wood production the wood is light and hard but with

poor durability Thus it is mainly used to produce plywood and for lightweight

construction besides as a source of pulp producing low- and medium- quality paper

(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it

useful remedies in the indigenous system of medicine A wide range of medicinal

activities in various parts of Kelampayan were reported by Gautam et al (2012) in their

pharmacological studies Leaf extracts of Kelampayan were shown to possess most

therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant

antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed

5

et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy

of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has

antidiabetic property In addition to its medicinal property Collins et al (as cited in

Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active

against some tumors The bark of Kelampayan also shows some similar activities found

in the leaves such as analgesic and anti-flammatory activities with addition of diuretic

and laxative activity Even the roots display medicinal property which is hypolipidemic

activity (Gaumat et al 2012)

22 Wood Formation in Forest Trees

221 Overview of the Development of Woody Stem

Wood is an irreplaceable natural product which holds a massive prospect lD global

industry with a multitude of applications Despite the fact that wood is an important

natural product knowledge about the structural and regulatory genes that govern its

fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough

understanding of the molecular biology of wood development therefore is imperative for

improvement of wood and fiber quality of forest trees

Despite the importance of the forest biome currently majority of wood is

harvested from natural forests destructively In addition the facts that forest trees require

naturally long generation times and lack of mutant lines have become obstacles for them

to un4ergo agricultural evolution of creating varieties of desirable traits like that lD

6

cultivation and domestication of crop species such as rice and soybean Therefore

improving the chemical composition of wood of forest trees becomes one of the main

applications of genes characterized in synthesis of traits superior to their wild ancestors

Wood formation has been focused on the anatomical level for decades According

to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to

genetic mechanisms that govern wood development and properties For example the

completed sequencing of tree genomes including that of Populus trichocarpa

(Wullschleger et al 2002) has made a significant impact on forest tree genomics

222 Chemical Composition of Woody Cell Walls

The process of wood fonnation also known as xylogenesis as described by Plomion et al

(2001) encompasses at least five major steps cell division (cambium cells divide to fonn

xylem and phloem) cell elongation cell wall thickening programmed cell death and

heartwood formation

The structure and composition of wood are influenced by cellular and

biochemical processes occurring in each of these steps The structure and composition of

wood in tum impact in the processing of wood In the development of woody stem of

trees of other wood-forming species the development of xylem and phloem from the

vascular cambium is expanded to a secondary level where they function to support and

transport Besides the secondary thickening of the cell wall is also one of the major

factQrs that determine the structure and composition of wood Secondary cell walls are

7

composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)

Hemicellulose and lignin are heteropolymeric compounds with variable compositions

(Ranik 2005) This gives rise to the variability of cell wall components between different

types of wood and wood from different species (Mellerowicz et al 2001) On the other

hand each phase of xylogenesis is regulated by the interaction of the differentiating cells

by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In

addition Friml (2003) has described that wood fonnation is also regulated by the plants

adaptability to the environmental changes

23 UDP-sugars

231 Biosynth esis and Biochemical Role of UDP-sugars

By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants

convert light energy to chemical energy which is stored in the bonds of sugar in forms of

monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are

building blocks of disaccharides and polysaccharides To form disaccharides and

polysaccharides a monosaccharide needs to be activated which is by the addition of a

nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide

sugar Nucleotide sugars are the universal sugar donors for the formation of

polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary

metabolites (Bar-Peled amp ONeill 2011)

8

The sugar component in nucleotide sugars are derived from a variety of sources

including the carbohydrate derived from photosynthesis the sugar generated by

hydrolysis of translocated sucrose the sugars released from storage carbohydrates the

salvage of sugars from glycoproteins and glycolipids the recycling of sugars released

during primary and secondary cell wall restructuring and the sugar generated during

plant-microbe interactions (Bar-Peled amp ONeill 2011)

Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent

nucleotide sugars which constituents include a monosaccharide and a nucleotide

Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher

plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the

starting substrate that is sequentially converted to UDP-sugars On the other hand in the

salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids

from cell wall These compounds then are incorporated into the cells and then converted

to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)

With the action of a variety of glycosyltransferases the sugar residue of

ucleotide sugars can be linked to other compounds such as carbohydrate protein and

pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases

0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to

tor molecules (aglycones) The conjugation leads to the formation of a range of

sylated molecules (Ross et ai 2001) Thus in plants being key precursors for

fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites

as sucrose structural components such as celulose hemicellulose and pectin as

as glycoproteins and glycolipids (Figure 21)

9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS

TREHALOSE CELLULOSE CALLOSE

RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I

Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)

An important product ofUSPase reaction UDP-Glc can be used in the formation

of disaccharides such as sucrose and trehalose as well as polysaccharides such as

cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and

stachyose which are the main carbon-transporting compounds In addition several other

UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized

by mechanisms involving USPase These UDP-sugars take part in the formation of pectin

and hemicellulose two of the most abundant biomolecules in nature Besides they are

also required for the glycosylation of proteins and lipids (Karr et al as cited in

Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass

production in plants (Kotake 2010)

10

24 UDP-sugar Pyrophosphorylase (USPase)

241 Metabolic Role of USPase

UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-

monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the

carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase

catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to

sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI

2011)

o 0 II IImonosaccharide UDP- +

~ O-p-Qr-p-O -I-phosphate monosaccharide I I

o 0

Diphosphate PPi UTP

Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)

It catalyzes the converSIOn of various monosaccharide I-phosphates to the

respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides

released during hydrolytic reactions involving polysaccharides and other glycoconjugates

(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita

IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence

that the salvage pathway plays a role in recycling monosaccharides released from

lysaccharides during cell wall synthesis and turnover

USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the

ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy

11

glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these

substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher

affinity towards USPase than pentose-l-phosphates

Previous studies in Arabidopsis have shown that USPase is essential in plant

reproductive processes USPase-knocked out plants show phenotype of pollen sterility

disabling transmission of the loss-of-function mutation through male gametophyte thus

Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and

Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull

pectocellulosic inner layer in the cell wall and has a shrunken shape

142 USPase Protein

Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB

(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or

tein of UDP-sugar pyrophosphorylase been characterized from any plant species of

In a study done by Kleczkowski et at (2011) they found that the USPase proteins

different plants share at least 60 identity at their amino acid sequence Based on

acid sequence identity of the derived proteins a comprehensive phylogenetic tree

ase has been constructed as shown in Figure 23

12

EucaryotllEUClll)OtiI

JliridiplantlleChlorophyta

krllpl_ _

~II(Jtllftm fAIsII_IIill_jor

Eucaryota rP- cruz EucaryotaEuglenozOfl

PItuIllOdi wwu Aiveolllta

Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )

As presented in the phylogenetic tree above in the Viridiplantae family to which

lampayan belongs only Populus trichocarpa is a woody plant and belongs to the

fimily Rubiaceae making it the only species closely related to Kelampayan for the

Characterization of wood formation gene

Although USPase has overlapping activities with some other UTP-dependent

aoptlOsphorylases it does not share significant homology at the amino acid sequence

with other plant UDP-sugar-producing pyrophosphorylases However they have

lIihnilar structural pattern which is inferred based on the only crystallized structure of

IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein

13

- ----~-----__-

Pusat Khidmat MakJumat Akademik lINJVIRSm MALAYSIA SARAWAK

PKHIDMAT MAKLUMAT AKADEMIK

1llIllIlIlfi~illllllllll 1000246611

Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)

Lee Vivian

A Thesis Submitted in Partial Fulfillment of The Requirement ofThe Degree of Bachelor of Science with Honours (Resource Biotechnology)

Supervisor Dr Ho Wei Seng Co-supervisor Dr Pang Shek Ling

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2013

ACKNOWLEDGEMENT
















DECLARATION




Lee Vivian






T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II



LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT 1

10 INTRODUCTION 2






23 UDP-sugars 8






31 Materials 16


32 Methods 16

III














3271 PCR of cDNA 28

3272 PCR ofgDNA 31



40 RESULTS 36




IV






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

ACKNOWLEDGEMENT
















DECLARATION




Lee Vivian






T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II



LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT 1

10 INTRODUCTION 2






23 UDP-sugars 8






31 Materials 16


32 Methods 16

III














3271 PCR of cDNA 28

3272 PCR ofgDNA 31



40 RESULTS 36




IV






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

DECLARATION




Lee Vivian






T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II



LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT 1

10 INTRODUCTION 2






23 UDP-sugars 8






31 Materials 16


32 Methods 16

III














3271 PCR of cDNA 28

3272 PCR ofgDNA 31



40 RESULTS 36




IV






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13


T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II



LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT 1

10 INTRODUCTION 2






23 UDP-sugars 8






31 Materials 16


32 Methods 16

III














3271 PCR of cDNA 28

3272 PCR ofgDNA 31



40 RESULTS 36




IV






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13














3271 PCR of cDNA 28

3272 PCR ofgDNA 31



40 RESULTS 36




IV






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13






50 DISCUSSIONS




54 PCRofgDNA 48




REFERENCES 52

v


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13


A

BLAST

BLASTn

bp

cDNA

CG

CTAB

cm

DEPC

DNA

DNase

dNTP

EMBL-EBI

g

gDNA

MgCh

min(s)

ml

mM

NCBI

ng

Ampere



Base pair


Cytosine guanine


Centimeter


Dietylpyrocarbonate





gram

Genomic DNA

Magnesium chloride

Minute(s)

Mi1i1itre

Milimolar


Nanogram

VI

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

V

PCR

PPi

RNA

RNAse

rpm

rRNA

RT-PCR

sec(s)

TAE

UDP

JlI

USPase

UTP

UV


Pyrophosphate

Ribonucleic acid



Ribosomal RNA


Second(s)

Tris-Acetate EDT A

Uridine diphosphate

Microlitre



Ultraviolet

Volt

Degree Celcius

VII

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

LIST OF TABLES


cDNA synthesis 28








VIII

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

LIST OF FIGURES


Page

10





















IX



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13



Lee Vivian



ABSTRACT



ABSTRAK





1

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

10 INTRODUCTION






















2























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13























3

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

t











4











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13











(Joker 2000)













5





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13





















6













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13













heartwood formation








7









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13









23 UDP-sugars











8
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13
























9

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13

SUCROSE

GLYCOLIPIDS

GLYCOPROTEINS


RAFFINOSE STACHYOSE

HEMICELLULOSE

PECTIN I












10








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13








2011)



o 0

Diphosphate PPi UTP











11










142 USPase Protein








12



krllpl_ _














13










142 USPase Protein








12



krllpl_ _














13



krllpl_ _














13

Documents

Isolation and Characterization of Partial Gene Sequence ... and Characterization of Partial... · 3.2.3 Nucleic Acids Extraction 21 3.2.3.1 Total RNA Isolation 21 3.2.3.2 Genomic