profiling and analysis of starch synthase … and analysis of...profiling and analysis of starch...

PROFILING AND ANALYSIS OF STARCH SYNTHASE AT DIFFERENT TRUNK HEIGHTS OF SAGO PALM

(Metroxylon sagu ROTTB)

George Deng Anyie

Master of Science 2012

PROFILING AND ANALYSIS OF STARCH SYNTHASE AT DIFFERENT TRUNK HEIGHTS OF

SAGO PALM (METROXYLON SAGU ROTTB)

GEORGE DENG ANYIE

A thesis submitted

In fulfillment of the requirements for the degree of Master of Science

(Biochemistry)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA W AK

2012

DECLARATION

I hereby declare that no portion of the work referred to in this thesis has been submitted in

support of an application for another degree or qualification to this or any other university or

institution of higher learning

George Deng Anyie

Matrik No 08-02-1330

Date ~VV) I)J L

ACKNOWLEDGMENT

First of all I would like to thank God for His grace and mighty in giving me the strength and

wisdom to complete this thesis A highly gratitude to my beloved supervisor and coshy

supervisor Associate Professor Dr Awang Ahmad Sallehin Awang Hussani Associate

Professor Dr MohdHasnainMdHussain and Associate Professor Dr HairulAzmanRoslan for

their dedication patience and guidance for the completion of my study The morale and

financial support from my beloved family are also highly appreciated thus enable me to bare

the challenges during the completion ofmy study Not to be forgotten Miss Eileen Debra for

her prayers patience and honesty in motivate me endlessly

I would also like to express my thankfulness especially to the Faculty of Resource Science

and Technology (FRST) for providing me good facilities and comfortable spaces to perform

my research The financial support from MOSTI is also greatly appreciated Nevertheless to

CRAUN Research Sdn Bhd for providing me good instruments and University Malaysia

Sarawak for providing me good facilities such as hostel library cafeterias bank and as well

as football field to release my tension after lab work Nevertheless great appreciation to

MdmSheilaUngau MrAzisAjirn and Ms Lim JataiKadin for their support and caring in

assisting me on the labs equipments I have work amongst trustworthy friends such as Frazer

ak Midot Wee ChingChing Jerry Gerunsing Lee Jong Jen Mohd Suhaib and Anastasia

Shera Their times and ideas spent for my research a highly appreciated God bless you all

Thank you

ii

ABSTRACT

Research on sago palm has been a main focus in Sarawak state as it has a great potential to

boost the economy Various studies have been performed on this starchy plant and one of it is

the study on its starch biosynthesis pathway Starch synthase was identified as one of the

enzymes that play vital role in biosynthesis of starch Therefore the profiling and

characterization of starch synthase has been studie Three palms were sampled from Bau

district specifically from KampungTanjong and KampungSidoh Targeted part for studies on

the palm wasthe base height middle height and top height of the trunk Initial studies were

the optimization of specific extraction protocol for starch synthase from sago palm withthe

GBSS buffer was identified as the most suitable buffer The best method for post extraction

purification and concentration was determined as the cold acetone precipitation method Then

total starch content in 1 gmL of sample and total protein concentration was measured and

undergoes ANOV A with the statistical analysis showed no significant difference (p lt 005)

oftotal starch content in 1 gmL of sample and total protein concentration among each palms

However ANOVA on the data for starch content between the inner part and outer part of the

trunk showed the outer part contained more starch than the inner part Presence of Starch

Synthase (SS) in optimization-treated sample and all samples were confirmed through HPLC

analysis as quantitative result and SDS-P AGE analysis as qualitative result Subsequently the

activities of SS were assayed through spectrophotometer The results showed no significant

different (p lt 005)between SS activity with the trunks height Developments of specific

primers have been done by few researchers on sago palm In this study a pairs of PCR

III

I primers were designed from cDNA library of sago palm and others starchy plants The studies

were initiated by total RNA isolation and RNAs conversion to cDNA The cDNAintegrity

was confirmed using polymerase chain reaction technique using in house gene primer called

elf-F and elf-R The cDNA was further amplified and sequenced Primer with labeled ssJ was

confirmed to be a specific primer for starch synthase as the BLAST resulted in percentage of

similarities with Zea mays full length cDNA clone (79) Zea mays starch synthase IIc

precursor (68) Triticumaestivllm starch synthase IIc precursor (77) and Oryza Sativa

soluble starch synthase II-I mRNA (77)Characterization of SS in sago palms trunk were

further analyzed through Western blotting and the results has confirmed the presence of SS

isoform at the size of 662 kDa 45 kDa 29 kDa 26 kDa and 177 kDa molecular

massAlthough Northern blot analysis was failed the specificity of the designed ssFI and

ssRI primer was confirmed Conclusively this research has successfully identified the

presence and size of SS isoform in sago palms trunk and its activity was observed to be

slightly gradually increased with the trunks height

Keywords Starch synthase Metroxylon sagll Rottb Western blottingPlawei HPLC

IV

A BSTRAK

PEMPROFILAN DAN ANALISIS KANJI SINTASE PADA KETINGGIAN BERBEZA

BATANG POKOK SAGU (METROXYLON SA GU ROTTB)

Kajian terhadap pokok sagu telah menjadi tllmpllan utama kerajaan negeri Sarawak kerana

mempunyai potensi untuk menjana ekonomi Pelbagai kajian telah dilakukan keatas

tumbuhan kanji ini dan salah satunya adalah kajian keatas kitar biosintesis kanji Kanji

sintase telah dikenal pasti sebagai salah satu enzim yang memainkan peranan penting dalam

kitur biosintesis kanji Oleh itu kajian lIntuk memprojilkan kanji sintase setelah dijalankan

Tiga pokok sagu dari daerah Bau iaitu di Kampung Tanjong dan Kampung Sidoh telah

diambi sebagai sampel Bahagian pokok yang dikaji ialah bahagian bawah batang tengah

batang dan atas batang Kajian awal ialah pengoptimaan kaedah pengekstrakkan kanji

sintase paling optimum dan hasilnya lanttan penimbal GBSS dipilih Kaedah terbaik untuk

langkah-langkah akhir proses penulinan dan pemekatan enzim telah dikenal pasti iaitu

pemekatan aseton sejuk Kemudian jumlah keseluruhan kanji didalam 1 gmL sampel dan

jumlah keselurllhan kepekatan protein telah diukllr and dianalisa menggunakan ANOVA dan

kajian statistic menunjukkan tiada perbezaan data untuk jumlah keselunthan kanji di dalam 1

gmL sampel dan jumlah keselunlhan kepekatan protein antara ketiga-tiga pokok sagu Selain

itu ANOVA keatas data jumlah keseluruhan kanji diantara bahagian tengah batang pokok

agu dan bahagian tepi batang pokok sagu menunjukkan bahagian tepi mengandllngi lebih

banyak kanji daripada bahagian dalam Kehadiran kanji sintase (SS) didalam semua sampel

serta sampel yang digunakan untuk pengoptimaan telah disahkan menggunakan analisis

kromatograji cecair berprestasi tinggi (HPLC) sebagai keputusan kuantitatif dan ana lis is

v

SDS-PAGE sebagai keplltllsan kualitatif Aktiviti SS dieseikan dengan menggunakan

spektropho tometer Keputusan menunjukkan tiada perbezaan (pltO05) data diantara aktiviti

SS dengan ketinggian batang pokok sagu Penghasilan primer spesijik untuk pokok sagu telah

dihasilkan oleh beberapa penyelidik sebelum ini Dalam kajian ini sepasang primer spesijik

untuk PCR telah dihasilkan daripada perpusatakaan eDNA pokok sagu dan juga eDNA

tumbuhan-tumbuhan berkanji lain Kajian awal dimulakan dengan pengasingan semua RNA

pokok sagu dan kemudian diterjemahkan kepada eDNA Ketulinan eDNA tersebut dipastikan

dengan menggunakan kaedah tindak balas polymerase berantai dan elf-F dan elf-R telah

digunakan sebagai primer-primer in-house Kemudian eDNA diamplijikasikan dan

dijlljUkkan Primer yang berlabel ssl telah berjaya dipastikan sebagai primer spesijik untuk

kanji sintase dimana keputusan BLAST memberikan peratus kesamaan dengan rantaian

penllh klon eDNAZae mays (79) prekursor kanji sintase lIe Zae mays (68) prelwrsor

kanji sintase lIe Triticum aestivum (77) dan mRNA kanji sintase lanlt Il-1 Oryza sativa

(77) Kajian memproilkan SS diteruskan dengan membuat analisa Western blot Keputusan

daripada analisis Western blot mengesahkan kehadiran ism SS pada berat molekul 662

kDa 45 kDa 29 kDa 26 kDa dan 177 kDa Analisa Northern blotting tidak berjaya

mencapai keputusan tetapi spesijikasi primer ssFl dan ssRl yang direka telah beljaya

dite1ltllkan Keseluruhannya kajian ini telah Beljaya mengesahkan kehadiran enzim SS

didalam batang pokok sagu serta ism-isrm enzim ss Selain itu kajian juga telah

berjaya menunjukkan aktiviti SS meningkat dengan perkadaran yang sedikit apabila

kedlldukan ketinggian pada batang pokok sagu meningkat

Kala kunci Kanji sintase Metroxylon sagu Rottb Western blotting Plawei HPLC

vi

Posat Khidmat Maldulllat Akademih UNIVERSm MALAYSIA SARAWA

TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures

List of Abbreviations

CHAPTER 1 - INTRODUCTION

11 Project Rationale

12 Objectives

CHAPTER 2 - LITERATURE REVIEW

21 Sago Palm (Metroxylon Sagu Rottb)

22 Starch Structure and Composition

23 Starch Biosynthesis

24 Spectrophotometer Assay of SS

25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17

18 26 High Perfonnance Liquid Chromatography

27 Northern Blotting 20

CHAPTER 3 - OPTIMIZATION OF PROTEIN EXTRACTION AND 22 PURIFICATION METHOD IN STARCH SYNTHASE

STUDY OF SAGO PALM (METROXYLON SAGU ROTTB)

31 Introduction 22

32 Materials and Methods 27

321 Sampling 27

322 Enzyme Extraction Using Different Buffer 27

323 Detennination ofTotal Protein Concentration 29

324 SDS-PAGE 30

325 Ammonium Sulphate Precipitation 30

326 Dialysis 31

327 Cold Acetone Precipitation 32

328 Enzyme Extraction Using Different pH 32

329 Spectrophotometer Assay of Starch Synthase 33

3210 HPLC Analysis 34

33 Results 35

33l Enzyme Extraction from Three Different Buffers 35

332 Ammonium Sulphate Precipitation and Dialysis 37

Vlll


334 Detection of ADP 40

334 Enzymatic activity at different pH 43

34Discussions 44

341 Extraction Buffers 44



344 Detection of SS 48

345 Spectrophotometer Assay of SS 49

35Conclusion and Future Direction 50

CHAPTER 4 PROFILING OF STARCH SYNTHASE ACTIVITY IN 51 PLAWEI GROWTH STAGES OF SAGO PALM (METROXYLON SAGU ROTTB)

41 Introduction 51

42Materials and Methods 54

421 Sampling 54

422 Iodine-starch Complex Colorimetric Method and Moisture 56 Content Measurement

423 Enzyme Extraction 57

424 Determination ofProtein Concentration 58

ix

58 425 Ammonium Sulphate Precipitation and Desalting

426 SDS-PAGE 58


428 Spectrophotometric Assay of Starch Synthase 57

429 Statistical Analysis 57

43Results 60 431 Iodine-Starch Complex Colorimetric Method 60

432 Moisture Content Measurement 61

433 Enzyme Extraction and Protein Quantification 63


435 Spectrophotometric assay of SS 72

44Discussion 75

45 Conclusion 80

CHAPTER 5 THE ESTABLISHMENT OF PCR-BASED SPECIFIC 81 MARKER FOR STARCH SYNTHASE IN SAGO PALM (METROXYLON SAGU ROTTB)

51 Introduction 81

52 Methodology 84

521 RNA Extraction 84

522 Spectrophotometric Measurement 85

523 Primer Design 85

x

I

524 Synthesis ofFirst Strand cDNA 86

525 PCR Amplification 87

5251 Polymerase Chain Reaction for cDNA Integrity 88

526 Native Agarose Gel Electrophoresis 89

527 cDNA Recovery From Agarose Gel 90

528 Sequencing ofPCR product 90

53Results 91


53 2 Synthesis of First Strand cDNA 93

53 3 PCR Amplification 94

534 Sequencing and analysis ofPCR Product 96

54Discussion 97


CHAPTER 6 WESTERN BLOT AND NORTHERN BLOT ANALYSIS 101 OF STARCH SYNTHASE IN SAGO PALM (METROXYLON SA GU ROTTB)

61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106

623 Western Blotting 106

624 Color Development of Expressed Protein 107

625 Probe Design for Northern Blotting 108


627 Chemiluminescent Detection ofNucleic Acid for Northern 110 Blotting

63ResuIts III

631 SDS-PAGE III

632 Spectrophotometer Quantification 112



64Discussions 117



65 Conclusion 125

CHAPTER 7 GENERAL CONCLUSION AND RECOMMENDATIONS 127

80 REFERENCES

APPENDIX A Standard Calibration Curve

APPENDIX B Calculation for Enzymatic Activity ofSS

xii

APPENDIX C Statistical Analysis For Spectrophotometer Assay

APPENDIX D Statistical Analysis for Starch Content and Total Protein Measurement

APPENDIX E Nucleotide Sequence for Primer Design

xiii

List of Tables

Table 21 The physiological growth stage of sago palm 10

Research Sdn Bhd

using different buffers on a sample of sago palms trunk (AngauMuda

stages of5g samples ofsago palm trunk using GBSS-buffer(n=3)

and dialysis was performed on Angaumuda samples purified by GBSS-

Table 31 The names of stages for each samples label received from Craun 27

Table 32 Protein concentration ofcrude enzyme of 5 g sago palms trunk extracted 35

stage) (n=3)

Table 33 Protein concentration of crude enzyme extracted from different growth 36

Table 34 Concentrated protein recovered after ammonium sulphate precipitation 37

buffer The 60 (wv) of the ammonium sulphate precipitation gives the highest value ofprotein concentration laquon = 3)

Table 41 Data of sago palms trunk at Plawei stage 54

Table 42 The amount of starch in 1 gmL of sago trunk 60

Table 43 One-way ANOYA on starch content in 1 glmL sample at different 61 trunks part in Plawei stage of sago palms

Table 44 Protein concentration of crude enzyme extracted from Plawei stage of 64 sago palm trunk using GBSS buffer

Table 45 Specific value of retention time height area and molarities for each 68 standard used in HPLC analysis

Table 46 The amount of ADP produced during spectrophotometer assay of SS 72 enzyme on three palms specifically at Plawei stage (n=3)

Table 47 Activity of SS observed at different heights of the sago palm at Plawei 73 stage (n=2)

Table 51 The sequence ofprimers that specifically designed for starch synthase 86

Table 52 The reaction mixture for RNA cleanup prior to cDNA synthesis 86

XIV

Table 53 The reaction parameters for the peR analysis set up 87

Table 54 The optimized volume of peR mixture for peR reaction using ssF 1 and 88 ssR2primers

Table 55 Molecular data ofelfgene for peR analysis 88

Table 56 The optimized volume of peR mixture for peR reaction using elf-F and 89 elfR primers

able 57 Purity and yield ofthe extracted RNA 92

Table 61 The optimal volume of samples mixture loaded into the formaldehyde 109 gels well

Table 62 The volume ofcomponents in prehybridization solution 110

Table 63 The volume ofcomponents in Formamide hybridization solution 110

Table 64 The concentration ofextracted RNA from each palm at different heights 112

Table 65 The occurrence of expressed SS within each sago palm The square root 117 symbol indicates the presence ofrespective sizes in the sample

Table 66 The percentage of different types of RNA in eukaryotic cells (Darling 124 ampBrickell 1996)

xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33

The estimated area of sago in Sarawak in 2003 (Courtesy of the Department of Statistics (http-wwwdoasarawakgovmystatistik07htm)

to 2007 Sarawak

2

The export of agricultural products 2007(http-www doasarawakgov my)

by Malaysia III year 3

Picture of MetroyxlonsaguRottb atPlawei stage captured at Bau (Singai area) district Sarawak

Picture was 9

The molecular structure of amylose and amylopectin (Shaw 1999)

11

Closed view of starch granules of sago palm (A) The SEM micrograph of starch granules in native sago palm 700x (Wong et at 2005) (B)amp(C) Optical microscope view ofstarch granule cultivated in mineral soil (Nozaki et at 2004)

12

The three steps of starch biosynthesis in higher plants (Martin amp Smith 1995)

15

The classical enzymatic glucose reaction that has been applied in producing formula for enzymatic assay (Illanes 2008)

16

The overall diagram of a HPLC system (Prichard et al 2003) 19

Picture ofa grater and grated sago pith samples 28

The bands of crude protein extracted using three different buffers (A) The samples extracted using the Wende1ampWeeden buffer (B) The samples extracted using GBSS buffer (C) The sample extracted using SS buffer Each lane contains 16 ~L of the sample Only one out of six samples is stained after SDSshyPAGE process for SS buffer and GBSS buffer Thus result shows only stained protein The molecular standard of A is the Kaleidoscope prestained protein ladder (Bio-Rad)

36

The ammonium sulphate precipitation of samples extracted using WendelampWeeden buffer The yellow color above the tube is the precipitation of BSA in which might have affected the reading of the protein concentration measurement through BSA standard method

38

XVI

Figure 34 Protein concentration of extracted sample from different growth stages after precipitated using cold acetone precipitation method

39

Figure 35A Graph shows the result of HPLC assay on precipitated enzyme extracted using GBSS-bufferat pH 4 There are six peaks was recorded in the graph above indicated that the sample was not purified even after cold acetone precipitation The highest peak at the retention time of 10161 minutes indicated the level of ADPGlc in sample

40

Figure 3SB Graph shows the result of HPLC assay on precipitated enzyme extracted using GBSS-buffer at pH 65 The number ofpeak that was recorded in graph was eight peaks indicated that the sample was not purified even after cold acetone precipitation The presence of ADPG1c was detected in peak number 4 (highest peak) with retention time of 10116 minutes

41

Figure 3Se Graph shows the result of HPLC assay on precipitated enzyme extracted using GBSS-buffer at pH 8 The number of peak that was recorded in graph was eight peaks indicated that the sample was not purified even after cold acetone precipitation The presence of ADPG1c was detected in peak number 4 (highest peak) with retention time of 10105 minutes

42

Figure 36 Figure above shows the progression activity of SS in spectrophotometric assay The most favourable condition of buffer pH was detected at pH 8 The progression was measured in minutes of time against the enzyme activity After 150 mins starch synthase enzyme activity is dropped to negative value therefore activity was only recorded within the 150 mins period oftime n=3

43

Figure 37 Approaches in order to avoid browning effect 37A Sago trunk was chopped into large cube immediately at the sampling site 37Bamp37C Sample was stored in Falcon tube and wrapped in aluminum foil before stored in 4degC freezer

46

Figure 41 A The flat top shape of the chopped tree (Palm 1) indicated it was in Plawei stage B the worker is in the middle of slicing the trunk into disc form using a chain saw C fresh look of the inner trunk after it was sliced D the disc form of the trunk after slicing process using chain saw

55

Figure 42 Fieldtrip on October 14 2009 for the sampling of sago palm 2 and sago palm 3 A complete image of the palms B an expert lab assistant of UNIMAS started to chop down the palm C measuring process on the diameter circumference and length of the trunk

55

XVll

Figure 43 The powder fonn ofsample after grounded in mortar 57

Figure 44 The percentage overview of total mass in 1 gram of the trunk of 62 Metroxylon sagu Rottb The number of 1 to 6 in the figure represents the sample from each high and part of sago trunk Specifically number 1 = base-centre 1 = base-side 3= middleshycentre 4- middle side 5= top-centre and 6= top-side

Figure 45 Picture on part of sago trunk that was chose for enzyme 63 extraction

Figure 46 The bands of crude enzyme protein from six different part of 65 palm 1 Lanes A to F in the Figure 4 represents the sample from each height and part of sago trunk A= base-centre B= base-side C= middle-centre D- middle side E= top-centre and F= top-side Arrows indicates the protein bands

Figure 47 The banding pattern of crude enzyme extracted from sago palm 66 2 (A) and sago palm 3 (B) Lane 7 and lane 8 contains the commercial protein Bovine serum albumin and ex - amylase respectively Lanes 1 = base-centre 2= base-side 3= middle-centre 4- middle side 5= top-centre and 6= top-side Arrows indicates the protein bands

Figure 48 The faint bands detected on SDS-P AGE gel after undergo 67 ammonium sulphate precipitation and desaltingThe alphabet of A to F in the figure represents the sample from each high and part of sago trunk Specifically alphabetA= base-centre B= base-side C= middle-centre D- middle side E= top-centre and F= top-side Arrow indicates the protein bands at size of 662 kDa and 45 kDa

Figure 49 Figure 49A- Figure 49F Sample GBSS 69amp 71

Figure 41 0 The comparison of SS activity between three sago palms by 74 estimated marginal means Briefly the graph indicates that the difference in SS activity between palms was increased as the height of trunk moved up from base to the top but insignificantly analyzed by ANOV A (n=2)

Figure 51 Native gel electrophoresis of RNA extracted samples from sago 91 palm The volume of sample loaded into the well is 5 ilL with 1 (wv) agarose gel All lane were loaded with three replicates ofsample A = Base height B = Middle height C = Top height Lane D is the 1 Kbp DNA ladder indicating a positive control for this analysis

xviii

Figure 52 Electrophoresis analysis shows that the single band in lane B 93 signify construction of first strand cDNA was successful The template RNA used to develop this cDNA was displayed on lane C Lane A is the 100 bp DNA ladder

Figure 53 Figure S3A shows the products of cDNA amplification before 94 undergoes purification Figure S3B is the purified PCR products obtained from sago palm cDNA after recovered from 1 (wv) agarose gel electrophoresis analysis in Figure S3A Lanes 1 and 3 indicates the purified PCR product at the size 376 bp molecular mass Meanwhile 2 is the 1 Kbp DNA ladder

Figure 54 Figure 54 Figure shows attempt for an optimization of ssF and 95 ssR primers at specific temperature using gradient peR The template is cDNA developed from RNAs sago palm Smearing occurrence was observed within all samples indicating the temperatures were not suitable for the primer Specifically number 1 = 50degC 2= 502dege 3= 509dege 4= 52degC 5= 532dege 6= 544dege 7= 556dege 8= 568dege 9= 579dege 10= 59degC 11= 598dege and 12= 60degC Lane A is the 100 bp DNA ladder

Figure 61 The diagram shows the overall process ofNorthern blotting The 102 pathway encompassed probes selection either from Oligonucleotides or cDNA and the labeling techniques are either using non-radioactive or radioactive (Trayhum 1996)

Figure 62 Two types of protein transfer in Western blotting Figure 62A 104 The outline of an electrophoresis transfer for a protein transfer system m wet transfer conditions (wwwmitosciencescom) Figure 62B The outline of an electrophoresis transfer for a protein transfer system in semi-dry transfer conditions

Figure 63 The analysis of Western blot for palm 1 The number of 1 to 6 113 represents the sample from each height and part (center amp side) ofsago trunk Specifically number 1 = base-centre 2= base-side 3= middle-centre 4= middle side 5= top-centre and 6= topshyside Arrows indicates the location of the expressed SS along the lane

Figure 64 The analysis of Western blot for palm 2 The number of 1 to 6 in 114 the figure represents the sample from each height and part (center amp side) of sago trunk Specifically number 1 = baseshycentre 2= base-side 3= middle-centre 4= middle side 5= topshycentre and 6= top-side

Figure 65 The analysis ofWestern blot for palm 3 The number of I to 6 in 115 the figure represents the sample from each high and part of sago trunk Specifically number I= base-centre 2= base-side 3=

XIX

Figure 66

Figure 67

Figure 68

middle-centre 4= middle side 5= top-centre and 6= top-side Arrows indicates the location of the expressed SS along the lane

Northern blotting analysis on sago palm 1 (A) sago palm 2 (B) sago palm 3 (C) No band was observed along all lanes while marker is transferred completely

116

The quality of the ssl probe illustrated by the dots brightness Numbers of 1 to 4 indicated the replicates of the prepared probe Replicate for number 4 shows the probes concentration can be viewed at the lowest concentration of30 pg

120

The two phenomenon of RNAs base pairing A Intramolecular base pairing of short region B Intennolecular base pairing between different molecules of RNA (Darling and Brickell 1994)

122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3


Adenosine Diphosphate

Adenosine Monophosphate

Adenosine Triphosphate

Glucose-6- Phosphate Dehydrogenase

Phosphoenolpyruvate Kinase

NicotinamideAdenine Dinucleotide Phosphate

Pyruvate Kinase

Hexokinase

High Performance Liquid Chromatography

Nanometer

Sodium Dodecyl Polyacrylamide Gel

Ribonucleic Acid

Deoxyribonucleic Acid

Complementary Deoxyribonucleic acid

Messenger Ribonucleic Acid

Micro Ribonucleic Acid

Deoxyribonucleotides

Polymerase Chain Reaction

Reverse transcriptase Polymerase Chain Reaction

Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters

EthylenediaminetetraaceticAcid

Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide

CetyltrimethylammoniumBromide

Polyvinylpyrrolidone

Lithium Chloride

Weight per Gram

Volume per Volume

Millimeters per Minutes

Gram per Millimeter

Gram per Liter

Milligram per Millimeter

Milligram per Gram

Nanomole per Milliliter

Grams

Milligrams

XXI

PROFILING AND ANALYSIS OF STARCH SYNTHASE AT DIFFERENT TRUNK HEIGHTS OF

SAGO PALM (METROXYLON SAGU ROTTB)

GEORGE DENG ANYIE

A thesis submitted

In fulfillment of the requirements for the degree of Master of Science

(Biochemistry)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA W AK

2012

DECLARATION




George Deng Anyie


Date ~VV) I)J L

ACKNOWLEDGMENT



















Thank you

ii

ABSTRACT





















III
















IV

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

DECLARATION




George Deng Anyie


Date ~VV) I)J L

ACKNOWLEDGMENT



















Thank you

ii

ABSTRACT





















III
















IV

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

ACKNOWLEDGMENT



















Thank you

ii

ABSTRACT





















III
















IV

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

ABSTRACT





















III
















IV

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI
















IV

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

A BSTRAK






















v























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI























vi


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI


TABLE OF CONTENTS

Declaration

Acknowledgements

Abstract

Abstrak

Table ofContents

List ofTables

List of Figures




12 Objectives






25 Western Blotting

vii

Page

11

Iii

v

VII

XlV

XVl

xx

1

6

7

8

8

11

13

16

17





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI





31 Introduction 22


321 Sampling 27



324 SDS-PAGE 30


326 Dialysis 31





33 Results 35



Vlll




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI




34Discussions 44








41 Introduction 51


421 Sampling 54




ix


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI


426 SDS-PAGE 58









44Discussion 75

45 Conclusion 80


51 Introduction 81

52 Methodology 84




x

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

I







53Results 91





54Discussion 97



61 Introduction 101

62 Methodology 106

621 Samples 106

xi

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

622 SDS-PAGE 106






63ResuIts III

631 SDS-PAGE III




64Discussions 117



65 Conclusion 125


80 REFERENCES



xii




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI




xiii

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

List of Tables


Research Sdn Bhd






stage) (n=3)













XIV












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI












xv

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

List of Figures

Figure 11

Figure 12

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 31

Figure 32

Figure 33


to 2007 Sarawak

2




Picture was 9


11


12


15


16




36


38

XVI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI


39


40


41


42


43


46


55


55

XVll










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI










xviii









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI









XIX

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

Figure 66

Figure 67

Figure 68



116


120


122

xx

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

ADP

AMP

ATP

G6PDH

PEP

NADP

PK

HK

HPLC

nm

SDS-PAGE

RNA

DNA

cDNA

mRNA

miRNA

dNTP

PCR

RT-PCR

f3








Pyruvate Kinase

Hexokinase


Nanometer


Ribonucleic Acid








Percentage

Beta

xx

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

mM

m

EDTA

OTT

mL

HCL

KOH

CTAB

PVP

LiCI

wv

vv

mlmin

glmL

gIL

MgmL

mgg

nmolmL-1

g

mg

Alfa

Degree Celsius

MilliMolar

Meters


Dithiothreitol

Millimeters

Hydrochloric Acid

Potassium Hydroxide



Lithium Chloride

Weight per Gram

Volume per Volume


Gram per Millimeter

Gram per Liter


Milligram per Gram


Grams

Milligrams

XXI

profiling and analysis of starch synthase … and analysis of...profiling and analysis of starch...

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