universiti putra malaysia production of specialized

UNIVERSITI PUTRA MALAYSIA

PRODUCTION OF SPECIALIZED TRANSFORMATION VECTORS FOR THE PRODUCTION OF BIODEGRADABLE PLASTICS IN TRANSGENIC

ARABIDOPSIS AND OIL PALM

ABDUL MASANI MAT YUNUS

FBSB 2006 33

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by Universiti Putra Malaysia Institutional Repository

https://core.ac.uk/display/42992071?utm_source=pdf&utm_medium=banner&utm_campaign=pdf-decoration-v1

PRODUCTION OF SPECIALIZED TRANSFORMATION VECTORS FOR

THE PRODUCTION OF BIODEGRADABLE PLASTICS IN TRANSGENIC


By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirement for the Degree of Master of Science

October 2006

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

PRODUCTION OF SPECIALIZED TRANSFORMATION VECTORS FOR

THE PRODUCTION OF BIODEGRADABLE PLASTICS IN TRANSGENIC


By


October 2006

Chairman: Ho Chai Ling, PhD

Faculty: Biotechnology and Biomolecular Sciences

Polyhydroxyalkanoates (PHAs), such as polyhydroxybutyrate (PHB) and

polyhydroxybutyrate-co-hydroxyvalerate (PHBV) are bacterial polyesters, which can

be used to produce biodegradable products. Since the mass production of PHAs in

bacteria via fermentation is expensive, the production of PHAs in plants may be an

attractive alternative. The production of PHB in plants required genetic engineering

of phbA, phbB and phbC genes of Ralstonia eutropha, whereas, the bktB, phbB,

phbC genes of R. eutropha and tdcB gene of Escherichia coli were required for

PHBV production. In this study, each of these gene was fused with the transit peptide

(Tp) of oil palm acyl-carrier-protein (ACP), and driven by the oil palm leaf-specific

promoter (LSP1), for targeting into the plastids of leaf cells. In total, four

transformation vectors, pLSP15 (PHB) and pLSP20 (PHBV), pLSP13 (PHB) and

pLSP23 (PHBV) were constructed for the transformation of Arabidopsis and oil

palm, respectively. Each vector contained the phosphinothricin acetyltransferase

gene (Bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin

promoter in pLSP13 and pLSP23, as plant selectable marker. Matrix attachment

iii

region of tobacco (RB7MAR) was also included, to stabilize the transgene

expression and to minimize gene silencing due to positional effects. Restriction

enzymes, polymerase chain reaction (PCR) and DNA sequencing were used to verify

all the constructed vectors. Arabidopsis transformation produced T1 transgenic

Arabidopsis plants with normal phenotypes at a transformation efficiency of

0.2%~1.0%. PCR and Southern analyses were used to confirm the insertion of the

transgenes. Nile blue A staining of these T1 plants demonstrated the accumulation of

PHB granules in the leaf. The initial screening of Basta-resistant oil palm

embryogenic calli transformed with pLSP13 using PCR demonstrated the presence

of Bar and PHB genes in transformed oil palm.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PENGHASILAN VEKTOR-VEKTOR TRANSFORMASI KHAS UNTUK

PENGHASILAN PLASTIK BIOMUDAHURAI DI DALAM ARABIDOPSIS

DAN SAWIT TRANSGENIK

Oleh


October 2006

Pengerusi: Ho Chai Ling, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Polihidroxialkanotes (PHAs) seperti polihidroxibutirat (PHB) dan polihidroxi-ko-

hidroxivalerat (PHBV) merupakan poliester bakteria yang boleh digunakan untuk

menghasilkan produk-produk biomudahurai. Oleh kerana penghasilan PHAs dalam

kuantiti yang besar menggunakan bakteria melalui kaedah fermentasi adalah mahal,

penghasilan PHAs di dalam tumbuhan mungkin alternatif yang menarik. Penghasilan

PHB di dalam tumbuhan memerlukan kejuruteraan genetik keatas gen phbA, phbB

dan phbC daripada Ralstonia eutropha, manakala gen bktB, phbB, dan phbC

daripada R. eutropha dan tdcB daripada Escherichia coli diperlukan untuk

menghasilkan PHBV. Melalui kajian ini, setiap gen disambung dengan peptid transit

(TP) daripada proten-pembawa-asil sawit (ACP) dan dikawal oleh promoter khusus

kepada daun (LSP1) untuk ditujukan ke dalam plastid sel daun. Sejumlah 4 vektor

transformasi iaitu pLSP15 (PHB) dan pLSP20 (PHBV), pLSP13 (PHB) dan pLSP23

(PHBV) telah dibina, masing-masing ditransformasi ke dalam Arabidopsis dan sawit.

Setiap vektor mengandungi gen pospinotirisin asetiltransferas (Bar) sebagai penanda

pemilihan tumbuhan yang dikawal oleh promoter CaMV35 di dalam pLSP15 dan

v

pLSP20 serta promoter ubikuitin di dalam pLSP13 dan pLSP23. Kawasan pelekatan

matrik daripada tembakau (RB7MAR) turut dimasukkan untuk mengstabilkan

pengungkap transgen dan meminimumkan penyeyapan gen hasil daripada kesan-

kesan posisi. Enzim penyekatan, tindakbalas berantai polimeras (PCR) dan

penjujukan DNA telah digunakan untuk mengesahkan vektor-vektor yang dibina.

Transformasi Arabidopsis menghasilkan pokok Arabidopsis transgenik T1 yang

normal dengan 0.2%~1.0% efisiensi transformasi. PCR dan pemblotan Southern

telah digunakan bagi mengesahkan penyelitan gen. Perwarnaan biru A nile terhadap

pokok-pokok T1 tersebut telah menunjukkan pengumpulan gumpalan-gumpalan

PHB di dalam daun. Penyaringan awal menggunakan PCR keatas kalus embriogenik

sawit yang ditransformasi menggunakan pLSP13 telah menunjukkan kehadiran gen

Bar dan PHB dalam sawit yang ditransformasikan.

vi

ACKNOWLEDGEMENTS

I sincerely thank my main supervisor, Dr. Ho Chai Ling, for her great support,

encouragement, friendship, and insight throughout the course of my study at the

Universiti Putra Malaysia. Without her thoughtful arrangements, instructions and

help, it would be impossible for me to complete this study. My thanks also go to

Assoc. Prof. Dr. Suhaimi Napis, for his advice, comments and guidance whenever

sought. I want to express my great gratitude to Dr. Hj. Ahmad Parveez Hj. Ghulam

Kadir, for his direct daily supervision and the helpful advices not only in science but

also in different aspects of life, and also for his useful comments, kind advises and

discussions during this study. I would like to present honest thanks to the Malaysian

Palm Oil Board (MPOB) for offering me full scholarship and financial support.

Special thanks to Dato‟ Dr. Mohd Basri Wahid (Director General), Dr Ahmad

Kushairi (Director of Biology) and Dr Ravigadevi (Head of Unit, ABBC) for

allowing me to completed my thesis. I would also like to thank Assoc. Prof. Datin

Dr. Siti Nor Akmar and Madam Chan Pek Lan, for providing the oil palm leaf-

specific promoter, Prof. Anthony J. Sinskey of MIT for PHB genes. Special thanks

extended to all staffs of Genetic Transformation Laboratory for their help and

assistance. Finally, I wish to express my deepest gratitude and appreciation to my

family, especially to my father (Mat Yunus Abas) and mother (Adiyah Hj Mokhtar),

for their continuous long-distance encouragement and support from Kumai, Pahang.

To my wife, Dayang Izawati, thank you for your love and encouragement during my

study.

vii

I certify that an Examination Committee has met on 17th

October, 2006 to conduct

the final examination of Abdul Masani Mat Yunus on his Master of Science thesis

entitled " Production of Specialized Transformation Vectors for the Production of

Biodegradable Plastics in Transgenic Arabidopsis and Oil Palm " in accordance with

Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian

Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the

candidate be awarded the relevant degree. Members of the Examination Committee

are as follows:

Datin Siti Nor Akmar Abdullah, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Janna Ong Abdullah, PhD

Lecturer

Faculty of Biotechnology and Biomolecular Sciences


(Internal Examiner)

Raha Abdul Rahim, PhD

Associate Professor



(Internal Examiner)

Ismanizan Hj. Ismail, PhD

Associate Professor

Faculty of Sciences and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

_______________________________

HASANAH MOHD GHAZALI, PhD

Professor/Deputy Dean,

School of Graduate Studies


Date: 21 DECEMBER 2006

viii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee are as follows:

Ho Chai Ling, PhD

Lecturer



(Chairman)

Hj. Ahmad Parveez Hj. Ghulam Kadir, PhD

Advanced Biotechnology and Breeding Centre

Malaysian Palm Oil Board

(Member)

Suhaimi Napis, PhD

Associate Professor



(Member)

_____________________

AINI IDERIS, PhD

Professor/Deputy Dean,

School of Graduate Studies


Date:

ix

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations

and citations which have been duly acknowledged. I also declare that it has not been

previously or concurrently submitted for any other degree at UPM or other

institutions.

____________________________


Date: 23 SEPTEMBER 2006

x

TABLE OF CONTENTS

Page

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

ii

iv

vi

vii

ix

xiii

xiv

xviii

CHAPTER

1 INTRODUCTION

2 LITERATURE REVIEW

2.1 Polyhydroxyalkanoates (PHAs)

2.1.1 Polyhydroxybutyrate (PHB)

2.1.2 Biosynthesis of PHB

2.1.3 Polyhydroxybutyrate-co-hydroxyvalerate

(PHBV)

2.1.4 Biosynthesis of PHBV

2.1.5 Commercialization of PHBV by Bacterial

Fermentation

2.2 Plant Transformation

2.2.1 Arabidopsis thaliana

2.2.2 Elaeis guineensis and E. oleifera (Oil palms)

2.3 Production of PHAs in Plants

2.3.1 Arabidopsis thaliana

2.3.2 Nicotiana tabacum (Tobacco)

2.3.3 Brassica napus (Rapeseed)

2.3.4 Gossypium hirsutum (Cotton)

2.3.5 Zea mays (Maize)

2.3.6 Medicago sativa (Alfalfa)

2.3.7 Linum usitatissimum (Flax)

2.4 Analysis of Transgenic Plant Producing PHB

2.5 Future Prospective

3 MATERIALS AND METHODS

3.1 DNA Manipulation

3.1.1 Determination of DNA Concentration

3.1.2 Phenol Extraction of DNA

3.1.3 Precipitation of DNA

3.1.4 Large Scale Preparation of Plasmid DNA

3.1.5 Small Scale Preparation of Plasmid DNA

3.1.6 Agarose Gel Electrophoresis

3.1.7 Isolation of DNA Fragments from Agarose

1

4

4

4

6

7

9

9

10

11

12

14

15

21

22

24

24

25

26

27

29

33

33

33

33

34

34

35

37

xi

Gel

3.1.8 Restriction Enzyme Digestion

3.1.9 Formation of DNA with Blunt Ends

3.1.10 Dephosphorylation

3.1.11 Ligation

3.1.12 Transformation of DNA into Compentent

Cells

3.1.13 Polymerase Chain Reaction (PCR)

3.2 Construction of Transformation Vectors

3.2.1 Plasmids

3.2.2 Construction of pLSP3

3.2.3 Construction of pLSP4, pLSP5, pLSP6 and

pLSP7

3.2.4 Construction of pLSP8

3.2.5 Construction of pLSP9, pLSP10 and pLSP11

3.2.6 Construction of pMB30

3.2.7 Construction of pLSP17 and pLSP18



3.3 Plant Transformation

3.3.1 Transformation of Arabidopsis thaliana

3.3.2 Transformation of Oil Palm

3.4 Analysis of Transgenic Plant

3.4.1 Extraction of Genomic Plant DNA

3.4.2 Screening of Transgenic Plant by PCR

Analysis

3.4.3 Screening of Transgenic Plant by Southern

Blot Analysis

3.4.4 Nile Blue A Staining Method

4 RESULTS

4.1 Construction of PHB and PHBV Transformation

Vectors

4.1.1 Construction of pLSP3 Vector

4.1.2 Construction of pLSP4, pLSP5, pLSP6,

pLSP7 and pLSP8 Single-gene Intermediate

Vectors

4.1.3 Construction of pLSP9, pLSP10 and pLSP11

Multiple-gene Intermediate Vectors

4.1.4 Construction of pMB30 Backbone Vector

4.1.5 Construction of pLSP17 and pLSP18 Single-

gene Transformation Vectors

4.1.6 Construction of pLSP13, pLSP15, pLSP20

and pLSP23 Transformation Vectors

4.2 Introduction of PHB and PHBV Transformation

Vectors into Arabidopsis thaliana

4.2.1 Transformation of pLSP15 and pLSP20

Transformation Vectors into Agrobacterium

tumefaciens strain LBA4404

37

39

39

40

40

41

43

47

47

47

52

52

58

61

61

61

64

64

64

70

73

73

74

74

78

79

79

82

84

91

95

95

98

103

103

xii

4.2.2 Agrobacterium-mediated Transformation

(Floral-dip) of Arabidopsis thaliana

4.2.3 Screening of Transgenic Arabidopsis Plants

by PCR Analysis

4.2.4 Characterization of Transgenic Arabidopsis

Plants by Genomic Southern Analysis

4.2.5 Visualization of PHB Granules in Transgenic

Arabidopsis Plants

4.3 Biolistics Transformation of Oil Palm with PHB

and PHBV Transformation Vectors

5 DISCUSSION

5.1 Development of PHB and PHBV Transformation

Vectors

5.2 Production of PHB and PHBV Transgenic Plants

6 CONCLUSIONS

BIBLIOGRAPHY

APPENDICES

BIODATA OF AUTHOR

105

108

117

124

127

130

130

135

140

143

150

169

xiii

LIST OF TABLES

Table

Page

1 Properties of SCL-PHAs

2 PHB production in transgenic plants

3 List of transgenic plants accumulating PHBV

4 Strains used in this study

5 Primers used in this study

6 Plasmids used in this study

7 Primers used for PCR screening

8 List of constructed vectors in this study

9 Selection of Basta-resistant Arabidopsis plants

5

30

31

42

45

48

75

81

110

xiv

LIST OF FIGURES

Figure

Page

1 The general structure and formula of SCL-PHAs and MCL-PHAs

2 Pathway of PHB and PHBV synthesis in R. eutropha

3 The structure of plant cell

4 Accumulation of PHB inclusions in the cytoplasm of transgenic

Arabidopsis plant

5 PHB granules in the plastid of transgenic A. thaliana

6 Construction of pLSP1









15 Construction of pLSP10 and pLSP11

16 Construction of pMB30

17 Construction of pLSP17 and pLSP18





22 The overall cloning strategy for the construction of PHB and PHBV

transformation vectors

5

8

16

16

18

49

50

51

53

54

55

56

57

59

60

62

63

65

66

67

68

80

xv

23 Selection of pLSP1 vector

24 Selection of pLSP3 vector

25 Restriction endonuclease analysis of pLSP1, pLSP2 and pLSP3

26 The use of cloning sites in pLSP3

27 Selection of pLSP4





32 Confirmation of sense orientation of inserted genes in pLSP4,

pLSP5 and pLSP7 by PCR analysis

33 Confirmation of sense orientation of inserted genes in pLSP6 and

pLSP8 by PCR analysis


35 Restriction endonuclease analysis of pLSP7 and pLSP8





40 Restriction endonuclease analysis of pMB35-46 and pMB30

41 Selection of pLSP17 and pLSP18




45 Confirmation of inserted genes in pLSP13, pLSP15, pLSP20 and

pLSP23 transformation vectors by PCR analysis

46 Physical map of pLSP13 transformation vector

83

83

85

86

87

87

88

88

89

89

90

92

92

93

93

94

94

96

96

97

99

99

101

101

xvi

47 Physical maps of pLSP15, pLSP20 and pLSP23 transformation

vectors

48 Selection of A. tumefaciens LBA4404 carrying pLSP15 and pLSP20

49 Digestion of plasmid DNA from A. tumefaciens LBA4404 carrying

pLSP15 and pLSP20

50 Arabidopsis plants of two-leaf stage of on Basta-wet compost

51 Basta selection on Arabidopsis plants

52 Phenotypes of T1 plants

53 Leaf phenotype‟s of Arabidopsis plant

54 Genomic DNA of T1 transgenic Arabidopsis plants

55 PCR analysis of T1PHB plants by using primers BAR2-F and

BAR2-R

56 PCR analysis of T1PHBV plants by using primers BAR2-F and

BAR2-R

57 Screening of phbA gene in T1PHB plants by using primers

PCRphbA2-F and PCRphbA2-R

58 Detection of phbB gene in T1PHB plants by using primers

PCRphbB2-F and PCRphbB2-R

59 PCR screening of T1PHB plants for phbC gene by using primers

PCRphbC2-F and PCRphbC2-R

60 PCR analysis of T1PHBV plants for the detection of bktB (A) and

phbB (B) genes

61 PCR analysis of T1PHBV plants for the detection of tdcB (A) and

phbC (B) genes

62 Dot blot hybridization of genomic DNA from T1PHB and T1PHBV

plants

63 Southern blot analysis of T1PHB plants

64 Southern blot analysis of T1PHBV plants

65 Integration of phbA gene in T1PHB plants by Southern analysis

66 Integration of bktB gene in T1PHBV plants by southern analysis

102

104

104

106

106

107

109

111

113

114

114

114

115

115

116

118

119

120

122

123

xvii

67 Epifluorescence microscopy of PHB granules in the leaf of plants

stained with Nile Blue A

68 Nile Blue A staining for 16 weeks old of T1PHB plant

69 PCR analysis of Basta-resistant oil palm embryogenic calli using

primers POR12 and POR38

70 PCR analysis of Bar gene in Basta-resistant oil palm embryogenic

calli

71 PCR analysis of phbA, phbB and phbC genes in Basta-resistant oil

palm embryogenic calli

125

126

128

128

129

xviii

LIST OF ABBREVIATIONS

ACP acyl carrier protein

btkB gene coding for 3-ketothiolase

Bar gene coding for phosphinothricin acetyltransferase

CaCl2 calsium chloride

CaMV35S cauliflower mosaic virus 35S

CoA coenzyme A

CTAB cetyltrimethylammonium bromide

2,4-D 2,4-dichlorophenoxyacetic acid

DIG digoxigenin

DMSO dimetyl sulfoxide

dNTP deoxynicotinamide triphosphate

dwt dry weight

EDTA ethylenediaminetetra acetic acid

fwt fresh weight

3HB 3-hydroxybutyrate

HCL hydrochloric acid

3HV 3-hydroxyvalerate

IBA indole-3-butyric acid

ilvA gene coding for threonine deaminase

KCL potassium chloride

KOH potassium hydroxide

LB left border

LSP1 leaf-specific promoter

MCL medium-chain-length

xix

MgC12 magnesium chloride

MgSO4 magnesium sulfate

MPOB Malaysian Palm Oil Board

MS murashige and skoog

NAA -naphthaleneacetic acid

NaCl sodium chloride

NaOH sodium hydroxide

Nos gene coding for nopaline syntase

PCR polymerase chain reaction

PDC puruvate dehydrogenase complex

PHA polyhydroxyalkanoate

phbA gene coding for 3-ketothiolase

phbB gene coding for acetoacetyl-CoA reductase

phbC gene coding for PHA synthase

PHB polyhydroxybutyrate

PHBV polyhydroxybutyrate-co-hydroxyvalerate

PRP pathogen-related protein

RB right border

RB7MAR RB7 matrix attachment region

RbCl2 rubidium chloride

SCL short-chain-length

SDS sodium dedocyl sulfate

TAG triacylglycerols

TCA tricarboxylic acid

tdcB gene coding for threonine dehydratase

xx

T-DNA transferable-DNA

Tp transit peptide

CHAPTER 1

INTRODUCTION

Palm oil is the second largest source of edible oil after soy oil in the world, which

contributes 23.18 million tonnes (19.8%) of the total world‟s production of oils and

fats (Pushparajah, 2001). Since 1990, Malaysia is the largest producer of palm oil,

contributing about 11.8 million tonnes or 50.9% of the total production, while

Indonesia produced about 7.5 million tonnes or 32.3%. Malaysia is also the world‟s

largest exporter of palm oil, accounting for about 61.1% or 10.62 million tonnes of

the total exports of 17.37 million tonnes in 2001. Palm oil has a wide range of

applications, about 80% are used for food industries such as cooking oil, shortenings,

margarines, ice creams and cookies while the rest are used as feedstock for a number

of non-food applications such a diesel fuel substitute, drilling mud, soaps and

epoxidised palm oil products, polyols, polyurethanes and polyacrylates (Salmiah,

2000). In addition to the cost benefit and its multiuse, palm oil was also proven to be

nutritious. Studies have indicated that palm oil lowers serum cholesterol levels to the

same degree as sunflower oil which is rich in unsaturated fatty acid (Manorama and

Rukmini, 1992). Palm oil does not increase the plasma cholesterol or low-density

lipoprotein (LDL), where increased level could be harmful but on the other hand, it

increases the high-density lipoprotein (HDL), which protects against heart disease

(Sundram et al., 1992). Furthermore, palm oil has anti-tumor effects especially with

the presence of high levels of vitamin E, tocopherols and tocotrienols (Nesretnam et

al., 1992).

2

The challenge that the oil palm industry will face in the 21st century is the ability to

maintain profitability in the face of labor shortage and limited land resources. World

production of palm oil was projected to double from the year 2000 to year 2020 with

a total production exceeding 40 million tones and it is expected that nearly 26% of

the world's oil and fat demand will be obtained from palm oil (Oil World, 2020:

www.mpob.gov.my). Due to this projected demand, it is important to increase the

yield of oil palm as well as to improve the palm oil quality at a better rate than that

has been achieved by conventional breeding. Accordingly, in early 1990s, the genetic

engineering programme has been initiated at Malaysian Palm Oil Board (MPOB),

then Palm Oil Research Institute of Malaysia (PORIM), to fulfill this demand (Cheah

et al., 1995). The primary strategy of this programme is to produce transgenic oil

palm with high oleic oil content (Cheah et al., 1995). Besides increasing oleic acid,

modification of oil quality and production of novel high value products have also

been targeted. These targets include increasing stearic acid, synthesizing palmitoleic

acid, synthesizing ricinoleic acid and producing biodegradable plastics (Parveez et

al., 1999). Currently, biodegradable plastics such as polyhydroxyalkanoates (PHAs)

are being produced under the trade name Biopol™ by Monsanto in a two-stage

glucose / propionate fed batch fermentation process using Ralstonia eutropha

(Kessler et al., 2001). The economics of the manufacturing process is still a major

barrier to the widespread use of PHAs. An alternative strategy for lowering

production costs which has been proposed is to develop transgenic plants that

produce PHAs. This strategy could lead to considerably cheaper production of PHAs

because production from plants does not require expensive fermentation equipments

and substrates (Poirier et al., 1992).

3

In the last 10 years, MPOB has made significant progress on the production of PHAs

[polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV)]

in transgenic oil palm, such as development of a transformation system using

biolistic-mediated method (Parveez, 1998), isolation of tissue-specific promoters

such as mesocarp and leaf specific promoters (Siti Nor Akmar et al., 2001),

construction of PHB and PHBV transformation vectors driven by constitutives and

mesocarp-specific promoters (Masani et al., 2001; Masani and Parveez, 2003),

transformation and production of transgenic oil palm (Parveez, 2003).

In this study, transformation of oil palm and Arabidopsis with the PHB and PHBV

genes with the goal of accumulating bioplastics in the leaves was initiated. The

objectives of this study are:

1. To construct PHB and PHBV transformation vectors driven by oil palm leaf-

specific promoter (LSP1).

2. To transform PHB and PHBV transformation vectors into Arabidopsis and oil

palm.

3. To confirm integration of PHB and PHBV genes in transgenic Arabidopsis

and oil palm by molecular analysis.

4

CHAPTER 2

LITERATURE REVIEW

2.1 Polyhydroxyalkanoates (PHAs)

Polyesters like polyhydroxyalkanoates (PHAs) are a large group of polymers of 3-

(R)-hydroxy fatty acids linked by an ester bond between the hydroxyl group and the

carboxy group of an adjacent monomer (Sudesh et al., 2000). PHAs are divided into

two groups, i.e. short-chain-length PHAs (SCL-PHAs) that is comprised of PHB and

the copolymer PHBV; and medium-chain-length PHAs (MCL-PHAs) that consists of

3-(R)-hydroxyhexanoate / 3-(R)-hydroxytetradecanoate monomers (Figure 1). PHAs

are osmotically inert compounds and they are optically active, biocompatible,

biodegradable and hydrophobic. The properties of PHAs vary with their

compositions (Table 1). PHAs are mainly composed of R-(-)-3-hydroxyalkanoic

acid monomers. Each type of PHA generally consists of 1000-10000 monomers,

but most are synthesized by SCL monomers. There are many different types of PHAs

that are characterized by chain length, type of functional group and degree of

unsaturated bonds. A higher degree of unsaturation increases the rubber qualities of

a polymer, and different functional groups change the physical and chemical

properties of a polymer (Madison and Huisman, 1999).

2.1.1 Polyhydroxybutyrate (PHB)

Most of the knowledge on bacterial production of PHB is generated from Ralstonia

eutropha (formerly known as Alcaligenes eutrophus) because it naturally produces

PHB and can produce up to 85% of its dry weight (dwt) when grown in media

containing excess glucose.

universiti putra malaysia production of specialized

Documents