UNIVERSITI PUTRA MALAYSIA

KHEW CHOY YUEN

FBSB 2013 15

FUNCTIONAL ANALYSES OF PUTATIVE BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 AND PUTATIVE ETHYLENE TRANSCRIPTIONAL COACTIVATOR FROM MALAYSIAN

INDICA RICE VARIETY (Oryza sativa L.) MR219

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FUNCTIONAL ANALYSES OF PUTATIVE BRASSINOSTEROID

INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 AND PUTATIVE

ETHYLENE TRANSCRIPTIONAL COACTIVATOR FROM MALAYSIAN

INDICA RICE VARIETY (Oryza sativa L.) MR219

By

KHEW CHOY YUEN

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

in Fulfillment of the Requirements for the Degree of Master of Science

December 2013

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COPYRIGHT

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Copyright © Universiti Putra Malaysia

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

of the requirement for the degree of Master of Science

FUNCTIONAL ANALYSES OF PUTATIVE BRASSINOSTEROID

INSENSITIVE 1- ASSOCIATED RECEPTOR KINASE 1 AND PUTATIVE

ETHYLENE TRANSCRIPTIONAL COACTIVATOR FROM MALAYSIA

INDICA RICE VARIETY (Oryza sativa L.) MR219

By

KHEW CHOY YUEN

December 2013

Chairman: Associate Professor Ho Chai Ling, PhD

Faculty: Biotechnology and Biomolecular Sciences

Phytohormones integrate metabolic and developmental signals in plants. Active

BRI1-Associated Kinase I (BAK1) is required to interact with Brassinosteroid

Insensitive 1 for brassinosteroid perception and signal transduction that regulate

plant growth and development while ethylene transcriptional co-activator (ETC) acts

on ethylene-responsive transcription factors, which positively control the expression

of ethylene-responsive plant genes to elicit a response appropriate to the stimulus.

The aim of this study is to annotate the functions of two putative phytohormone-

related transcripts, OsI-BAK1 and OsI-ETC from a Malaysian rice variety, MR219.

Functional annotation of phytohormone-related genes, which are closely related to

yield, may offer new solution to increase rice yield in the country. In the present

study, the function of OsI-BAK1 and OsI-ETC were investigated using a transgenic

approach through gene overexpression and RNAi silencing. In this study, one

overexpression vector, pMDC32-BAK1 and two RNAi vectors, pANDA-BAK1 and

pANDA-ETC were constructed. These vectors, together with an existing

overexpression vector pMDC32-ETC were transformed into rice MR219 through

Agrobacterium-mediated transformation, respectively. After plant transformation,

the phenotypic and molecular changes on the transgenic rice plants were analyzed.

The transgenic rice plants overexpressing OsI-BAK1 had corrugated and twisted

leaves at the early stage of growth. Histological analysis of these leaves showed

some differences in their cell arrangement in the vascular tissue compared to that of

the untransformed plants. The bulliform cells which were enlarged and plugged deep

into leaf epidermis in these leaves could have contributed to the abnormal

morphology of the leaf blades. Silencing of OsI-BAK1 in rice plants produced about

two folds higher number of undeveloped green and unfilled grains compared to

untransformed plants. Histological analyses were conducted on the rice hull, culms

and leaves in OsI-BAK1RNAi plants. The longitudinal section of rice hull from OsI-

BAK1RNAi plants showed that the embryo was either absent or retarded in its

development. Taken together, OsI-BAK1 gene may play an important role in the

developmental processes of seeds and leaf cell arrangement. Embryogenic calli

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transformed with the overexpression vector harboring OsI-ETC could not regenerate.

The constitutive expression of OsI-ETC in rice might increase the production of

ethylene and inhibit shoot regeneration. The transgenic plant containing a construct

designed for RNAi silencing of OsI-ETC showed no obvious morphological

difference compared to that of untransformed plant. As it is possible that this

construct did not actually function and the plant may not have been RNA silenced to

give obvious phenotypic changes. Functional analysis of OsI-BAK1 and OsI-ETC in

this study has shed light on their biological functions in rice.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

ANALISIS FUNGSI PUTATIF “BRASSINOSTEROID INSENSITIVE 1-

ASSOCIATED RECEPTOR KINASE 1” DAN PUTATIF “ETHYLENE

TRANSCRIPTIONAL COACTIVATOR” DARIPADA PADI MALAYSIA

INDICA VARIETI (Oryza sativa L.) MR219

Oleh

KHEW CHOY YUEN

Disember 2013

Pengerusi: Profesor Madya Dr. Ho Chai Ling, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Fitohormon mengintegrasikan isyarat metabolik dan perkembangan dalam tumbuhan.

Brassinosteroid Sensitif 1 (BRI1) yang aktif diperlukan untuk berinteraksi dengan

BRI1-Associated kinase I (BAK1) untuk persepsi brassinosteroid dan transduksi

isyarat yang mengawalatur pertumbuhan dan perkembangan tumbuhan sementara

ko-aktivator transkripsi etilena (ETC) bertindak ke atas faktor transkripsi yang

respon terhadap etilena, dan mengawal ekspresi gen secara positif terhadap etilena

untuk mendapatkan tindak balas yang sesuai dengan rangsangan. Tujuan kajian ini

adalah untuk menganotasikan fungsi dua transkrip yang berkaitan dengan fitohormon

iaitu, OsI-BAK1 dan OsI-ETC daripada padi Malaysia indica varieti MR219. Anotasi

fungsi gen yang berkaitan dengan fitohormon yang mempengaruhi hasil padi

menawarkan penyelesaian baru untuk meningkatkan penghasilan padi di negara ini.

Dalam kajian ini, penyelidikan atas fungsi OsI-BAK1 dan OsI-ETC telah dijalankan

menggunakan pendekatan transgenik melalui pengzahiran gene berlebihan dan RNAi

perendam gen. Dalam kajian ini, satu vektor pengzahiran gen berlebihan, pMDC32-

BAK1 dan dua vektor RNAi, pANDA-BAK1 dan pANDA-ETC; telah dibina.

Vektor-vektor ini bersama satu vektor pengzahiran gene berlebihan yang sedia ada,

iaitu pMDC32-ETC telah ditransform ke dalam padi MR219 menggunakan kaedah

perantaraan Agrobacterium. Selepas transformasi tumbuhan, perubahan fenotip dan

molekul atas tanaman padi transgenik telah dianalisis. Tanaman padi transgenik yang

menghasilkan OsI-BAK1 berlebihan menunjukkan fenotip daun berkedut dan

berpintal pada peringkat awal pertumbuhan. Analisis histologi daun menunjukkan

susunan sel dalam tisu vaskular yang tidak teratur berbanding dengan tumbuhan

tidak ditransform. Sel-sel bulliform didapati bersaiz besar dan terbenam di dalam

epidermis daun yang berkemungkinan menyebabkan ketidaknormalan morfologi

pada bahagian daun. Perendam gen OsI-BAK1 dalam padi menghasilkan bilangan

biji padi yang hijau dan tidak berisi iaitu dua kali ganda tinggi berbanding dengan

tumbuhan yang tidak ditransformkan. Analisis histologi telah dijalankan untuk

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mengkaji anatomi biji, batang dan daun padi OsI-BAK1RNAi. Keratan melintang

biji padi menunjukkan bahawa biji tersebut tidak mempunyai embrio atau

mempunyai embrio yang perkembangannya terbantut. Secara keseluruhannya, gen

OsI-BAK1 mungkin memainkan peranan yang penting dalam proses perkembangan

biji dan susunan sel daun. Penghasilan OsI-ETC yang berlebihan dalam padi

menyebabkan kegagalan kalus embriogenik untuk menghasilkan pucuk. Ekspresi

konstitutif OsI-ETC dalam padi mungkin meningkatkan pengeluaran etilena dan

membantutkan regenerasi pucuk. Tanaman padi transgenik yang mengandungi RNAi

konstruk untuk perendam gene OsI-ETC tidak menunjukkan perbezaan morfologi

yang jelas berbanding dengan tumbuhan yang tidak ditransformkan. Ini mungkin

disebabkan konstruk ini tidak berfungsi dan perendaman ekspresi gen tidak berlaku

pada pokok OsI-ETCRNAi untuk memberi perubahan fenotip yang jelas. Analisis

fungsi OsI-BAK1 dan OsI-ETC dalam kajian ini telah memberi penjelasan mengenai

fungsi biologi mereka dalam padi.

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ACKNOWLEDGEMENTS

I would like to express the deepest appreciation to my committee chairman,

Associate Professor Ho Chai Ling for the useful comments, remarks and engagement

through the learning process of this master thesis. Her continually encouragement

and guidance in this study have materialized this project. Furthermore I would like to

thank my committee members, Associate Professor Parameswari and Associate

Professor Wong Hann Ling, whose contribution in stimulating suggestions and

support in regard to research. I wish to thank the Institute of Tropical Agriculture

(ITA) for providing space and facilities in the transgenic green house and I would

like to extend my gratitude to my lab mates in Biotech 3: Shahanaz, Rouh San, Yung

Chie, Keat Ai, Anis, Syin Yiing and Ee leen for their technical assistance and the

brainstorming discussion we had together. Last but not least, I would like to dedicate

this thesis to my family for their endless love and trust to make this thesis a reality.

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I certify that a Thesis Examination Committee has met on 9 December 2013 to

conduct the final examination of Khew Choy Yuen on her Master of Science thesis

entitled “Functional Analyses of Putative Brassinosteroid Insensitive 1- associated

Receptor Kinase 1 and Putative Ethylene Transcriptional Coactivator from

Malaysian Indica Rice Variety (Oryza sativa L.) MR219” in accordance with the

Universities and University Colleges Act 1971 and the Constitution of the Universiti

Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that the

student be awarded the degree of Master of Science.

Members of the Thesis Examination Committee were as follows:

Tan Wen Siang, PhD

Professor

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Janna Ong Abdullah, PhD

Associate Professor

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal examiner)

Rozi Mohamed, PhD

Associate Professor

Faculty Perhutanan

Universiti Putra Malaysia

(Internal examiner)

Jennifer Ann Harikrishna, PhD

Professor

Faculty of Science

Universiti Malaya

(External examiner)

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 17 February 2014

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

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

members of the Supervisory Committee were as follows:

Ho Chai Ling, PhD

Associate Professor

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Parameswari Namasivayam, PhD

Associate Professor

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Wong Hann Ling, PhD

Associate Professor

Faculty of Science

Universiti Tunku Abdul Rahman

(Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other degree

at any other institutions;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published in book

form;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: Date: 2 JUNE 2014

Name and Matric No.: KHEW CHOY YUEN (GS27030)

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the wiritng of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: Signature:

Name of

Chairman of

Supervisory

Committee:

Ho Chai Ling, PhD

Name of

Member of

Supervisory

Committee:

Parameswari

Namasivayam, PhD

Signature:

Name of

Member of

Supervisory

Committee:

Wong Hann Ling, PhD

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xvii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Rice plant 3

2.2 The importance of rice as a global food crop 3

2.3 The importance of rice to plant research 4

2.4 Malaysian rice cultivar-MR219 5

2.5 The role of plant hormones in plant 5

2.5.1 Brassinosteroids 5

2.5.2 Brassinosteroid Insensitive 1-associated Receptor Kinase 1

(BAK1) 6

2.5.3 The effects of brassinosteroids in cell division 8

2.5.4 The effects of brassinosteroids in grain filling 9

2.6 Ethylene 10

2.6.1 The nature, occurrence and effects of ethylene 10

2.6.2 Ethylene Transcriptional Coactivator (ETC) 10

2.7 Genetic engineering and tissue culture of rice 12

2.7.1 Agrobacterium tumefaciens-mediated gene transformation 12

2.8 Gateway technology 14

2.8.1 Overexpression of transgene 15

2.8.2 Gene silencing (RNAi) 16

3 MATERIALS AND METHODS

3.1 Isolation of total RNA 18

3.2 Isolation of complete coding sequence of (CDS) of OsI-BAK1 19

3.2.1 cDNA synthesis 19

3.2.2 PCR amplification of OsI-BAK1 19

3.2.3 Purification of PCR product 20

3.2.4 Cloning of PCR product 20

3.2.5 Verification of positive transformants 21

3.2.6 Plasmid DNA extraction 21

3.2.7 Sequencing 21

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3.3 Construction of a plant expression plasmid overexpressing

OsI-BAK1 with LR recombination reaction 21

3.4 Construct preparation for RNAi studies 22

3.5 Transformation of Agrobacterium tumefaciens LBA4404 by

electroporation 23

3.5.1 Preparation of Agrobacterium competent cells 23

3.5.2 Transformation into Agrobacterium competent cells by

electroporation 23

3.5.3 Plasmid isolation from Agrobacterium cells 24

3.6 Rice planting 24

3.7 Seeds sterilization and callus induction 25

3.8 Basal medium and optimization of plant growth hormones 25

3.9 Rooting and acclimatization of regenerated plantlets 25

3.10 Agrobacterium-mediated transformation into rice 25

3.10.1 Determination of minimal inhibitory concentration

(MIC) of hygromycin B for MR219 rice calli 25

3.10.2 Agrobacterium infection and selection of transgenic calli 26

3.11 Screening of the putative transformants 26

3.11.1 Small scale genomic DNA extractions from rice leave 26

3.11.2 PCR verification of the putative transgenic rice 27

3.12 Reverse transcription polymerase chain reaction (RT-PCR)

analysis of OsI-BAK1RNAi transformants 27

3.12.1 Synthesis of first strand cDNA 27

3.12.2 RT-PCR 28

3.13 Phenotypic analyses of transgenic rice 28

3.14 Lamina joint assay 29

3.15 Histological analysis 29

3 RESULTS AND DISCUSSION

4.1 Functional analysis of putative brassinosteroid insensitive 1-associated

kinase 1 (OsI-BAK1) 30

4.1.1 Sequence analysis 30

4.1.2 Overexpression of a putative OsI-BAK1 36

4.1.2.1 Preparation of an overexpression construct 36

4. 1.2.2 Agrobacterium-mediated transformation of

overexpression OsI-BAK1 into rice variety Oryza

sativa indica variety MR219 38

4.1.2.3 Molecular analysis of OsI-BAK135S transformants 40

4.1.2.3.1 PCR screening of putative OsI-BAK135S

transformants 40

4.1.2.4 Morphological analysis of OsI-BAK135S plants 42

4.1.2.5 Histological analysis of OsI-BAK135S plants 44

4. 1.3 RNA interference of OsI-BAK1 46

4.1.3.1 Agrobacterium-mediated transformation of RNAi

OsI-BAK1 into Oryza sativa indica rice variety

MR219 48

4.1.3.2 Molecular analysis of OsI-BAK1RNAi transformants 49

4.1.3.3 Semiquantitative RT-PCR analysis of OsI-BAK1RNAi

plants 51

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4.1.3.4 Lamina joint assay 51

4.1.3.5 Morphological analysis of OsI-BAK1RNAi plants 53

4.1.3.6 Histological analysis of OsI-BAK1RNAi 56

4.2 Functional analysis of Ethylene Transcriptional co-activator

(OsI-ETC) 63

4.2.1 Sequence analysis 63

4. 2.2 Agrobacterium-mediated transformation of pMDC32-ETC

into rice variety Oryza sativa indica variety MR219 67

4.2.3 Construct preparation for RNAi studies of pANDA- ETC 69

4.2.3.1 RNAi construct 69

4.2.4 PCR screening of putative OsI-ETCRNAi transformant

at T0 generation 70

4.2.5 Morphological analysis of OsI-ETCRNAi plant 71

5 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR

FUTURE STUDIES 73

REFERENCES 75

APPENDICES 102

BIODATA OF STUDENT 114