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Construction of Plant Transformation Cassettes and

Heterologous Expression of Genes Encoding Cyanamide

Hydratase and Antifungal Proteins

Thesis submittedto the Pondicherry University for the Degree of

Doctor of Philosophy

S. ISAAC KlRUBAKARAN

Departmentof Biotechnology

School of Life Sciences

Pondicherry University

Puducherry

605

014

INDIA

MAY

-m

PRY

%,,L

PONDICHERRY UNIVERSITY

DEPARTMENT OF BIOTECHNOLOGY

SCHOOL OF LIFE SCIENCES

PUDUCHERRY

605

014,

INDIA

Dr.

Sakthivel

Reader

Head

CERTIFICATE

Cen~fiedthat th~srhesik entltlcd

"Construction of plant trvnsformation

cassettes and heterologous expression of genes encoding cyanamide hydrahue

and antifungal proteins"

record CISresearch work done by ~hccandldulc,

Mr.

Isaac Kirubakaran,

durlng the perlod

h~rrtudy In lhc Dcpalinlent of

Blchechnolopy. Pondicherry llni\err~ty,Puduchcrry, under

aupervlrion atid thnl it

has not formed prevlourly the bakik lor the awud

any degree, dlplorna.

asrnciateship or fellowrh~p.

Puducherry

Date:

LL-/

(N.

Sakthivel)

Phone:91413.2654430

(off.)

Fax:

91.413-2655715/2655265

Etnnil:[email protected]

hereby declare that the work prescirted in

his

Ihesis has ken c:~rriedour

me under the guidance and

supervision

Dr.

SAKTHIVEI..

Dcpann~cnl

Biotechnology. School of Life Sciences, Pondicherry Clniversily,Puducherry

b0.5

014

and this work ha%not been whrnitted elsewhere for any other degrce.

Puducherry

Date:

DEDICATED

BELOVED

PARENTS

ACKNOWLEDGEMENT

During the years as a Ph.D. btudent, rirany ~xoplehaye

contributed

to\vanls ;I

111e:lnin~fill:lnd succeshflll ourccvnr of

I~IS

rcsc:~rcl~l)~oje~.t.tlo\vevc.r :I \,cry I'cw Ii;~v'

stood :~pi~rtby sheer virtue ol'cxccllrnce

ill

~hcirlieltl nritl

have Iwcn fortuli:~lcto

bcncfit iron] their guidance.

hereby express my deep sense of gratitudr to niy guide

Dr.

Sakthivcl,

!'or his

expert guidance. constant encour:lgement and ide;~swhich rchulted in the culrnini~tion

of this thesis.

reserve my special thanks fro111the hottom

my he:lrt to Prof.

Jayarhandran

for the support and encouragement.

am grateful to

Dr.

Arul, Dr.

Sudharani, Dr.

Arurnugarn,

Dr. Lata Shukla,

and

Dr.

Prashant

for their co-oprition.

wish to record my slncere thanks to the doctoral committee members

Pruf.

Jayachandran

and

Dr.

Parthasarathy.

express my deep sense of gratitude to

Prof.

Muthukrishnan.

Kansas State

University. Department of Biochemistry. Kansa~,USA for providing me the harley

and wheat cDNA clones and cnh and chitinase antiserum and

Prol. Tranquet Olivier,

Scientific Leader. Antibody production unit, Institut

National de la Recherche Agronomique

(INRA).

Unite Biopolymeres, Interactions,

Assemblages, Nantes. Cedex, France for providing me wheat LTP antiserum.

am indebted to

Dr.

Ulaganathan,

Associate Professor. Centre for Plant

Molecular Biology, Osmania University. Hyderahad, for helping

with the plant

transformation work.

really grateful to

Ms.

Mubarak Begum.

Ms.

Snehalatha Rani and

Mr.

Shashikanth Research Scholm. Centre for Plant Molecular Bicioloyy.

Osnlania University. tlyderah:~d,for helping Ine in le;~rning;\lid prcweetling with plant

tr;~nsforlnation.rtuJies.

My ainccrt tli;lnks to hIr.

11.

Iialachantlran. Dioi~lfor~~i;~tirsCcntre. llnivcrsity of

h1adr.d~:11iJ >IS.

Shanthi, Bicli~~formatlcrC'entrc. Pt~ndiclicrryll~~ivcrsityfor

helping me in molecular modeling studics.

thank all my fellow collengues, seniors and juniors, whoni

might have left out due

to oversight. hut whose best wishes have ;~lwaysencclur~gednlc.

would like to thank our office staffs Mr.

('.

Iiul~~krishnan,Mr. P. Rnn~alinguni,

Mr. Rarnarnuorlhy, Mr.

Kannuyiram, hlrs.

Valli and

Mrs.

Kalivnnl, for their

co-operation and help. My speci:~lthanks goes to all the non-teaching staff menlhcrs

of Pondicherry University who helped me throughout the study.

gratefully acknowledge Department of Science and Technology (1)ST). New

Delh~for providing me with Junior Research Fellowship.

Last and definitely by no means the least.

would like to rnake a mention of

parents, all my family members and well wishers, who have ken eagerly looking

forward to this personal milestone. However, they have alstr been very patient with

my failures

and

have always extended their support

providing

me with a driving force

for the successful completion of this research work.

Date:

jl,i,l

j)ph*

(S. ISAAC KIRUBAKAKAN)

CONTENTS

CHAPTER

INTRODUCTION

REVIEW OFLITERATURE

MATERIALS ANDMETHODS

RESULTS

DISCUSSION

SUMMARY

REFERENCES

Page

No.

LIST OFABBREVIATIONS

BAP

BClP

BLAST

BSA

cDNA

DNA

DTT

dNTP

EDTA

EPSPS

GlcNAc

GLP

IAA

IPTG

kDa

kHz

LTP

MEGA

three-dimenaion:~l

two-dimension:~l

6-henzyl-a11iirio-pur1ne

5-bromo-4-clilorc)-3-indolyl

pli~ispl~;rtc

basic local ;ilignmcnt s'n~ch

t<r>l

base pair

bovine scrum albumin

complementmy DNA

centimeter

deoxyrihonucleic acid

Dithiothreitol

2'deoxynucleotide S'trlphosphate

ethylenediaminetetraacetic

arid

5-enol pyruvyl shikimate-.%phosphate synthase

endoplasmic reticulum

N-acetyl-D-glucosamine

gerrnin-like protein

hour

hygromycin phosphotransferase

indole-3-aceticacid

isopropyl P-D-thiogalactoside

jasmonic acid

kilo

base

kilo Dalton

kilo hertz

kilo volt

Luria Bertani

lipid transfer protein

milli Ampere

molecular evolutionary genetics and sequence alignment tool

milligram

min

mhl

%IS

NAA

NBT

nni

nmol

NMR

npt

ns-LTP

par

PBS

PCR

PDA

RMSD

ROS

rl'

SARE

SDS

SDS-PAGE

SEM

SSC

TAE

T-DNA

TEMED

minutes

milliliter

niilli~iieter

niilli~iiolar

Murilhhige Skwg

naphthalene acetic :wid

nitrohlue terr~zuliuln

nanogram

neighhour-jolning

nanometer

nanornole

nuclear magnetic

resonance

neornycin phosphorri~n~icr;~~

non-specific lipid rr;in,lcr protein

optical density

phosphinothricin N-acetyl-rr:insferase

phosphate huffer saline

polynierase chain reaction

potato dextrose agar

pathogenesis-related

root mean square dev~alion

reactive oxygen species

revolulions per mlnute

salicylic

acid

SA rehponsive element

sodium dodecyl sulphate

sodium dodecyl sulphate-polyacrylamidc gel electrophoresis

scanning electron microscope

saline sodium citr:~te

Tris acetate EDTA

transfer DNA

Tris EDTA

tetmethylelhylenediamine

tumor

inducing

TLP

UPGMA

\\,/v

WGA

YEB

thaumatin-like protein

unweighted pair gmup

cuithl~lcticmean analysis

lllt~l~i~ll~t

vcrlt

volil~nc

vc>lumc

weight

volume

\vhc;rt gcrm :~:glutiriin

yeast extract hrorh

microgram

microlitre

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