Final Report Nbri.maniSH SINGH

8/8/2019 Final Report Nbri.maniSH SINGH

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A

Project Report

On

Cloning of MYB11 transcription factor from Arabidopsis

and development of construct for plant transformation

Submitted To

PUNJAB TECHNICAL UNIVERSITY

In the partial fulfilment of the requirement for the award of

Degree of

Master of Science

In

Biotechnology

Under The Guidance Of

Dr. P.K. TRIVEDI

Work carried out at:

Molecular Biology and Genetic Engineering Division

National Botanical Research Institute,Council of Scientific and Industrial Research, ,

Lucknow-226001



DE L T B CANDIDATE

I, Manish Kumar Singh hereby declare that this dissertation report entitled, ³Cl i of

MYB transcri tion factor from Arabi opsis and development construct for plant

transformation´ is carried out by me at National Botanical Research Institute, Lucknow,

under the guidance of Dr. P.K. Trivedi, Scientist, Molecular Biology and Genetic

Engineering Division, for partial fulfilment for the award of the degree of Master of Science

in Biotechnology at Department of Biotechnology, Agra institute of Management &

Technology , Agra (U.P)

DATE Manish Kumar Singh

PLACE:



CONTENTS

1-Introduction

2-Review of literature

3-Material and method

3.1-isolation of MYB11 cDNA

3.1.1-RNA isolation

3.1.2-RT PCR of total RNA

3.1.3-Purification of PCR products

3.2-Digestion of Plasmid vector

3.3-Digestion of AtMYB11cDNA

3.4-Ligation of vector and purified At MYB11 CDNA

3.5-Transformation of ligation mixture

3.5.1- Preperation of E.Coli DH5 competent cells

3.5.2-Transformation of E.coli with ligation mixture

3.6-Positive colony selection through colony PCR

3.7-Clone confirmation

3.7.1-Plasmid DNA isolation from positive colonies

3.7.2-PCR amplification

3.8-Introduction of AtMYB11 cassette in agrobacterium cells

3.8.1-Preperation of Agrobacterium competent cells

3, 8, 2- Freeze-thaw transformation of agrobacterium competent cells with MYB11

construct



3.8.2-Screening of positive colonies through colony PCR

3.9-Transformation of tobacco through AtMYB11 construct

4-Results

5-Conclusions

6-HPLC analysis

7- References



ACKNOWLEDGEMENTS

This project training is all meaningless without paying gratitude to the people who made a

great support to me and without them it is impossible to complete my work.

I am extremely grateful to Dr. K.C Gupta, Director, NBRI, for accepting me as a project

trainee.

I would like to make a sincere confession and at the same time express gratitude to Dr. P.K.

Trivedi for granting me permission to undertake my dissertation for partial fulfillment of

Master of science degree in Biotechnology.

I am greatly thankful to Dr . Pravendra Nath Head, Plant Gene Expression Lab, Centre

for Plant Molecular Biology (CPMB), National Botanical Research Institute (NBRI),

Lucknow for his excellent guidance, constant support, and encouragement throughout my

work.

I am also greatly thankful to Dr. A.P. Sane Scientist and Dr. Vidhu Sane, Scientist, Plant

Gene Expression Lab for providing constant source of encouragement at each and all stepsof this work.

I am highly obliged and owe my special thanks to my guide Mr. Ashutosh Pandey for his

able guidance, valuable support, keen interest, constant painstaking efforts, meticulous

supervision, constructive criticism and innovative scientific orientation in my dissertation

work.

I would like to thanks to Mr.Prashant Mishra, Mr.Devesh Shuk la Mr. Nehal Akhtar,

Mr. Manish Ti ari,, who helped me in my practical work and understanding the problem

through discussions.

Special thanks to Mr. Rajesh Kumar Singh, Ms Aparna Mishra, Mr. Rakesh Kumar

Upadhayaya, Ms.Parul Gupta, Ms Smita Kumar, Ms. Saumya Pathak, Ms.Depika

Sharma, Mr. Amar Pal Singh, Mr. Saurabh Pandey, Ms. Rajluxmi, and Ms. Samatha

Reddy.

Mr. Ram Avadh is highly acknowledged for autoclaving, routine lab works and

maintaining plants in glass house and Mr. Ram Kumar is acknowledged for his glassware

washing.

Finally, special and cordial thanks to my beloved respected parents . I have no words to

thank them for their care, support, encouragement, blessings and prayers that help me

accomplishing this work. Their presences in my mind always inspire me to do better with full

devotion.

MANISH SINGH

M.Sc. biotechnology



ABBBR EVIATION

bp Base pair

CIA Chloroform:Isoamylalcohol (24:1)

DNA Deoxyribonucleic acid

dNTP Deoxyribonu clioside triphosphate

EDTA Ethylene diamine tetra acetic acid

EtBr Ethidium bromide

Kbp Kilo base pair

LA Luria agar

LB Luria broth

RNA Ribonucleic acid

RNAse Ribonuclease -A

DNAse Deoxyribonuclease

TAE Tris acetate EDTA

CTAB N-cetyl N, N, N-trimethyl ammonium bromide

SDS Sodium dodecyl sulphate

BME -marceptoethanol

YEB Yeast extract broth

CHS Chalcone Synthesis

CHI Chalcone Isomerase

F3H Flavonoied -3- Hydroxelase



Introduction

Flavonoids are the low molecular weight secondary metabolites found throughout the plant

kingdom. These secondary plant product play active role in various developmental processes,

biochemical processes as well as environmental response. These compounds synthesize

through phenyl propanoid pathway. The general phenyl propanoid pathway leads from

phenyl alanine to coumaroyl-coA and this conversion is initiated by the enzyme phenyl

alanine ammonia lyase (PAL). Phenyl propanoid pathway produces thousands of compounds

many of which are species-specific. An important branch leads to the production of

flavonoids including flavonols, anthocyanins and tannins. Chalcone synthase (CHS) is the

first committed enzyme of this pathway.

Flavonoids play an important role in plant as structural components (such as lignin),

protectants against biotic as well as abiotic stresses as they are antipathogenic,phytoalexins,

antioxidants and UV-absorbing compounds. They also paint flowers and fruits. Flavonoids

act as signalling molecule in plants. Recent studies indicate that flavonols are required for

male fertility and more specifically pollen tube growth in maize, petunia and tobacco, but

they are not essential in Arabidopsis.

Co-ordinate transcriptional control of biosynthetic genes emerges as a major mechanism

dictating the final levels of secondary metabolites in plant cells. This regulation of

biosynthetic pathways is achieved by specific transcription factors. Transcription factors are

sequence specific DNA-binding proteins that interact with the promoter regions of target

genes, and modulate the rate of initiation of mRNA synthesis by RNA polymerase II. These

proteins regulate gene transcription depending on tissue type and/or in response to internal

signals, for example plant hormones, and to external signals such as microbial elicitors or UV

light. External signals may induce production of internal signals. Transcription factors have

been isolated and characterized for two plant metabolic pathways, leading to biosynthesis of

flavonoids and of terpenoid indole alkaloids (TIA), respectively. End products of the

flavonoid biosynthesis pathway include the anthocyanin pigments. In various plant species it

has been shown that tissue-specific regulation of the structural genes involved in anthocyanin

biosynthesis is directly controlled by a combination of two distinct transcription factor

families with homology to the protein encoded by the vertebrate proto-oncogene c-MYB,

and the vertebrate basic-Helix-Loop-Helix (bHLH) protein encoded by the proto-oncogene c-

MYC, respectively .



Review of literature

Flavonoids form a large group of polyphenolic compounds that occur naturally in plants.

Based on their core structure, the aglycone, they can be grouped into several classes such as

chalcones, flavonones, isoflavones, flavonols, dihydroflavonols and anthocyanins. To date

more than 7000 flavonoids have been identified (Ververidis et al.2007 ). The large diversity

is attributable to single or combinatorial modification of aglycone such as glycosylation,

methylation, and acylation. As a group, flavonoids are involved in many aspects of plant

growth and development, such as pathogen resistance, pigment production, UV light

protection, pollen growth and seed coat development (Harborne1986). Several new roles of

these compounds are being established. Plant flavonoids are considered as natural regulator

of auxin efflux and consequent auxin polar transport (Brown et al 2001).

There is increasing evidences that flavonoids, in particular those belonging to the class of

flavonols (Such as Kaempherol and quercitin), are potentially health protecting compounds as

result of their high antioxidant activity (Rice Evans et al 1995), and their ability, in vitro to

induce human protective enzyme systems (Shih et al 2000). Based on these observations it

was postulated that flavonoids may offer protection against coronary diseases and cancer

(Trevisanto et al 2000). In addition several epidemiological studies have suggested a direct

correlation between cardioprotection and consumption of flavonols from dietary sources like

onion, apple and tea (Keli et al 1996).

Based on these studies there are growing interests to develop food crops with elevated levels

of these flavonoids as many commonly consumed food stuffs are deficient in these

phytoceuticals. Tight temporal and spatial regulation of different genes of the

phenypropanoid pathway is responsible for lower and tissue specific accumulation of these

compounds. As these compounds get their origin from phenylpropanoid pathway (Figure1),

different genes can be targeted to be modified in their expression. Chalcone Isomerase (CHI)

is an important enzyme for biosynthesis of Naringenin, a substrate upstream for production of

flavonols. Overexpression of Petunia CHI in tomato resulted a 78-fold increase in flavonol

contents of fruits (Muir et al 2001). Fukusaki et al (2004) and Nishihara et al (2005) have

reported that structural genes in the flavonoid biosynthetic pathway, the CHS and CHI genes

were strongly suppressed by RNAi in Transgenic Torenia and Tobacco plants, respectively

and displayed a decrease in Flower color intensity.



Within plant cells, some genes are expressed constitutively whereas others respond to

specific stimuli. Both patterns depend on the interaction of transcription factors required for

gene expression, and they are important in the regulation of cell activities. Therefore,

alteration in the expression of transcription factor genes normally results in dramatic changes

to a plant and structural changes to these genes may represent a significant evolutionary

force. As a practical consequence, engineering of transcription factor genes provides a

valuable means for manipulation of plants.

A typical plant transcription factor contains, with few exceptions, a DNA binding region,

oligomerization site, a transcription-regulation domain and a nuclear localization signal.

Transcription factors-which can be activators, repressors or both-display a modular structure.

They often control multiple enzymatic steps in natural product pathways in plant system and

their ectopic over expression may provide simple mean of up regulating a whole biosynthetic

pathway (Broun P. 2004). Over 25 different transcription factors belonging to different

protein families have been identified to control flavonoid biosynthesis (MYB, bHLH, WD40,

WRKY, WIP, Homeodomain, bMADS) (Broun, 2005). There are several success stories for

engineering flavonoid biosynthesis through homologous and heterologous overexpression of

these transcription factors. Members of MYB transcription factor super family are

characterized by the presence of an amino acid motif structurally and functionally related to

the product of the retroviral oncogene v-MYB and its animal cellular counterpart c-MYB.

MYB proteins have been identified in a large number of eukaryotic organisms ranging fromfungi (Stober-Grasser et al., 1992, Ohi et al, 1994, Tice-Baldwin et al., 1989) and to

vertebrates (Gonda et al 1985. Slamon et al 1986, Nomura et al 1988). While the MYB

domain of c-MYB consists of three imperfect repeats (referred to as R1, R2 and R3), proteins

with other numbers of MYB repeats have also been identified (Riechmann et al, 2000,

Stracke et al 20001, Jiang et al 2004). In contrast to the situation in animals, R2R3-MYB

genes in plants comprise a large gene family. In Arabidopsis, 126 MYB genes of the R2R3

types have been described (Stracke et al 2001).

Up to now, no or only few functional data are available for the overwhelming majority of

plant MYB genes .The functional data available indicate that MYB transcription factors are

involved in a wide array of cellular processes. These include development (Oppenheimer et al

1991,), signal transduction (Bendar and Fink 1998), Plant disease resistance (Daniel et al

1999), Cell devision (Hirayama and Schinozaki, 1996) and secondary metabolism (Borevitz

et al 2000)



PAP1 belongs to MYB transcription factor and known to regulate most of the genes of

phenylpropanoid pathway. It was initially identified by activation tagging from Arabidopsis

(Borevitz et al; 2001, Tohge et al 2005), its heterologous overexpreession in Tobacco resulted

in activation of similar target genes as in Arabidopsis (De-Yu Xie et al 2006). MYB11

belongs to similar family of plant transcription factors and have been found to upregulate

several genes of phenylpropanoid pathway. It has been shown that it is a transcriptional

regulator of Chalcone synthase and Flavonol synthase in planta. Because of strong amino

acid sequence similarity of the Arabidopsis R2R3-MYB factor MYB11 to the maize MYB

protein Zmp (84% identity within the MYB domain, 67% overall similarity) that controls

phlobaphene synthesis in floral organs, Mehertens et al (2005) selected MYB11 to investigate

its role as a part of the regulatory network controlling phenylpropanoid metabolism in

Arabidopsis. They studied MYB11 function by transient coexpression using protoplast of

cultured Arabidopsis cells. Knockout mutants and ectopic overexpression plants were

generated and investigated with respect to a flavonoid phenotype. MYB-12 was found to be a



flavonol specific activator of flavonoid biosynthesis with the two flavanoid biosynthesis

genes CHS and IFS and its primary targets. Transcriptional activation by MYB11 is

coactivator-independent but requires the presence of functional MREs within target

promoters.

Thus overexpression of MYB11 in other plant systems may be a good approach to

simultaneously upregualte several genes of flavonoid biosynthesis and metabolic engineering

of flavonoids, Present work is a step towards that direction where heterologous expression of

Arabidopsis MYB11 transcription factor may be used to upregulate flavonoid biosynthesis

genes and enhancement of flavonoid pool in concerned plant system. Further engineering of

other genes could lead to the accumulation of other novel flavonoids in such systems.

MYB11 have already been identified as a flavonol specific transcription factor in

Arabidopsis. At NBRI PGEL, this transcription factor was cloned in order to overexpress in

some heterologous system to find out if it could function same in other system like tomato,

tobacco .Earlier MYB11 overexpressing transformed lines were generated by the same

laboratory. My disseration work was focused on the following objectives:

Cloning of MYB11 transcription factor from Arabidopsis.

Development of construct for plant transformation.

Agrobacteriummediated transformation of N.tobaccum.



Material and Method

3.1Isolation of MYB11 cDNA:

3.1.1 RNA Isolation

Total RNA from Arabidopsis flowers was isolated according to protocol of Mehar et

al(2000).

RNA extraction buffer:

CTAB (10%) 2 %(

w/v)

EDTA (0.5M, pH8.0)

20mM

NaCl (5M) 1.4mm

Tris (2M, pH8.0) 100mM

BME 10µl/ml

y Flowers of Arabidopsis plants were taken and crushed under liquid nitrogen to fine

powder and extracted with 10ml of extraction buffer with gentle vortexing.

y The homogenate was placed in a water bath at 65ºC for 1 hr with frequent mixing.

y After the incubation an equal volume of chloroform was added to each sample and the

tubes were centrifuged at 10000rpm for 20min in SS34 rotor, sorvall.

y The aqueous phase was re-extracted with equal volume of chloroform.

y The aqueous phase was collected in a clean SS34 tube and lithium chloride 10M was

added to it to a final concentration of 3M.This enables selective Precipitation of RNA

while DNA stays in solution. The tubes were kept overnight at 4ºC.

y The samples were centrifuged at 10000rpm for 30min at 40C to pellet the RNA.

y The pellet was washed with 70% ethanol, air dried and then dissolved in 500µl of sterile

water.

y The dissolved RNA was then extracted with equal volume of water-saturated phenol and

centrifuged at 10000 rpm for 5min.

y To the aqueous phase, 0.5 volume of phenol and 0.5 volume of chloroform (chloroform

/IAA mixture 24:1) was added to extract and centrifuged as described above.



y The aqueous phase was finally extracted with an equal volume of chloroform isoamyl

alcohol mixture. This process of phenolization is carried out to denature and remove the

proteins from nucleic acids.

y The RNA in the aqueous phase was precipitated with 0.1-volume of 3M sodium acetate

(pH 5.0) and 2.5 volumes of ethanol overnight at -70ºC.

y The RNA pellet was obtained by centrifugation at 10000rpm for 30min and then washed

with 70% ethanol.

y The pellet was dried in a speed-vac and dissolved in 50-µl of sterile water.

y RNA was analysed on 1% agarose gel in 1X TBE buffer (Figure2)

3.1.2 R T PCR OF Total RNA

Total RNA was subjected to first strand cDNA synthesis according to prescribed protocol of

Fermentas.cDNA thus synthesized was amplified with designed gene specific primers

(modified at 5¶end to introduce sites for cloning) .Reaction mixture for PCR is as follows

RT (cDNA) 0.5 µl

Buffer (10X) 2µl

dNTP (0.8mM) 1.5l

Forward Primer (At MYB11 For) 1l(5 picomole/l)

Reverse Primer (At MYB11 Rev) 1l (5 picomole/l)

High fidelity Polymerase (Fermentas) 0.2l (1 unit)

Water 12.8l

Total Volume 20l

y Denaturation step was performed for 2 min at 94ºC.

y Primers were allowed to anneal to denatured template at 52º C for 15 sec.

y Primers were extended by High Fidelity DNA polymerase at 72º C for 1.5 minute.

y The amplification was carried out for 35 cycles followed by a final extension of

7min at 720C were carried out under 9700 Perkin Elmer automated PCR.

After amplification, the PCR product was analyzed on the 0.8% gel agarose.



3.1.3 Purif ication of PCR Product

PCR products of At MYB 11 was subjected to gel electrophoresis on 0.8% agarose gel with

TAE buffer along with marker.Band of expected size was cut from the gel under a

transilluminator.From this the At MYB11 cDNA was purified by using Amersham column

according to following method

y Add 1 volume of capture buffer to 1 volume of sliced gel. Tubes were Kept at 60ºC

till the gel melts completely.

y Transfer to a GFX spin column & kept at room temperature for 1 min.

y Placed a GFX spin column in a provided 2 ml collection tube.

y For binding of DNA, the sample was applied to the GFX spin column and centrifuged

for 30-60 sec.

y Discarded flow-through. Placed the GFX spin column into the same tube.

y To wash, added 0.5 ml wash buffer to the GFX spin column and centrifuge for 30-60

sec

y Discarded flow-through and placed the GFX spin column back in the same tube.

Centrifuge the column for an additional 2 min at maximum speed.

y GFX spin column was placed in a 1.5 ml microfuge tube.

y To elute DNA add 50Ql autoclaved double distilled water to the center of the GFX

spin column membrane and centrifuged the column for 1 min at room temperaturey Column centrifuged at full speed for 1min to recover the purified DNA.

3.2 Digestion of plasmid vector

Plant expression vector pBI121 was digested with Xba1 and Sac1 restriction enzymes to

remove its GUS region so that desired fragment can be ligated at these sites. Digestion

reaction was as follows

Water 16µl

Vector pBI 121 40µl (800ng)

Restriction enzyme ( X ba I ) 3µl

Restriction enzyme (Sac I ) 4µl

Restriction Buffer 10X (Tango) 7µl

Total reaction volume 70µl

The digestion was carried out for 5 hrs at 370C



After digestion the digested mixture was purified with amersham column as described earlier.

3.3 Digestion of At MYB11 cDNA

At MYB 11 cDNA was amplified with modified gene specific primers having sites of Xba1

and Sac1 at Forward and reverse primers respectively.Thus obtained purified At MYB11

cDNA was subjected to restriction digestion with Xba1 and Sac1 restriction

enzymes.Digestion reaction was as follows

Water 16µl

Vector pBI121 40µl (400ng)

Restriction enzyme ( X ba I ) 3µl

Restriction enzyme (Sac I ) 4µl

Restriction Buffer 10X (Tango) 7µl

Total reaction volume 70µl

The digestion was carried out for 5hrs at 370C.

After digestion the digested mixture was purified with amersham column as described earlier.

3.4Ligation of vector and purif ied At MYB11 CDNA

Ligation reaction was as follows and carried out at 160C overnight on a water bath.

Vector pBI121 1µl

Insert MYB11 gene 6µl

10X Ligase buffer 2µl

T4 DNA Ligase 2µl

Water 9µl

Total 20µl



3.5Transformation of ligation mixture

3.5.1 Preperation of E.Coli DH5 competent cells

y A single colony of E.coli (DH5E) was inoculated in 5 ml LB and grown overnight at

37ºC in an incubator shaker set at 200 rpm.

y A small aliquot (200µl) of overnight grown culture was added to 100 ml of LB. It was

incubated in an incubator shaker at 200 rpm till the culture OD at 600nm reached 0.4

to 0.6.

y The culture was chilled on ice and centrifuged at 4000 rpm in a SS34 rotor (Sorvall)

for 4 min at 4ºC to harvest the cells.

y

The supernatant was discarded and the pellet was gently suspended in 10ml of icecold 0.1 M CaCl2.

y Bacterial suspension was kept on ice for 30min.

y Tubes were centrifuged at 3000 rpm (rotor) for 3 min.

y Supernatant was discarded and pellet was resuspended in 10ml of 0.1M MgCl2 kept

on ice for 30min and then centrifuged at 3000 rpm for 3 min.

y The supernatant was discarded and pellet was resuspended in 2ml 0.1M CaCl2

containing 10% glycerol.

y Aliquots of 100µl were stored at -70ºC until further use.

3.5.2 Transformation of E . coli with ligation mixture

y Ligated mixture i.e. At MYB11 and pBI121 vector, was transformed into competent

E.coli cells.

y For transformation, 20 µl of ligated product was added to 100µl of competent cells.

y Cells were chilled in ice for 30min.

y Cells were given heat shock at 42ºC for 90sec and then chilled for 5min immediately

after heat treatment.

y Then 4 volumes of LB was added to the cells and kept for incubation at 37ºC for 1 hr.

y 100µl culture was plated on LA plate containing 100µg/ml of kanamycin as the vector

(pBI121) has a gene for Kanamycin resistance. The plate was incubated overnight at

37ºC.



y The colonies obtained were picked on fresh LA plates containing Kanamycin for

preparation of master plate.

3.6-Positive Colony selection through colony PCR

The colonies having positive inserts were confirmed by PCR amplification. After

the confirmation the plasmid was isolated from positive colonies.

PCR amplif ication

Colony PCR was carried out with Vector primers to check the presence of At MYB11 gene

insert in the pBI121vector. About 10 colonies from master plate were inoculated in 1 ml L.B

separately in 10 eppendrof tubes. These tubes were incubated at 370C overnight. Next day

PCR reaction mixtures were prepared for the two clones. The following primer combinations

were used for PCR-

(i) Forward primer specific to CaMV Promoter (CaMVFor)

(ii) Reverse Primer specific to Nos terminator (Nos Ter)

Reaction mixture for colony PCR (10 samples)

E.coli culture having insert 1l

Buffer (10X) 2µl

dNTP (0.8mM) 1.5l

Forward Primer 1l(5 picomole/l)

Reverse Primer 1l (5 picomole/l)

Taq polymerase 0.8l (1 unit)

Water 12.7l

Total Volume per sample 20l



y Primers were extended by Taq DNA polymerase at 72º C for 1.5 minute.





After amplification, the PCR product was analyzed on the 0.8% agarose gel and colonies

showing amplification in colony PCR were marked on master plates.

3.7-CLONE CONFIRMATION

Clone confirmation was done by isolation of the constructs and then its PCR amplification

with vector primers and a combination of gene specific and vector primer.

3.7.1-Plasmid isolation from positive colonies:.

Plasmid isolation by alkaline lysis method (Sambrook et al. 1989) was done and plasmid

2A11-CHI was isolated from positive colonies.

Solution-I

Glucose 50mM

Tris.Cl 25mM

EDTA 10mM

Solution-II NaOH 0.2 N

SDS 1 %( w/v)

Solution-III

Potassium Acetate 3M (Final)

Glacial Acetic Acid 5M (Final)

Method:

y The positive colonies of both clones were inoculated in LB having Kanamycin drug

for selection. Culture was incubated at 37ºC overnight at 200 rpm in an incubator

shaker.

y The cells were harvested at 6000rpm centrifuging 5 minutes 4ºC.



y The supernatant was completely discarded and cells were suspended in 4ml cold TE

solution and mixed by vortexing.

y To the suspension 8ml of freshly prepared solution II was added and cells were lysed

by inverting the tube several times and kept for 3 min at room temperature.

y To the lysate 6 ml of ice-cold solution III was added and contents were mixed

properly and kept on ice for 10 minutes.

y Then centrifuged the tube at 10000 rpm for 15 min at 4ºC.

y To the supernatant 0.6 volumes of isopropanol was added.

y The tube was kept on ice for 10 min and then centrifuged at 10000 rpm for 10 min.

y The supernatant was discarded and nucleic acid pellet was washed with 70% ethanol.

y The pellet was dried in a DNA speed vacuum apparatus (Savant) and dissolved in 4ml

of water. Equal volume of phenol-chloroform was added to remove protein

contamination.

y Aqueous phase was collected and 0.1 volume of 3M-sodium acetate (pH 5.2) and two

volumes of ethanol were added. The tube was kept for 30 min at-20ºC.

y The tube was centrifuged at 10000 rpm for 15 min.

y Supernatant was discarded and the pellet was washed with 70% ethanol.

y Pellet was dried in DNA speed vacuum apparatus and dissolved in 400µl of water and

stored at -20ºC.

y Isolated plasmids were analyzed on agarose gel.3.7.2-PCR amplif ication

Isolated pBI121MYB11 plasmids were subjected to PCR with CaMV35S For and NoS Rev

primers. In another reaction PCR was performed with CaMV For and MYB11 Rev gene

specific primers to finally confirm cloning of MYB11 insert in pBI121 vector. Reactions was

as follows

Plasmid DNA 1 µl (4ng)

Buffer (10X) 2µl

dNTP (0.8mM) 2 l

Forward Primer (CaMV For) 1l(5 picomole/l)

Reverse Primer (Nos Rev) 1l (5 picomole/l)

Taq DNA polymerase 0.5l (1 unit)



Water 12.5l

Total Volume 20l

Other reaction-

Plasmid DNA 1 µl (4ng)

Buffer (10X) 2µl

dNTP (0.8mM) 2 l

Forward Primer (CaMV For) 1l (5 picomole/l)

Reverse Primer (At MYB11 Rev) 1l (5 picomole/l)

Taq DNA polymerase 0.5l (1 unit)

Water 12.5l

Total Volume 20l






After amplification, the PCR product was analyzed on the 0.8% gel agarose.

3.8-Introduction of MYB11 cassette in Agrobacterium Cells:

3.8.1-Preparation of Agrobacterium competent cells

y A loop full of agrobacterium LBA4404 strain from the master plate was inoculated in

5 ml YEB medium containing streptomycin (250µg/ml) and rifampicin (50µg/ml) and

grown over night at 280

C at 220 rpm in an incubator shaker.

y A 20l aliquot from this 5 ml of primary culture was re-inoculated in to a fresh 50 ml

YEB media containing streptomycin (250µg/ml) and rifampicin (50µg/ml) and grown

over night at 280C at 220 rpm in an incubator shaker.

y The overnight grown culture were taken in a 40 ml centrifuge tube and chilled for 10

min.



y After 10 min, the agrobacteruim cells were harvested by centrifuging it on a RC-5C

centrifuge at 4000 RPM, for 5 min at 40C.

y After centrifugation, the supernantant was discarded and bacterial pellet was

suspended in 10 ml of 20 mM CaCl2.

y After suspension, the cells were again centrifuged on a RC-5C centrifuge at 4000

RPM, for 5 min at 40C.

y After centrifugation, the supernantant was discarded and pellet was suspended in 2 ml

of 20 mM CaCl2.

y After suspension, the 100-100 µl of the cell suspension was aliquoted in eppendorf

tube and kept on ice till the transformation.

3.8.2- Freeze-thaw transformation of agrobacterium competent cells with

YB11 constructs.

y 5 µl of plasmid DNA containing approximately 2 µg of plasmid was mixed to one

tube of agrobacterium competent cell.

y The tube was closed and freeze using liquid nitrogen. After freezing the cells was

allowed to thaw at room temperature for 10 min.

y After thawing of the cells, 400 µl of the YEB media was added to the tube and

kept in an incubator shaker with shaking at 220 RPM and allowed to grow at 220

RPM for 3 hr.

y After 3 hr cells were plated on the LA plate containing streptomycin (250, µg/ml)

rifampycin and kanamycin (50µg/ml of each). The plates were incubated at 28 ºC

for 48 hrs.

y The colonies obtained were streaked on a fresh LA plate containing streptomycin

(250µg/ml) rifampycin and kanamycin (50µg/ml of each) for preparation of

master plate. The plates were incubated at 28 ºC for 48 hrs and stored at 4 ºC until

the further use.

3.8.2-Screening of positive colony through colony PCR

The colonies for construct were confirmed by PCR amplification using vector specific

CaMV35S forward and Nos reverse primer. Two colonies from the master plate were

inoculated in 100 µl of YEB median containing streptomycin rifampycin(250µg/ml) and



kanamycin (50µg/ml of each) in 2 separate eppendorf tubes. The tubes were incubated at

280C for overnight with shaking at 220 rpm. A common PCR reaction mixture was prepared

for all the PCR reactions using two primers as follows:

PCR reaction Mix

D.D water 39 µl

10X PCR Buffer 6 µl

DNTPs mix (10 mM) 3 µl

Primer CaMV35S 3 µl (5 pM/l)

Primer NosT 3 ul (5 pM/l)

Taq DNA polymerase 3 µl (3 U)

Total Volume 57 µl

For the PCR, 1 µl culture from each of the eppendorf tubes were placed in 2 different

PCR tubes. An extra PCR reaction was also set to work as a negative control. In this negative

control PCR reaction, 1µl water was added as the template. After that, 19 µl of PCR reaction

mix 1 or 2 were added in these three PCR tubes. PCR reaction was carried out in Perkin

Emler PCR machine as follows:






After amplification, the PCR product was analyzed on the 0.8% gel agarose

3.9- Transformation of Tobacco Plants through pBI121 MYB11 construct:

Tobacco leaf discs were transformed using Ag robact erium mediated transformation

according to the protocol as described by Prashant et al (Unpublished).Following

methodology was adopted-



Preperation of bacterial suspension

y A single colony of Agrobacteria harbouring pBI121 35S MYB11 plasmid was

inoculated in 5ml LB having selective antibiotics(50µ/ml Rifampicin,50 µ/ml

Kanamycin and 250 µ/ml Streptomycin) and incubated at 28C with shaking at 200

rpm for 24hrs.

y From the above mentioned culture an aliquot of 100µl was inoculated in 25 ml of

YEB with selective antibiotics and incubated at 28C with shaking at 200 rpm till OD

of bacterium becomes 1 at 600nm

y Agrobacteria were harvested at 6ooorpm for 5min at 4C and resuspended in similar

volume of YEB.The bacterial suspension thus obtained was diluted four times with

YEB.In that bacterial suspension acetosyringone was added at concentration of

100µM and left at room temperature till transformation.

Preperation of Leaf discs

y Young leaves of N icotiana tabacum var Petit Havana were taken and washed in

running water for 30 mins and then surface sterilized with 0.1% Mercuric chloride

solution for 2 minutes.Leaves were thoroughly washed with sterile distilled water

under laminar air flow.

y Surface sterilized leaves were cut into small squares of about 1cm×1cm aseptically

under laminar air flow.These squares were kept in sterile petriplates.

Agroinfection and Cocultivation

y Leaf pieces of Nicotiana were submerged in Agrobacterim suspension and wrapped in

an Aluminium foil swirled occasionally for 20 minutes.

y Leaves were blotted dry and plated over MS medium (Murashige and Skoog 1962)

and then kept at 26C in dark for 2 days.



Plantlet Regeneration and selection

After Co cultivation the leaf Discs were plated on regeneration medium (Modified MS

medium with 2mg/liter BAP and 0.2mg/liter IAA+100mg/liter kanamycin+250mg/liter

Cifotaxime). Subculturings were done after every two weeks.

Rooting

After three subculturing serviving regenerated plantlets were cut and put into rooting medium

(half strength MS with 100mg/ml kanamycin).



Result and Discussion

RNA isolation: Good qualit R (Fi 2) from Arabidopsis Flowers was isolated as

evidenced by Gel Electrophoresis and spectrophotometr ic analysis.

Fi .2 R A isolated from Arabidopsis f loral tissue

RT PCR of RNA from Arabidopsis Flower: R PCR of Arabi¡

¢ £ ¤ is f loral tissue R A

with desi ned gene specif ic pr imers resulted in to amplif ication of a fragment of about 1.15

Kb. Its si e corresponds to expected si e of at M B11 cDNA (Fig.3).

Fig.3 R ¥ PCR of R NA isolated from Arabidopsis seedling. Lane 1- PCR product. Lane 2- Hind III/EcoR I

double digest DNA molecular weight marker.



Colony PCR for positive colony screening

10 colonies were selected from the plates for PCR amplif ication for Positive colony screening

with Vector pr imers. Af ter gel electrophoresis (Fig3) of PCR products colonies 5th

and 8th

were found to have an amplif ied band of about 1.35 Kb. Therefore these colonies may be

positive colonies having desired recombinant vector.

Fig. 4 Colony PCR to screen positive colonies having M¦

B11 inser t

1st lane HE marker, Lane 2 to11 amplif ied PCR products. Colonies 6 and 9 being putative positive

colonies.

Clone Confirmation

Af ter PCR amplif ication of isolated plasmid from transformed colony an amplif ied band of

approximately 1.35 Kb was obtained with vector pr imers (Fig.5).In another PCR reaction

with one vector pr imer and M B11 R ev, a band of approximately 1.25 kb was obtained(Fig.5). These results conf irm cloning of M B11 cDNA in pBI121.

Fig.5 PCR of plasmid DNA from putative transformed colony. Lane 1- Hind III/EcoR I double digest DNA

molecular weight marker. Lane 2- PCR with CaMV For and M¦

B 11R ev, Lane 3- PCR with CaMV For and

Nos R ev

1 2 3 4 5 6 7 8 9 10 11



Plantlet Regeneration and Development of Transgenics

Af ter 2 weeks of co cultivation small green structures star ted emerging from the leaf surface

and rest of the por tion got yellow colored due to selection (f igure 6 and 7).R egenerated

plantlets were sub jected to repeated subcultur ing af ter 2 weeks. Plantlets surviving af ter 3

subcultur ings were mentioned as putative transgenics of M B11.In rooting medium plantlets

developed roots af ter 7-8 days. Af ter rooting (Figure8 and 9) were sub jected to PCR with

vector pr imers to conf irm transgene.

Fig. 6 and 7 Putative transgenic tobacco plants regenerating on selective M § medium with 50 mg/l kanamycin.

Fig. 8 and 9 rooted i¨ © it ro grown putative M

B 11 transgenic tobacco plants on half strength M

medium



5-Conclusions

A cDNA of M B11 transcr i ption factor of Arabidopsis thaliana was successfully cloned in

plant expression vector pBI121(Fig9) under the control of constitutive 35Spromoter.Binary

vector thus obtained were mobili ed into Agrobacter ium background.Tobacco transgenics

overexpressing M B11 were raised and their fur ther analysis is under progress.

Fig.10 R epresentation of plant expression construct for over-expression of AtM B11



High Performance Li uid Chromatography

Apart from my dissertation I have also learnt High Performance liquid chromatography

technique for qualitative as well as quantitative analysis for secondary metabolites present in

plant material. Most of the herbal medicines and food items like grapes and wines contain a

range of antioxidant phenolics with HPLC being the most preferred method for their analysis

and standardization. Polyphenols are important antioxidants, because of their high redox

potentials, which allow them to act as reducing agents, hydrogen donors and singlet oxygen

quenchers. Flavonoids are chemical moieties widely distributed in the plants that are

important biologically active constituents of daily human diet with significant

pharmacological potential viz. antihepatotoxic, antiallergic, anti-inflammatory,

antiosteoporotic and antitumor activities.

A HPLC method for the separation and quantification of phenolics in a single run with a total

of 4 different phenolics has been developed (Fig. 11). The spectra of each of the compounds

was also recorded and analysed to study the method precision and also for the easy

identification of the compounds (Fig, 11).

Prepration of Herbal Samples-

Individual flavanols in the plants were determined either as aglycones or as flavanol

glycosides by preparing acid-hydrolyzed or nonhydrolyzed extracts,respectively.

For preparation of acid-hydrolyzed extract- plant material was extracted with 80% ethanol

overnight at room temperature with brief agitation. The filtrates were evaporated to 1.0 mL,

and 3 volumes of HCl (1 M) was added followed by incubation at 94_C for 2 h to hydrolyze

any conjugate forms of flavanoids. After hydrolyzation, samples were extracted with ethyl

acetate, evaporated to dryness, and resuspended in 80% methanol. (Fig.11)

For non-hydrolyzed extracts- plant material was extracted in methanol:water (70:30)

overnight at room temperature with brief agitation. Extracts were filtered through a 0.2-mm

filter (Millipore) before separation and quantitation of flavonols using a liquid chromatograph

(Fig.12) and a Merck Purospherstar (250 3 4.6 mm, 5-mm pore size) C18 column with guard

column of the same chemistry. Elution of flavonols was carried out at a flow rate of 0.8 mL

min21 with 0.5% phosphoric acid as solvent A and methanol as solvent B, using a gradient

elution with 75% to 70% A (0±5 min), 70% to 50% A (5±10 min), 50% to 20% A (10±15

min), and 20% to 80% A (15±25 min). Flavonols were quantified by calculating the area of



an individual peak and comparing this with a standard obtained from Sigma-Aldrich.(Misra

et al. 2010)

Fig.- HPLC Chromatogram of standard ( R = Rutin, Q = Quercetin, G = Genistein, K =

Kaempferol ) and non-hydrolysed plant material.



Fig.12 - HPLC Chromatogram of standard ( R = Rutin, Q = Quercetin, G = Genistein, K

Kaempferol ) and non-hydrolysed plant material.



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