Final Report Nbri - maniSH SINGH

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I, Manish Kumar Singh hereby declare that this dissertation report entitled

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)
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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 DH5Į 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
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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 # # " 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 ###
for granting me permission to undertake my dissertation for partial fulfillment of
Master of science degree in Biotechnology.
I am greatly thankful to Dr# 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 # ## Scientist and # $ , Scientist, Plant
Gene Expression Lab for providing constant source of encouragement at each and all steps
of this work.
I am highly obliged and owe my special thanks to my guide # % 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 # # & # &
# , who helped me in my practical work and understanding the problem
through discussions.
Special thanks to # ' ( # ' &
) % % # " # % & # &
# # % # ' ( * #
'%#

# ' is highly acknowledged for autoclaving, routine lab works and
maintaining plants in glass house and #' is acknowledged for his glassware
washing.
Finally, special and cordial thanks to my beloved . 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.

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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 ȕ -marceptoethanol
YEB Yeast extract broth
CHS Chalcone Synthesis
CHI Chalcone Isomerase
F3H Flavonoied -3- Hydroxelase

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 .
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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, a a 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 a 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 from
fungi (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 ca
a, 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 ca
a
(Borevitz et al; 2001, Tohge et al 2005), its heterologous overexpreession in Tobacco resulted
in activation of similar target genes as in ca
a (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 ca
a R2R3-MYB factor MYB11 to the maize MYB
protein
(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
ca
a. They studied MYB11 function by transient coexpression using protoplast of
cultured ca
a 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
ca
a 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

ca
a. 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:

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Total RNA from Arabidopsis flowers was isolated according to protocol of Mehar et
al(2000).
'* -
CTAB (10%) 2 %(
w/v)
EDTA (0.5M, 20mM
pH8.0)
NaCl (5M) 1.4mm
Tris (2M, pH8.0) 100mM
BME 10µl/ml
þ Flowers of Arabidopsis plants were taken and crushed under liquid nitrogen to fine
powder and extracted with 10ml of extraction buffer with gentle vortexing.
þ The homogenate was placed in a water bath at 65ºC for 1 hr with frequent mixing.
þ 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.
þ The aqueous phase was re-extracted with equal volume of chloroform.
þ 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.
þ The samples were centrifuged at 10000rpm for 30min at 4 0C to pellet the RNA.
þ The pellet was washed with 70% ethanol, air dried and then dissolved in 500µl of sterile
water.
þ The dissolved RNA was then extracted with equal volume of water-saturated phenol and
centrifuged at 10000 rpm for 5min.
þ 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.
þ 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.
þ 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.
þ The RNA pellet was obtained by centrifugation at 10000rpm for 30min and then washed
with 70% ethanol.
þ The pellet was dried in a speed-vac and dissolved in 50-µl of sterile water.
þ RNA was analysed on 1% agarose gel in 1X TBE buffer (Figure2)
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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) 1.5ȝl
Forward Primer (At MYB11 For) 1ȝl(5 picomole/ȝl)
Reverse Primer (At MYB11 Rev) 1ȝl (5 picomole/ȝl)
High fidelity Polymerase (Fermentas) 0.2ȝl (1 unit)
Water 12.8ȝl
Total Volume 20ȝl
þ Denaturation step was performed for 2 min at 94ºC.

þ Primers were allowed to anneal to denatured template at 52º C for 15 sec.
þ Primers were extended by High Fidelity DNA polymerase at 72º C for 1.5 minute.
þ 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.
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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
þ Add 1 volume of capture buffer to 1 volume of sliced gel. Tubes were Kept at 60ºC
till the gel melts completely.
þ Transfer to a GFX spin column & kept at room temperature for 1 min.
þ Placed a GFX spin column in a provided 2 ml collection tube.
þ For binding of DNA, the sample was applied to the GFX spin column and centrifuged
for 30-60 sec.
þ Discarded flow-through. Placed the GFX spin column into the same tube.
þ To wash, added 0.5 ml wash buffer to the GFX spin column and centrifuge for 30-60
sec
þ 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.
þ GFX spin column was placed in a 1.5 ml microfuge tube.
þ To elute DNA add 50§ l autoclaved double distilled water to the center of the GFX
spin column membrane and centrifuged the column for 1 min at room temperature
þ Column centrifuged at full speed for 1min to recover the purified DNA.
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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 () 3µl
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.
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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 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.
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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

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þ A single colony of a (DH5) was inoculated in 5 ml LB and grown overnight at

37ºC in an incubator shaker set at 200 rpm.
þ 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.
þ 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.
þ The supernatant was discarded and the pellet was gently suspended in 10ml of ice
cold 0.1 M CaCl2.
þ Bacterial suspension was kept on ice for 30min.
þ Tubes were centrifuged at 3000 rpm (rotor) for 3 min.
þ 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.
þ The supernatant was discarded and pellet was resuspended in 2ml 0.1M CaCl2
containing 10% glycerol.
þ Aliquots of 100µl were stored at -70ºC until further use.
,#1#. E + *
þ Ligated mixture i.e. At MYB11 and pBI121 vector, was transformed into competent
a cells.
þ For transformation, 20 µl of ligated product was added to 100µl of competent cells.
þ Cells were chilled in ice for 30min.
þ Cells were given heat shock at 42ºC for 90sec and then chilled for 5min immediately
after heat treatment.
þ Then 4 volumes of LB was added to the cells and kept for incubation at 37ºC for 1 hr.
þ 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.
þ The colonies obtained were picked on fresh LA plates containing Kanamycin for
preparation of master plate.
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The colonies having positive inserts were confirmed by PCR amplification. After
the confirmation the plasmid was isolated from positive colonies.
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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)
' * %'56
a culture having insert 1ȝl
Buffer (10X) 2µl

dNTP (0.8mM) 1.5ȝl
Forward Primer 1ȝl(5 picomole/ȝl)
Reverse Primer 1ȝl (5 picomole/ȝl)
Taq polymerase 0.8ȝl (1 unit)
Water 12.7ȝl
Total Volume per sample 20ȝl

þ 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.
,#7 $!/'

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#

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)
-
þ 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.
þ The cells were harvested at 6000rpm centrifuging 5 minutes 4ºC.
þ The supernatant was completely discarded and cells were suspended in 4ml cold TE
solution and mixed by vortexing.
þ 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.
þ To the lysate 6 ml of ice-cold solution III was added and contents were mixed
properly and kept on ice for 10 minutes.
þ Then centrifuged the tube at 10000 rpm for 15 min at 4ºC.
þ To the supernatant 0.6 volumes of isopropanol was added.
þ The tube was kept on ice for 10 min and then centrifuged at 10000 rpm for 10 min.
þThe supernatant was discarded and nucleic acid pellet was washed with 70% ethanol.
þ 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.
þ 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.
þ The tube was centrifuged at 10000 rpm for 15 min.
þ Supernatant was discarded and the pellet was washed with 70% ethanol.
þ Pellet was dried in DNA speed vacuum apparatus and dissolved in 400µl of water and
stored at -20ºC.
þ Isolated plasmids were analyzed on agarose gel.
,#7#. '
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 ȝl
Forward Primer (CaMV For) 1ȝl(5 picomole/ȝl)
Reverse Primer (Nos Rev) 1ȝl (5 picomole/ȝl)
Taq DNA polymerase 0.5ȝl (1 unit)
Water 12.5ȝl
Total Volume 20ȝl
Other reaction-
Plasmid DNA 1 µl (4ng)
Buffer (10X) 2µl

dNTP (0.8mM) 2 ȝl
Forward Primer (CaMV For) 1ȝl (5 picomole/ȝl)
Reverse Primer (At MYB11 Rev) 1ȝl (5 picomole/ȝl)
Taq DNA polymerase 0.5ȝl (1 unit)
Water 12.5ȝl
Total Volume 20ȝl

After amplification, the PCR product was analyzed on the 0.8% gel agarose.
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þ Aloop fullofagrobacterium LBA4404strain from the master plate was inoculated in

5 ml YEB medium containing streptomycin (250µg/ml) and rifampicin (50µg/ml) and
grown over night at 280C at 220 rpm in an incubator shaker.
þ A 20ȝl 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 28 0C at 220 rpm in an incubator shaker.
þ The overnight grown culture were taken in a 40 ml centrifuge tube and chilled for 10
min.
þ After 10 min, the agrobacteruim cells were harvested by centrifuging it on a RC-5C
centrifuge at 4000 RPM, for 5 min at 4 0C.
þ After centrifugation, the supernantant was discarded and bacterial pellet was
suspended in 10 ml of 20 mM CaCl2.
þ After suspension, the cells were again centrifuged on a RC-5C centrifuge at 4000
RPM, for 5 min at 40C.
þ After centrifugation, the supernantant was discarded and pellet was suspended in 2 ml
of 20 mM CaCl2.
þ After suspension, the 100-100 µl of the cell suspension was aliquoted in eppendorf
tube and kept on ice till the transformation.
,#8#. /9 + + YB11

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

tube of agrobacterium competent cell.
þ The tube was closed and freeze using liquid nitrogen. After freezing the cells was
allowed to thaw at room temperature for 10 min.
þ 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.
þ 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.
þ 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.
,#8#. % %'

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:
' *

D.D water 39 µl
10X PCR Buffer 6 µl
DNTPs mix (10 mM) 3 µl
Primer CaMV35S 3 µl (5 pM/ȝl)
Primer NosT 3 ul (5 pM/ȝl)
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
,#: .

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Tobacco leaf discs were transformed using c a mediated transformation
according to the protocol as described by Prashant et al (Unpublished).Following
methodology was adopted-

þ 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.
þ 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
þ 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.
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þ Young leaves of aa 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.
þ Surface sterilized leaves were cut into small squares of about 1cm×1cm aseptically
under laminar air flow.These squares were kept in sterile petriplates.

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

an Aluminium foil swirled occasionally for 20 minutes.
þ Leaves were blotted dry and plated over MS medium (Murashige and Skoog 1962)
and then kept at 26C in dark for 2 days.
'
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.
'
After three subculturing serviving regenerated plantlets were cut and put into rooting medium
(half strength MS with 100mg/ml kanamycin).

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2 $ %
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).
2

Individual flavanols in the plants were determined either as aglycones or as flavanol
glycosides by preparing acid-hydrolyzed or nonhydrolyzed extracts,respectively.
/ % %9* - 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)
/ % %9 * - 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)
/.- HPLC Chromatogram of standard ( R = Rutin, Q = Quercetin, G = Genistein, K =
Kaempferol ) and non-hydrolysed plant material.

/#.- HPLC Chromatogram of standard ( R = Rutin, Q = Quercetin, G = Genistein, K
Kaempferol ) and non-hydrolysed plant material.

'!/!'!!
Broun,P.(2005)Transcription control of flavonoid biosynthesis: a complex network of

conserved regulators involved in multiple aspects of differentiation in
Arabidopsis.Curr,Opin.Plant Biol.8,272-279
Broun P. (2004Transcription factors as tools for metabolic engineering in plants .Curr.Opin

.Plant biol.7, 202-209.
Brevets JO, Xia YJ, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a
conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383-2393
BrownDE, Tice-Baldwin K, Fink G R, Arndt KT (1989) BAS1 has a Myb motif and activates
HIS4 transcription only in combination BAS2.Science 246:931-935
DanielX, LacommeC,Morel JB, roby D(1999) A novel myb oncogene homologue in

Arabidopsis thaliana related to hypersesnsitive cell death. Plant j 20:57-66
De-yu Xie , Shashi B Sharma,Elane Wright, Zeng-Yu Wang and R.A. Dixon(2006)Metabolic
engineering of proanthocyandins through co-expression of anthocyanidin reductase and the
PAP1 MYB transcription factor.The Plant Journal 45,895-907
Frank M,Harald K,Pawel B,Weisshaar B(2005).The Arabidopsis transcription Factor MYB11

is a flavonol ±specific regulator of Phenylpropanoid biosynthesis. Plant Physiology: 138
1083-1096
Gonda TJ, Gough NM, Dunn AR, de Blaquiere J (1985) Nucleotide sequence of cDNA
clones of murine myb proto-oncogene.EMBO j 4:2003-2008
Harborne JB. (1986)The flavonoids.Advances in Research Since 1986, 1st ed.

(London:Chapmand hall).
Hirayama T,Li SF, Parish (2003)A cdc+homolog of higher of a higher plant,Arabidopsis

thaluana.Proc Natl Acad Sci USA 93:13371-13376.
Mehar H, Dhawan P,Nath P(2000) A simple procedure for isolation of high quality RNA
from ripening Banana fruit. Plant Molecular Biology Reporter 18:109-115.
Misra P, Pandey A, Tiwari M, Chandrashekar K, Sidhu OP, Asif MH, Chakrabarty D,

Singh PK, Trivedi PK, Nath P, Tuli R (2010) Modulation of Transcriptome and
Metabolome of Tobacco by Arabidopsis Transcription Factor, AtMYB11, Leads to Insect
Resistance Plant Physiology Vol. 152, pp. 2258±2268,
Muir SR,Collins GJ ,Robinson S,Hughes S,Bovy A,De Vos CHR.van Tumen AJ,Verhoeyen
ME.2001.Overexpression of petunia chalcone isomerase in tomato results in fruits containing
increased levels of flavonols,Nature Biotechnology 19,470-474
Murashige T, Skoog F(1962) Plant Physiology 15:473-497
Nomura N, Takahashi M, Matsui M,Ishii S,Date T,Sasamoto S,Ishizaki R(1988)Isolation of

human cDNA clones of myb related genes,A-myb and b-myb.Nucleic Acid
Resarch16:11075-11089
Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD (1991) a myb gene is
required for leaf trichome differentiation in Arabidopsis is expressed in stipules.
Rashotte AM, Murphy AS, Normanly J,Tague BW,Peer WA,Taiz Land Muday
GK(2001).flavonoids act as negative regulators of Auxin transport in arabidopsis.Plant
physiology.126:524-535
Reichmann JL,Heard j,Martin G,Reuber L,jiang CZ,Keddie J,Adam L,Pineda O,Ratcliffe

OJ,Samaha RR,et al (2000) Arabidopsis transcription factors:genome ±wide comparative
analysis among eukaryotes.Science290:2105-2110
Rice Evans, CA Miller, NJ, Paganga G, and Miller N (1997).Antioxidant properties of

phenolic compounds:The polyphenolic content of fruit and vegetables and their antioxidant
activities.What does a serving constitute?Trends Plant Sci 2,152-159
Stober-Grasser U, Brydolf B, BinX, grasser F, Firtel RA, Lipsick JS (1992) The Myb DNA
binding domain is highly conserved in Dictyostelium discoideum.Oncogene 7:589-596.
Sambrook,J,Fritsch EF and Maniatis T.(1989).Molecular Cloning:A laboratory Manual.(Cold

spring harbour,NY:Cold spring Harbour Laboratory Press).
Sebastien Besseau,Laurent Hoffmann,Pierrette Geoffroy.Catherine Lapierre,Brigitte Pollet

and Michel Legrand(2007). Flavonoid accumulation in Arabidopsis repressed in lignin
synthesis affects auxin transport and plant growth, Plant Physiology
Slamon DJ,BooneTC,Murdock DC,Keith DE,Press MF,Larson RA,Souza LM(1986) Studies

of the human c-myb gene and its product in human acute leukemias.Science 233:347-351.
Stacke R, Werber M, Weisshaar b (2001) The R2R3 gene family in Arabidopsis thaliana.
Curr Opin Plant Biol 4:447-456
Tice-Baldwin K, Fink G R, Arndt KT (1989) BAS1 has a Myb motif and activates HIS4
transcription only in combination BAS2.Science 246:931-93
Ververidis F, Trantas E, Douglas C, Vollmer G, Kretzschmar G, Panopoulos N (2007)

Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I.
Chemical diversity, impacts on plant biology and human health. Biotechnol J 2: 1214±1234

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3-Material and method

3.1-isolation of MYB11 cDNA

3.1.2-RT PCR of total RNA

3.1.3-Purification of PCR products

3.2-Digestion of Plasmid vector

3.4-Ligation of vector and purified At MYB11 CDNA

3.5-Transformation of ligation mixture

3.5.1- Preperation of E.Coli DH5Į competent cells

3.5.2-Transformation of E.coli with ligation mixture

3.6-Positive colony selection through colony PCR

3.7.1-Plasmid DNA isolation from positive colonies

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

3.9-Transformation of tobacco through AtMYB11 construct

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

RNA Ribonucleic acid

TAE Tris acetate EDTA

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

SDS Sodium dodecyl sulphate

YEB Yeast extract broth

CHS Chalcone Synthesis

CHI Chalcone Isomerase

F3H Flavonoied -3- Hydroxelase

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

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

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

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

Buffer (10X) 2µl

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

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

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

Total Volume 20ȝl

þ Denaturation step was performed for 2 min at 94ºC.

Restriction Buffer 10X (Tango) 7µl

The digestion was carried out for 5 hrs at 370C

,#,   

Vector pBI121 40µl (400ng)

Restriction enzyme () 3µl

Restriction enzyme () 4µl

Restriction Buffer 10X (Tango) 7µl

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

,#0$      

Vector pBI121 1µl

Insert MYB11 gene 6µl

10X Ligase buffer 2µl

T4 DNA Ligase 2µl

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

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

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Restriction enzyme () 3µl

Restriction enzyme () 4µl

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þ A single colony of a (DH5) was inoculated in 5 ml LB and grown overnight at

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þ Denaturation step was performed for 5 min at 94ºC.

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3.7.1-Plasmid isolation from positive colonies:#

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

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þ Aloop fullofagrobacterium LBA4404strain from the master plate was inoculated in

,#8#. /9 + + YB11

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

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

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

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

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