Expression analysis of water stress related genes in tomato plant 2019

DECLARATION
I hereby declare that the work is presented in this summer training “Expression analysis of water
stress related genes in Tomato plants” submitted to the CSIR-NEIST, Jorhat is a record of an
original work done by me under the guidance of Dr Ratul Saikia, Sr. Principal Scientist of
Biological Sciences And Technology Division(BSTD), CSIR-NEIST. The results embodied in
this report have not been copied from any other Department/Institute/University.
Ron Hazarika
M.Sc Biotechnology
2nd
semester

ACKNOWLEDGEMENT
I would like to express my immense gratitude to my supervisor Dr Ratul Saikia , Sr.
Principal scientist of Biological Sciences And Technology Division(BSTD), CSIR-NEIST for
his motivating guidance and constant encouragement to carry out my work.
I am truly thankful to Dr Hari Prasanna Deka Boruah, Sr. Principal Scientist (HOD) of
BSTD for his support and guidance.
I take this opportunity to put forward my sincere thanks to the Director, Dr G.N. Sastry,
CSIR-NEIST, Jorhat, Assam for giving me this opportunity to undergo summer training at this
institute.
I would like to acknowledge and extend my gratitude to Ms Archana Yadav,Technical
Officer of BSTD for extending her help in performing the experiments.
I would also like to thank Miss Parismita Gogoi and Miss Priyanka Kakoti for their guidance
and support throughout the experiments.
I would like to express my gratitude to Dr. Santanu PalChaudhuri(Professor, Head Of
Division) and Dr. Swatilekha Ghosh, (Associate Professor) of Amity Institute of Biotechnology,
Kolkata (AIBNK), Amity University, Kolkata, West Bengal for their continuous inspirations
during carrying out this training.
Finally I would like to express my warmest thanks to my parents and my friends for their
encouragement, help and support to complete my project successfully.
I express my sincere gratitude to all concerned.
Date: Ron Hazarika
Amity University Kolkata

ABSTRACT
Gene expression analysis involves the determination of the pattern of genes expressed at
the level of genetic transcription, under specific circumstances or in a specific cell. The
measurement of gene expression is a critical tool employed across drug discovery, life science
research and the optimization of bioproduction. Expression analysis involves several techniques
ranging from whole genome gene expression analysis such as microarrays or RNA sequencing,
to more specific target gene expression techniques such as qPCR techniques.
Gene expression analysis typically involves the isolation or capture of transcribed RNA
within a sample, followed by amplification and subsequent detection and quantitation.
This project work started with the collection of leaf sample from Tomato plants grown
under stress condition noting the water logging level, isolation of RNA and purification is done.
Extraction of RNA was performed using NucleoSpin® RNA plant isolation kit. Estimation of
cDNA and gene expression analysis is done using RT-PCR.

CONTENTS
1. Introduction
i. Plant stress and its cause.
ii. Plants taking in consideration.
iii. Abiotic stress.
iv. Abiotic stress inducible genes.
v. Transcriptional factor genes involved in abiotic stress.
vi. Transcriptional factor involved in response to flooding
stress.
vii. Ribonucleic acid.
viii. Complimentary DNA.
2. Materials and methods.
3. Results and discussion.
4. Conclusion.
5. Implications for future research.
6. References.

1. INTRODUCTION
In recent years, the world has experienced significant challenges from Mother Nature.
Tragic wildfires, severe droughts, heavy rains, massive flooding, hurricanes, and more have
wreaked havoc throughout the states. These environmental threats have ruined crops, harmed
livestock, destroyed vegetations and even normal ecosystem, but especially those in key
agricultural regions. Lately, these natural disasters have taken the news stage due to their
intensity and frequency and represent the impacts of a changing climate. Farmers around the
world often bear the brunt of these disasters and feel the impact of climate change especially
close to home and in their business bank accounts. External environmental impacts like those
plaguing news headlines as of late pose significant risks to plant and crop health and often stress
plants beyond their tolerance limits and can lead to diminished marketable yields. Natural
disasters are an obvious cause of plant stress, even to the naked eye. But, did you ever realize
that plant stress comes in many other forms, some even invisible to the naked eye.
Plant Stress and its causes
Plant stress is a state where a plant is growing in non-ideal growth conditions and has
increased demands put on it. Plant stress refers to any unfavourable condition or substance that
affects a plant’s metabolism, reproduction, root development, or growth. Plant stress can come in
different forms and durations. Some plant stressors are naturally occurring, like drought or wind,
while others may be the result of human activity, like over irrigation or root disturbance.
Plant stress is caused by a variety of factors, some of which are obvious (like natural
disasters), while others occur on a micro scale in the soil. Recent natural disasters represent one
type of plant stress factors, called abiotic factors, which usually occur above ground. A second
type of plant stress factors are called biotic factors, which mostly occur underground, and can
cause plant stress through pathogens and pests.
Abiotic stresses originate from the surrounding environment of the plant. One of the
most important abiotic factors affecting plants is water stress. A plant requires a certain amount
of water for optimal growth, too much water can cause plant cells to swell and burst, whereas too
little water can lead to desiccation. Temperature stresses can also negatively impact a plants
growth and livelihood. Cold weather may affect the amount and rate of uptake of water and

nutrients, and hot weather can affect the permeability of plant membranes. Abiotic factors come
in other forms as well such as wind, toxins, and light.
Biotic stresses can cause damage to plants through living organisms that may cause
disease. In agriculture, biotic stresses are most often responsible for pre or post-harvest losses.
Soil is filled with fungi and bacteria – 1 teaspoon of soil can hold billions of microorganisms.
Just like microorganisms found in humans, some can be beneficial, and others can be
detrimental. Healthy soil biological systems showcase an appropriate, harmonious balance
between beneficial microorganisms that protect against biotic stresses and detrimental
microorganisms – that if not held in check can result in biotic plant stresses. Examples of
common biotic plant stress factors include pathogens, insects, and weeds but the exact types of
factors depend on the environment and differ from region to region.
About the plants
1. Tomato - The tomato is the edible, often red, berry of the plant Solanum
lycopersicum, commonly known as a tomato plant. The species originated in
western South America and Central America. Tomatoes are a significant source
of umami flavor. Numerous varieties of the tomato plant are widely grown in temperate
climates across the world, with greenhouses allowing for the production of tomatoes
throughout all seasons of the year. Tomato plants typically grow to 1–3 meters (3–10 ft)
in height. They are vines that have a weak stem that sprawls and typically needs
support. Indeterminate tomato plants are perennials in their native habitat, but are
cultivated as annuals. Determinate, or bush, plants are annuals that stop growing at a
certain height and produce a crop all at once. The size of the tomato varies according to
the cultivar, with a range of 0.5–4 inches (1.3–10.2 cm) in width.

2. Bhut Jolokia: The Bhut jolokia , also known as ghost pepper. ghost chili pepper, ghost
chili and ghost jolokia, is an interspecific hybrid chili pepper cultivated in the Northeast
Indian states of Arunachal Pradesh, Assam, Nagaland and Manipur. It is a hybrid
of Capsicum chinense and Capsicum frutescens and is closely related to the Naga
Morich of Nagaland and Bangladesh. Bhut jolokia mainly belongs to the
species Capsicum chinense Jaqc. It was earlier thought to be a hybrid of Capsicum
frutescens and Capsicum chinense on the basis of randomly amplified polymorphic DNA
(RAPD) analysis. However, it has recently been described as a distinct species (Capsicum
assamicum) on the basis of morphological properties, molecular phylogeny of the internal
transcribed spacer (ITS) region and differential proteomic analysis. Bhut jolokia is a self-
pollinated plant, however, considerable cross pollination (up to 10%) may occur when
insect population is high. It behaves as a semi-perennial herb if grown under optimal
condition. The plant grows to a height of 57-129 cm at 6 months. Under semi perennial
situation it may grow even taller.
ABIOTIC STRESS
Abiotic stress is the negative impact of non-living factors on the living organisms in a specific
environment. The non-living variable must influence the environment beyond its normal range of
variation to adversely affect the population performance or individual physiology of the
organism in a significant way.
Whereas a biotic stress would include living disturbances such as fungi or harmful insects,
abiotic stress factors, or stressors, are naturally occurring, often intangible and inanimate factors
such as intense sunlight, temperature or wind that may cause harm to the plants and animals in

the area affected. Abiotic stress is essentially unavoidable. Abiotic stress affects animals, but
plants are especially dependent, if not solely dependent, on environmental factors, so it is
particularly constraining. Abiotic stress is the most harmful factor concerning the growth
and productivity of crops worldwide. Research has also shown that abiotic stressors are at their
most harmful when they occur together, in combinations of abiotic stress factors.
Stress Consequences Plant Responses
Heat stress
High temperature leads to high
evaporation and water deficit.
The consequent increased
turnover of enzymes leads to
plant death.
Efficient protein repair systems
and general protein stability
support survival, temperature can
lead to acclimation.
Chilling and cold stress
Biochemical reactions proceed
at slower rate, photosynthesis
proceeds, carbon dioxide
fixation lags, leading to oxygen
radical damage. Indeed,
freezing lead to ice crystal
formation that can distrupt cells
membranes.
Cessation of growth in adaptable
species may be overcome by
changes in metabolism. Ice
crystal formation can be
prevented by osmolyte
accumulation and synthesis of
hydrophilic proteins.
Drought
Inability to water transport to
leaves leads to photosynthesis
declines.
Leaf rolling and other
morphological adaptations. Stoma
closure reduces evaporative
transpiration induced by ABA.
Accumulation of metabolities,
consequently lower internal water
potential and water attracting

Flooding and submergence
Generates anoxic or micro
aerobic conditions
Interfering with mitochondrial
respiration.
Development of cavities mostly
in the roots that facilitate the
exchange of Oxygen and ethylene
between shoot and root
(aerenchyma).
Heavy metal accumulation
and metal stress
In excess, detoxification
reactions may be insufficient or
storage capacity may exceed.
Excess of metal ions may be
countered by export or vacuolar
deposition but metal ions may
also generate oxygen radicals.
High light stress
Excess light can lead to
increased production of highly
reactive intermediates and by-
products that can potentially
cause photo-oxidative damage
and inhibit photosynthesis
Exposure of a plant to light
exceeding what is utilized in
photochemistry leads to
inactivation of photosynthetic
functions and the production of
reactive oxygen species (ROS).
The effects of these ROS can be
the oxidation of lipids, proteins,
and enzymes necessary for the
proper functioning of the
chloroplast and the cell as a
whole.
Abiotic stress-inducible genes
The complex plant response to abiotic stress involves many genes and biochemical
molecular mechanisms. The analyze of the functions of stress-inducible genes is an important
tool not only to understand the molecular mechanisms of stress tolerance and the responses of
higher plants, but also to improve the stress tolerance of crops by gene manipulation. Hundreds
of genes are thought to be involved in abiotic stress responses. Many drought-inducible genes are

also induced by salt stress and cold, which suggests the existence of similar mechanisms of stress
responses.
These genes are classified into three major groups:
1.Those that encode products that directly protect plant cells against stresses such as heat stress
proteins (HSPs) or chaperones, LEA proteins, osmoprotectants, antifreeze proteins,
detoxification enzymes and free-radical scavengers.
2.Those that are involved in signalling cascades and in transcriptional control, such as Mitogen-
activated protein kinase (MAPK), Calcium-dependent protein kinase (CDPK) and SOS kinase,
phospholipases and transcriptional factors.
3.Those that are involved in water and ion uptake and transport such as aquaporins and ion
transporters.
Transcriptional factor genes involved in abiotic stress
Plant growth and productivity are under constant threat from environmental changes in
the form of various stress factors. The most common abiotic stresses are drought, flooding or
submergence, salinity, extreme temperatures (heat and freezing) and high light. Furthermore, the
continued modification of the atmosphere by human activities lead to increase in the
concentration of ozone in the troposphere and this can generate oxidative stress, which leads to
the destruction of proteins and cells, premature ageing and reduced crop yields.Tolerance or
susceptibility to these abiotic stresses is a very complex phenomenon, both because stress may
occur at multiple stages of plant development and more than one stress simultaneously affects
the plant. Therefore, the perception of abiotic stresses and signal transduction to switch on
adaptive responses are critical steps in determining the survival and reproduction of plants
exposed to adverse environments.
During the past few years, transcriptome analysis has indicated that distinct environmental
stresses induce similar responses. Overlap between stress responses can explain the phenomenon
known as cross-tolerance, a capability to limit collateral damage inflicted by other stresses
accompanying the primary stress.
Responses to abiotic stresses require the production of important metabolic proteins such as
those involved in synthesis of osmoprotectants and regulatory proteins operating in signal
transduction pathways that are kinases or transcription factors (TFs). The regulation of these
Fig. 1. Transcriptional network of abiotic stress responses.

genes requires
proteins operating
in the signal
transduction pathways, such as transcriptional factors, which regulate gene expression by
binding to specific DNA sequences in the promoters of respective target genes. This type of
transcriptional regulatory system is called regulon. At least four different regulons that are active
in response to abiotic stresses have been identified. Dehydration-responsive element binding
protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 regulons function in abscisic
acid (ABA)-independent gene expression, whereas the ABA responsive element (ABRE)
binding protein (AREB)/ABRE binding factor (ABF) regulon functions in ABA-dependent gene
expression.
In addition to these major pathways, other regulons, including the NAC (or NAM, No
Apical Meristem) and Myeloblastosis-Myelocytomatosis (MYB/MYC) regulons, are involved
in abiotic stress-responsive gene expression (Fig. 1). Particularly, NAC- type TF OsNAC6 is
induced by abiotic stresses, including cold, drought and high salinity.
Transcriptional factor involved in response to waterlogging stress

Flooding and submergence are two conditions that cannot be tolerated by most plants for
periods of time longer than a few days. These stresses lead to anoxic conditions in the root
system. At a critical oxygen pressure, mitochondrial respiration that provides the energy for
growth in the photosynthetically inactive roots will decrease, and then cease and the cells will
die. Recent reviews on gene expression analysis performed by microarray tools reported as the
expression of several transcription factors, such as heat shock factors, ethylene response binding
proteins, MADS-box proteins, AP2 domain, leucine zipper, zinc finger and WRKY factors,
increases in response to various regimes of oxygen deprivation in Arabidopsis and rice. Recently
using a qRT-PCR platform has identified TFs that are differentially expressed by hypoxic
conditions. Among the TFs that have been characterized, members of the AP2 ⁄ ERF-type family
are the most commonly represented in the set of up-regulated TFs, followed by Zinc-finger and
basic helix-loop-helix (bHLH-type) TFs, while TFs belonging to the bHLH family are the most
commonly represented in the set of down-regulated TFs, together with members from the bZIP
and MYB families. In silico experiments and trans-activation assays shown that some TFs active
in flooding stress are able to regulate the expression of hypoxia responsive genes. Particularly,
five hypoxia-induced TFs (At4g29190; LBD41, At3g02550;HRE1, At1g72360; At1g69570;
At5g66980) from different TF families [Zinc Finger, Ligand Binding Domain (LBD) or Lateral
Organ Boundary Domain, ERF, DNA binding with one finger (DOF), ARF] showed this ability.
Accumulation of ROS is a common consequence of biotic and abiotic stresses, including oxygen
deprivation. There is evidence of redox-sensitive TFs, at least one of which might be involved in
the adaptive response to low oxygen. ZAT12, a putative zinc finger-containing TF, is recognized
as a component in the oxidative stress response signalling network of Arabidopsis, promotes
expression of other TFs and the upregulation of cytosolic ascorbate peroxidase 1, a key enzyme
in the removal of H2O2 .
RNA (Ribonucleic
acid )

RNA is a polymeric molecule essential in various biological roles
in coding, decoding, regulation and expression of genes. RNA and DNA are nucleic acids, and,
along with lipids, proteins and carbohydrates, constitute the four major macromolecule essential
for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike
DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired
double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information
(using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters G,
U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic
information using an RNA genome. Some RNA molecules play an active role within cells by
catalyzing biological reactions, controlling gene expression, or sensing and communicating
responses to cellular signals. One of these active processes is protein synthesis, a universal
function in which RNA molecules direct the synthesis of proteins on ribosome. This process
uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal
RNA (rRNA) then links amino acids together to form coded proteins.
Structure of RNA
RNA is a ribonucleic acid that helps in the synthesis of proteins in our body. This nucleic acid
is responsible for the production of new cells in the human body. It is usually obtained from the
DNA molecule. RNA resembles same as that of DNA, the only difference being that it has a
single strand unlike the DNA which has two strands and it consists of only single ribose sugar
molecule in it. Hence is the name Ribonucleic acid. RNA is also referred to as a enzyme as it
helps in the process of chemical reactions in the body. The ribonucleic acid has all the
components same to that of the DNA with only 2 main differences within it. RNA has the same

nitrogen bases called the adenine, Guanine, Cytosine as that of the DNA except the Thymine
which is replaced by the uracil. Adenine and uracil are considered as the major building blocks
of RNA and both of them form base-pair with the help of 2 hydrogen bonds.
RNA has two major and basic functions as given below-
• Firstly it assists the DNA and acts as a messenger between the DNA and the ribosomes.
• Secondly it helps the ribosomes to choose the right amino acid which is required in
building up of new proteins in the body.
Complementary DNA (cDNA)
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded
RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the
enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes.
When scientists want to express a specific protein in a cell that does not normally express that
protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to
the recipient cell. cDNA is also produced naturally by retroviruses(such as HIV-1, HIV-2, simian
immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates
a provirus. The term cDNA is also used, typically in a bioinformatics context, to refer to an
mRNA transcript's sequence, expressed as DNA bases (GCAT) rather than RNA bases (GCAU).
cDNA is derived from mRNA, so it contains only exons but no introns.
1. From the hairpin loop, a DNA polymerase can then use it as a primer to transcribe a
complementary sequence for the ss cDNA.
2. Now, you should be left with a double stranded cDNA with identical sequence as the
mRNA of interest.

Applications of cDNA
Complementary DNA is often used in gene cloning or as gene probes or in the creation of
a cDNA library. When scientists transfer a gene from one cell into another cell in order to
express the new genetic material as a protein in the recipient cell, the cDNA will be added to the
recipient (rather than the entire gene), because the DNA for an entire gene may include DNA
that does not code for the protein or that interrupts the coding sequence of the protein
(e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence
tags (EST).
2.MATERIALS AND METHODS
2.1 SAMPLE PREPARATION
Nutrient Broth was prepared and the three bacterias RJ12, RJ15 and RJ46
was inoculated in the broth and kept at 36o
C for 24 hrs. These bacterial broth was

then inoculated to the 10 day old tomato seedlings and allowed to grow. After 10
days waterlogging treatment was performed in the 20 days old seedlings of tomato.
Normal watering was done in the control plants and the waterlogging pots were
filled with water upto 5 cm from the soil level. Then after 7 days of waterlogging
leaves from the plants were collected and further crushed with the help of liquid
nitrogen and RNA was isolated.
Fig: Waterlogging treated plants
Fig: Control plants
2.2 RNA ISOLATION FROM PLANTS
KIT USED- NucleoSpin® RNA plant isolation kit.
PRINCIPLE- One of the most important aspects in the isolation of RNA is to prevent
degredation of RNA during the isolation procedure.
With NucleoSpin RNA plant Method,

1. The cells are first disrupted by grinding in the presence of liquid N2.
2. Complete denaturation is achieved by incubation in a solution containing large
amount of chaotropic ions.
(a chaotropic molecule is a molecule in water solution that can disrupt the
hydrogen bonding network between the water molecules)
3. The lysis buffer immedietly inactivates the RNases- which are present in virtually
all biological materials – creates appropriate binding conditions which favors
adsorption of RNA to silica membrane.
4. Contaminating DNA is removed by an rDNase solution wgich is directly applied
onto the silica membrane during the preparation
5. Washing steps with 2 different buffers removes salts, metabolites and macro
molecular cellular components.
6. Pure RNA is finally eluted under low ionic strength conditions with RNase free
H2O.
PROTOCOL
1. Grind up to 100mg tissue under liquid N2. Grind with mortar and pestle – grind the
sample to a fine powder in the presence of liquid N2. Make sure the sample does not thaw
during or after grinding. Add some frozen powder containing β-marceptaethanol
immediately.
2. Add 350 µl Buffer RA1 + 350 µl β-marceptaethanol to 100mg of tissue and vortex.
(If lysate solidifies upon addition of RA1, use 350 µl of RAP buffer)
3. Reduce the viscosity and clear the lysate by filtration through NucleoSpin filter (violet
ring). Place the filter in 2ml collection tube and centrifuge for 1 min at 11000 rpm.
Transfer the filtrate to new 1.5 m; microcentrifuge tube.
4. Discard the NucleoSpin filter. Add 350 µl ethanol (70 %) and mix it by pipetting up and
down or by vortexing
5. For each preparation, take one NucleoSpin RNA plant column (light blue ring). Place it
in a collection tube and load the lysate. Centrifuge for 30 sec at 11000 rpm.
6. Add 350 µl MDB (membrane desalting buffer) and centrifuge at 11000 rpm for 1 min to
dry the membrane.
7. Preparation of DNase reaction mixture
For each isolation---- 10 µl rDNase +90 µl reaction buffer for rDNase.

Now add 95 µl DNase reaction mixtures directly onto the centre of the silica membrane
of the column. Incubate in room temperature for 15 mins.
1st
wash
200 µl buffer RAW2 to the plant column. (Centrifuge for 30sec at 11000rpm). Place the
column in a new collection tube.
2nd
wash
600 µl buffer RA3 to the column. (Centrifuge for 30sec at 11000rpm). Place the column
in a new collection tube.
3rd
wash
250 µl buffer RA3 to the column. (Centrifuge for 2 mins at 11000rpm). Place the column
in a nuclease collection tube.
8. Elute RNA in 60 µl RNase free H2O and centrifuge at 11000 rpm for 1 min. For higher
concentration elute with 40 µl.

Reagents
• 96–100 % ethanol (to prepare Wash Buffer RA3)
• 70 % ethanol (to adjust RNA binding conditions)
• Reducing agent (ß-mercaptoethanol, or DTT (dithiothreithol), as supplement for Lysis Buffer
RA1 Consumables
• 1.5 mL microcentrifuge tubes
• Sterile RNase-free pipette tips Equipment
• Manual pipettors
• Centrifuge for microcentrifuge tubes.

2.3 cDNA SYNTHESIS
KIT USED- GeneSure First Strand cDNA Synthesis Kit
The kit uses Reverse Transcriptase, which lowers RNase H activity compared to AMV reverse
transcriptase. The enzyme maintains activity at 42-50° C and is suitable for synthesis of cDNA
up to 13 kb. The recombinant RNase Inhibitor, effectively protects RNA from degredation at
temperatures up to 55° C. First strand cDNA synthesized with this system can be directly used as
a template inPCR or Real-time PCR. It is also ideal for second strand cDNA synthesis . all the
components should be stored at -20° C.
PROTOCOL
1) After thawing, the components of the kits are centrifuged and mixed and stored in ice.
2) The following reagents are added into sterile, nucleasefree tube on ice in indicated order.
Template RNA Total RNA or
specific RNA
0.1 ng- 5 µg
0.01 pg-0.5 µg
Primer Oligo(dT)
primer or
random
Hexamer
Primer or
gene-specific
Primer
1µl
1µl
15-20 pmol
Water nuclease-free To 12 µl
Total volume 12 µl
3) Incubate at 65o
C for 5 mins.
4) Now these components are added in indicated order:

5X Reaction Buffer 4 µl
RNase inhibitor (200/µl) 1 µl
10 mM dNTP Mix 2 µl
M-MuL V RT (200/µl) 1 µl
Total volume 20 µl
5) They are mixed gently and centrifuged.
6) Now the samples are incubated for 60 mins at 42° C followed by termination of the reaction
by heating at 70°C for 5 mins.
2.4 RT-PCR Amplification
The product of the first strand cDNA synthesis can be used directly in RT-PCR or qPCR. was
performed in the Applied Biosystems Step One Plus Real-time PCR System.
Reaction setup- 10pm- 100µm
Preparation of the sample :

ACTINE
GENE(housekeeping)
CATALASE GENE
Master Mix 10.00 µL 10.00 µL
Forward Primer 0.20 µL 0.20 µL
Reverse Primer 0.20 µL 0.20 µL
Sample 2.00 µL 2.00 µL
H2O 7.60 µL 7.60 µL
Dilution of the Sample :
cDNA Water Total volume
Control (WL) 9.00 µL 5.63 µL 14.63 µL
RJ12 (WL) 8.00 µL 6.06 µL 14.06 µL
Sample plating
C
actine
C
actine
C
catalase
C
actine
C
catalase
C
catalase
WL
actine
WL
catalase
WL
actine
WL
actine
WL
catalase
WL
catalase

3.RESULTS AND DISCUSION
3.1 Isolation of RNA using NucleoSpin Plant kit of the water logging Bhut Jolokia Leaves
1st
set Concentration Absorbance (A260/A280)
Sample 1 130.3 ng/µl 1.89
Sample 3 200 ng/µl 1.95
2nd
set Concentration Absorbance(A260/A280)
3.2 Isolation of RNA using NucleoSpin Plant kit of the water logging Tomato Leaves
Concentration Absorbance (A260/A280)
RJ15 Control 7.7 ng/µl 1.55
RJ15 water logging 55.5 ng/µl 2.06
RJ46 control 92.7 ng/µl 2.05
Normal control 94.6 ng/µl 2.04
Water logging control 12.3 ng/µl 2.00

3.3 Quantification of the cDNA of the isolated samples of RNA of the Bhut Jolokia with the
help of Takara Kit.
1st
set Concentration Absorbance(A260/A280)
2nd
set Concentration Absorbance (A260/280)
3.4 Quantification of the cDNA of the isolated samples of RNA of the Tomato Plant with the
help of GeneSure Kit.
Concentration Absorbance(A260/280)
RJ15 Control 275.5 ng/µl 1.20
Normal control 93.0 ng/µl 1.72
Water logging control 74.0 ng/µl 1.75

3.5 Results of the samples run in RT-PCR
Applied BiosystemsStepOne Software v2.3ExperimentsWaterlogg.eds
Target
Name
Reporter CT CT Mean CT SD Δ CT
Mean
CT
Threshold
Tm 1
actin SYBR 33.46772003 33.97212601 2.920469 0.043820911 80.65464
actin SYBR 37.11194611 33.97212601 2.920469 0.043820911 80.80377
actin SYBR 31.33671761 33.97212601 2.920469 0.043820911 83.19183
catalase SYBR Undetermine
d
0.530779801 65.14435
d
0.530779801 66.34305
d
0.530779801 65.59743
actin SYBR 33.94418716 33.83980942 0.143072 0.043820911 80.65464
actin SYBR 33.89851379 33.83980942 0.143072 0.043820911 80.50551
actin SYBR 33.67671967 33.83980942 0.143072 0.043820911 81.10369
d
36.96339798 0.530779801 80.05962
d
36.96339798 0.530779801 97.96591
catalase SYBR 36.96339798 36.96339798 3.123591 0.530779801 68.43078

Block Type – 96 well
Chemistry SYBR_GREEN
Experiment D:Applied BiosystemsStepOne Software v2.3ExperimentsWaterlogg.eds
Instrument: StepOnePlus
Sample Name Target Name Task CT Mean
c actin Unknown 33.97213
c catalase Unknown
wl actin Unknown 33.83981
wl catalase Unknown 36.9634
Analysis – SinglePlex
Endogenous gene – actin
RQ Min/ Max – 95.0
Reference - c

4. CONCLUSION
Gene expression profiling simultaneously compares the expression levels of
multiple genes between two or more samples. This analysis can help to identify the
molecular basis of phenotypic differences and select gene expression targets for in-
depth study. Real-time PCR is the gold-standard technique for verification of
differential gene expression profiles. Plant gene expression, in response to stress
cues, is tightly controlled by transcriptional regulators. Posttranslational
modifications are a key mechanism to control the activities of transcription factors
(TFs). The regulation of gene expression in plants, as in other higher eukaryotes, is
a subject of daunting complexity. Nevertheless, even a partial understanding of
how plant genes work, in conjunction with the methods of molecular biology and
plant tissue cultures, opens the door to a dazzling array of techniques for
manipulating various aspects of the phenotypes of plants.
In this project, we had analysed the expression of the water logging gene
that is involved in the water logging stress of the tomato plant. Catalase gene
expression study of the waterlogging plant was performed by keeping Actin gene
as the housekeeping gene. After the extraction of RNA from the leaves of the plant
and estimation of the cDNA , the sample is run in the Real-time PCR system. At
present we are at a very early stage of realizing many of the goals that have been
ambitiously (and, in some cases, unrealistically) proposed for manipulating gene
expression in plants, such as nitrogen-fixing cereals, because the systems are not
sufficiently understood at the molecular and genetic levels. For plant biochemists,
study of plant genes, their functions and regulation, is resulting in a quantum leap
in the understanding of familiar biochemical pathways, and the elucidation of less
familiar ones.

REFERENCE
❖ Gene expression in plant https://www.sciencedirect.com/topics/biochemistry-genetics-
and-molecular-biology/gene-expression-in-plant
❖ Plant genes for abiotic stress https://www.intechopen.com/books/abiotic-stress-in-plants-
mechanisms-and-adaptations/plant-genes-for-abiotic-stress
❖ RNAhttps://en.wikipedia.org/wiki/RNA
❖ Complementary DNA
https://www.sciencedirect.com/topics/neuroscience/complementary-dna
❖ Plant stress. What causes it –how to reduce it https://www.coolplanet.com/blog/plant-
stress-what-causes-plant-stress-and-how-to-reduce-it/
❖ Introduction to plant stress https://link.springer.com/chapter/10.1007/978-3-319-59379-
1_1
❖ Water Stress in Plants: Causes, Effects and Responses
https://www.researchgate.net/publication/221921924_Water_Stress_in_Plants_Causes
_Effects_and_Responses
❖ Response of plants to water stress
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952189/
❖ NucleoSpin®
RNA Plant https://www.mn-net.com/tabid/1327/default.aspx
❖ Manual http://www.genetixbiotech.com/manual.php

Expression analysis of water stress related genes in tomato plant 2019

Expression analysis of water stress related genes in tomato plant 2019

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