Call Girl Raipur 📲 9999965857 ヅ10k NiGhT Call Girls In Raipur
Expression analysis of water stress related genes in tomato plant
1. Expression analysis of water stress
related genes in Tomato plants
under the guidance of
Dr Ratul Saikia,
Sr. Principal Scientist of Biological Sciences
And Technology Division(BSTD)
Ron Hazarika
M.Sc. Biotechnology
2nd semester
2. OBJECTIVES
The purpose of this training was to acquire knowledge in the field of biotechnology. The topic given was “Expression
analysis of water stress related genes in Tomato plants”. The project was done through RNA isolation and cDNA
synthesis by the use of certain kits and preparations of samples for RT-PCR.
Here we performed:-
1. RNA isolation from Bhut Jolokia and Tomato plants.
2. cDNA synthesis of the obtained RNA samples.
3. Preparing the samples to run on RT-PCR
3. 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?
4. 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.
5. Materials and methods
For RNAisolationfrom the plant.
• 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.
8. RESULTS AND DISCUSION
Isolation of RNA using NucleoSpin Plant
kit of the water logging Bhut Jolokia
Leaves
First set
Concentration Absorbance
(A260/A280)
Sample 1 130.3 ng/µl 1.89
Sample 2 225.0 ng/µl 1.90
Sample 3 200 ng/µl 1.95
Quantification of the cDNA of the isolated samples
of RNA of the Bhut Jolokia with the help of Takara
Kit.
First set
Concentration Absorbance(A260/A28
0)
Sample 1 190.5 ng/µl 1.48
Sample 2 149.6 ng/µl 1.70
Sample 3 201.3 ng/µl 1.81
9. Concentration Absorbance(A260/A280)
Sample 1 150.4 ng/µl 1.70
Sample 1 210.3 ng/µl 1.88
Sample 1 110.6 ng/µl 1.99
Concentration Absorbance (A260/280)
Sample 1 191.7 ng/µl 1.65
Sample 1 155.4 ng/µl 1.83
Sample 1 189.9 ng/µl 1.82
Isolation of RNA using NucleoSpin Plant kit of the
water logging Bhut Jolokia Leaves
Second set
Quantification of the cDNA of the isolated samples of
RNA of the Bhut Jolokia with the help of Takara Kit.
Second set
10. 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
RJ46 water
logging
39.1 ng/µl 2.05
RJ12 control 78.8 ng/µl 2.09
RJ12 water
logging
28.9 ng/µl 2.06
Normal control 94.6 ng/µl 2.04
Water logging
control
12.3 ng/µl 2.00
Isolation of RNA using NucleoSpin Plant kit
of the water logging Tomato Leaves
11. Quantification of the cDNA of the isolated samples of RNA of the Tomato Plant
with the help of Gene Sure Kit.
Concentration Absorbance(A260/280)
RJ15 Control 275.5 ng/µl 1.20
RJ15 water logging 184.7 ng/µl 1.34
RJ46 control 81.2 ng/µl 1.75
RJ46 water logging 131.8 ng/µl 1.64
RJ12 control 81.3 ng/µl 1.74
RJ12 water logging 87.9 ng/µl 1.83
Normal control 93.0 ng/µl 1.72
Water logging control 74.0 ng/µl 1.75
12. 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. 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. We
concluded that the gene responsible here for water logging stress in the Catalase 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 and the responsible gene is identified and analysed.
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.
13. 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_Effect
s_and_Responses
14. NOTE OF THANKS
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 Parishmita 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.
