This is my dummy proposal for for research design and development course from which I learned a lot about the structure of research proposals and their requirements....

1. TRANSCRIPT EXPRESSION
PROFILING OF ANNEXIN MULTIGENE
FAMILY IN RICE Oryza sativa GROUP
indica cv. IR64 , N22, PRH 10 and Pusa
Sugandh UNDER DROUGHT STRESS
By:
Prathu Raj Singh Kushwah

2. Problem Statement
 Research has shown an up-regulation of plant
Annexins under different abiotic stress responses as
they mediate in Calcium signalling pathways.
 Expression profiling study of Arabidopsis Annexin
genes demonstrated the differential expression under
various abiotic stresses (Barthakur et al., 2006).
 Recently Jami et al. (2008) showed that ectopic
expression of an annexin from Brassica juncea
confers drought tolerance in transgenic tobacco
plants.
 However, no direct research has been conducted
using RT-PCR based expression analysis of different
members of indica rice annexin gene family under
normal growth conditions and abiotic stresses.

3. Significance
 Present Study aims at establishing role of annexin
multigene family under drought stress.
 The identification and isolation of genes associated
with drought stress tolerance is of major importance in
order to better understand this trait and increase the
efficiency in developing drought stress resistant
varieties.
 Some of the varieties used in my study are already
drought resistant so their study could help me confirm
the role of annexins in drought stress.
 This study could go deeper into study of vrious abiotic
stresses in plants such as Temperature, Chemical
Stresses and high wind exposure.

4. Literature Review
 Abiotic Stress effects
 Genetic elements involved
 Annexins

5. ENVIRONMENTAL STRESS
(ABIOTIC) (BIOTIC)
STRESS RESPONSE
RESISTANCE SUSCEPTIBILITY AVOIDANCE
ACCLIMATION SENESCENCE
SURVIVAL
GROWTH DEATH
SURVIVAL

6. Genetic Involvement
Allelic variation in genes that underlie the adaptive responses should be useful
in breeding programs for improved stress tolerance (Lafitte et al., 2004)

7. Annexins
 Annexins are multigene, multifunctional family of proteins with a
broad taxonomic distribution capable of Ca2+ -dependent and Ca2+ -
independent binding to endomembrane and plasma membrane.
 Members of the annexin protein family have been continuing to
grow since their association with intracellular membranes was first
reported in 1977. Their ability to “annex” or aggregate membranes
at certain Ca2+ concentrations led to the initial interest in these
proteins as components in Ca2+ - mediated secretion.
 First discovery of plant annexins was made by Boustead et al.
(1989) from suspension culture of tomato (Lycopersicon
esculentum) cells.
 Over 200 unique annexin sequences have been described in more
than 65 species covering plants, fungi, protists, higher vertebrates,
and also in a prokaryote (Morgan et al., 2006).

8. Location of Annexins
Structure of Annexins

9. Functions of Annexins
Anticipated functions of plant annexins are
 Sensor and channel activity
 Exocytosis
 Growth and development
 Actin binding
 Peroxidase activity
 ATPase and GTPase activity
 Annexins and reactive oxygen species
 Light responses and nyctinastic movement
 Responding to stress stimuli and pathogens

10. Expression Profiling
 Expression profiling based on RT-PCR, Northern blotting
and quantitative real time PCR is one of the major aspect
to study the expression of various genes in response to
various abiotic and biotic factors.
 These factors result in down-regulation and up-regulation
of genes in various phases of the life cycle of the plant.
Expression profiling help us in finding the level of various
genes expressed in different parts of the plants, in different
time period.
 Expression profiling study of Arabidopsis annexin genes
demonstrated the differential expression under various
abiotic stresses (Barthakur et al., 2006). Recently Jami et
al. (2008) showed that ectopic expression of an annexin
from Brassica juncea confers drought tolerance in

11. Objective Hypothesis
To perform transcript As annexins are
expression profiling by supposed to have role
Reverse Transcriptase in calcium mediated
Polymerase Chain
Reaction (RT-PCR) of signal transduction
seven members of cascades, in drought
indica rice annexin stress we should have
gene family from a diffrential expression of
drought sensitive
cultivars N22, Pusa different members of
sugandh, PRH10 and Annexin multigene
IR64 under normal family.
growth conditions and
drought stress.

12. Work Plan
 Grow different species of rice uptill 10 day old seedling stage.
 Give them drought stress for 3, 6, 9 and 12 days respectively
(keeping 0 day as Control and also different controls for 3, 6,
9 and 12 days respectively.
 Isolate RNA from Different Samples and perform RT-PCR to
get required cDNAs.
 Perform end point expression profiling using seven Annexin
primers for each sample (Use 3 tubes for each reaction)
 Compare the band intensity for characterizing Up Regulation
or Down Regulation of different annexins.
 Confirm the validity of Hypothesis and try to establish roles of
different annexins.

13. Evaluation of
Drought Stress
quality of RNA
Treatment and
Harvesting of RNA by gel
electrophoresis
Rice Seedlings Isolation
Process Removal of DNA
Checking of
Descriptio from RNA
samples (via
Various PCR
products for up
n DNAse
treatment)
regulation and Amplification of
down regulation of target cDNA by
various gene PCR, with 7
primers different primers
for different
cDNA
members of Synthesis
AnnOSI gene

14. Experimental Design
Rice pots (2x4x9) cDNA Samples
(72)
Significance Treatments
Variety
PCR Reactions
(72x7)
Final Results (4x2)

15. Timeline:
Job Time
Pots Growth (Start line) 2 Weeks
Treatment & Collection 2 weeks
RNA Isolation & DNAse 2 Weeks
Treatment
RT PCR, & cDNA quantification 1 Week
Primer Standardization 3 Weeks
PCR Reactions 6 Weeks
Data Collection 2 Weeks
Reporting & literature review 3 Weeks
Total 19 Weeks (~5 Months)

16. Costs:
Reagents Cost (US$)
Seeds and Soil 200.00
Qiagen RNAeasy plant minikit 620.00
One Step RT PCR Kit 527.00
Primers 98.00
Promega PCR MM 1212.00
Lab Supplies 400.00
Agarose 137.69
Total 3194.69

17. Limits
 Use of Drought stress only for Oryza Sativa
Indica Varieties.
 No direct analyses of loss or gain of function.

18. References
 Boustead C M Smallwood M Small H Bowels D J and Walker J H
(1989) Identification of calcium-dependent phospholipid- binding proteins
in higher plant cells. Federation of European Biochemical Societies.
244(2): 456-460.
 Lafitte H R Ismail A Bennett J (2004) Abiotic stress tolerance in
rice for Asia: progress and future. Proceedings of the 4th
International Crop Science Congress, 26th September- 10th
October 2004, Brisbane, Australia.
 Levitt J (1972) Responses of plants to environmental stresses. Academic
Press, New York
 Morgan R O Martin Almedina S Garcia M Jhoncon- Kooyip J
Fernadez M (2006) Deciphering function and mechanism of
calcium-binding proteins from their evolutionary
imprints. Biochemica et Biophysica Acta 1763: 1238-
1249.
 Price A Cairns J Horton P et al (2002) Linking drought-resistance
mechanisms to drought avoidance in upland rice using a QTL
approach: progress and new opportunities to integrate stomatal and
mesophyll responses. Journal Experimental Botany 53:989–1004.

19. Thank
You!
