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

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.

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.

Literature Review

Abiotic Stress effects Genetic elements involved Annexins

ENVIRONMENTAL STRESS

(ABIOTIC) (BIOTIC)

STRESS RESPONSE

RESISTANCE

ACCLIMATION

GROWTH

SURVIVAL

SUSCEPTIBILITY

SENESCENCE

DEATH

AVOIDANCE

SURVIVAL

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)

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).

Structure of Annexins

Location of Annexins

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

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 transgenic tobacco plants.

To perform transcript expression profiling by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) of seven members of indica rice annexin gene family from a drought sensitive cultivars N22, Pusa sugandh, PRH10 and IR64 under normal growth conditions and drought stress.

As annexins are supposed to have role in calcium mediated signal transduction cascades, in drought stress we should have diffrential expression of different members of Annexin multigene family.

Objective Hypothesis

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.

Drought Stress

Treatment and

Harvesting of Rice

Seedlings

RNA Isolatio

n

Evaluation of quality of RNA

by gel electrophoresi

s

Removal of DNA from RNA samples (via

DNAse treatment)

cDNA Synthesi

s

Amplification of target cDNA by PCR, with 7

different primers for

different members of

AnnOSI gene family

Checking of Various PCR

products for up regulation and

down regulation of various gene

primers

Process Descripti

on

Experimental Design

Rice pots (2x4x9)

Significance

Variety

Treatments

cDNA Samples(72)

PCR Reactions(72x7)

Final Results (4x2)

Timeline:

Job Time

Pots Growth (Start line) 2 Weeks

Treatment & Collection 2 weeks

RNA Isolation & DNAse Treatment

2 Weeks

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)

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

Limits

Use of Drought stress only for Oryza Sativa Indica Varieties.

No direct analyses of loss or gain of function.

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.

Thank You!