TRANSCRIPT EXPRESSIONPROFILING OF ANNEXIN MULTIGENE FAMILY IN RICE Oryza sativa GROUPindica 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
Genetic InvolvementAllelic 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).
Functions of AnnexinsAnticipated 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
Objective HypothesisTo perform transcript As annexins areexpression profiling by supposed to have roleReverse Transcriptase in calcium mediatedPolymerase ChainReaction (RT-PCR) of signal transductionseven members of cascades, in droughtindica rice annexin stress we should havegene family from a diffrential expression ofdrought sensitivecultivars N22, Pusa different members ofsugandh, PRH10 and Annexin multigeneIR64 under normal family.growth conditions anddrought stress.
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.
Evaluation ofDrought Stress quality of RNATreatment and Harvesting of RNA by gel electrophoresisRice 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
Costs: Reagents Cost (US$) Seeds and Soil 200.00Qiagen 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.