DIFFERENTIAL GENE EXPRESSION ANALYSIS OF DURUM WHEAT UNDER SALT STRESS CONDITION Olimpia D’Onofrio 1 , Filomena Carriero 1 , Pasquale De Vita 2 , Teresa Mango 1 , Giovanni Sozio 1 , Paolo Riccardi 3 and Rosa Anna Cifarelli 1 . 1 Metapontum Agrobios – S.S. Jonica 106 km 448,2 – 75010 Metaponto (MT), Italy 2 C.R. E.N.E.A. Trisaia – S.S. Jonica 106 km 419,2 – 75026 Rotondella (MT), Italy 3 University of Basilicata – Biotechnology Department – C.da Macchia Romana – 85100 Potenza, Italy E-mail:odonofrio@agrobios.it 5 th Plant GEMs MEETING Venice 11/14 October 2006 Introduction The monitoring of gene expression levels in cells for thousands of genes simultaneously is the power of the DNA microarrays. DNA arrays can be used for many different purposes, most prominently to measure levels of gene expression (messenger RNA abundance) for tens of thousands of genes simultaneously (1). The present study was designed to target the early responses of genes to salinity stress at seedling stage, using a homemade durum wheat microarray. It is essential to study the functions of stress-inducible genes to understand the molecular mechanisms of stress tolerance and the responses of higher plants (2, 3). It was compared unstressed vs. salt stressed durum wheat (Triticum durum Desf. cv. Adamello) plants, since this species has considerable economic importance in salinity-affected arid and semiarid regions of the world. In wheat many genes are, probably, involved in abiotic stress responses, so it was applied the durum wheat cDNA microarray to identify new salt stress inducible genes and to examine the differences between their expression profile in three different salt concentrations. Preliminary results showed that there are differentially expressed genes between the stressed and unstressed samples. Material and methods The flow chart below outlines the experimental steps: References 1. R.A.Cifarelli et al. Macroarray Expression Analysis Of Transgenic And Wild Type Durum Wheat Plants. (2004) SIGA Joint Meeting Lecce, Italy – 15-18 September 2. Seki M. et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high- salinity stresses using a full-length cDNA microarray. (2002) Plant J. Aug;31(3):279-92 3. Hwang EW, Kim KA, Park SC, Jeong MJ, Byun MO, Kwon HB. Expression profiles of hot pepper (Capsicum annum) genes under cold stress conditions. J Biosci. 2005 Dec;30(5):657-67. 4. Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868. Acknowledgements We thank Mr. G. Lauria for technical support, Dr. F. Cellini for scientific support, Dr. S. Errico and ENEA Biotech laboratory for microarray customer services. Samples: Stressed Unstressed Replicas: Labeling: Arrays: Design: R G RNA1 RNA2 RNA3 RNA4 A 1 A 2 B 1 B 2 R G R G R G A 1 B 1 A 2 B 2 Isolated total RNA from leaves of salt stressed and unstressed durum wheat Performed first-strand cDNA synthesis using SuperScript'' III RT and amino-modified dNTPs. (Invitrogen) Purified the amino-modified cDNA Performed the fluorescent dye coupling reaction. (Amersham) Purified the labeled cDNA Fig.1 Experimental design Fig.2 A normalized MA-plot for one microarray Fig. 3 Clustered display of gene expression data from three experiments of salinity stress. The dendrogram and coloured image were produced using a clustering program (4), so that genes with similar expression patterns could be grouped together. The colour scale ranges from saturated green for log ratios -2.0 and below (repression) to saturated red for log ratios 2.0 and above (induction). Each gene is presented by a single row of coloured box and each experiment is represented by a single column. Cluster containing repressed genes is shown enlarged at right along with the protein showing the highest similarity from a BLASTX comparison to the non-redundant EMBL database. Results and discussion 9248 sequenced cDNA clones of a “totipotent” durum wheat expression library were amplified and each PCR fragment was spotted on glass slide by using the SpotArray 24 (Perkin Elmer). cDNA microarrays were hybridised with Cy3 and Cy5 fluorescently labelled probe pairs of salt-treated plus unstressed plants. The analysis were performed with total RNA prepared from three different salt (NaCl) concentrations: a) 50 mM, b) 100 mM and c) 150 mM. Every experiment was made in dye-swap with biological replica. The images, scanned by a ScanArray Express scanner (Perkin Elmer), were visually inspected to flag and exclude the abnormal spots with irregular shape or dirt. The signal on each slide was normalized using within-print-tip-group lowess normalization with default parameters. Analyses were carried out on log2- transformed intensities and ratios. Genes that were considered as differential expressed have a |log2-ratio|>2, that is with intensity value 4-fold greater. Hierarchical clustering with the average linkage method was employed only on those genes that showed different expression in one or more salt concentration. The cluster data were visualized by the Treeview program (4). This study showed that salt stress has a different effect on expression patterns of known, novel or unknown genes, in the three concentrations studied, specially in 100 and 150 mM (tab.1). That work is useful to next research about the genetic traits involved in grow up of wheat under salt stress conditions. The results will be confirmed by RT-PCR. Hybridisation A: Unstressed B:150mM salt stressed A B Tab.2 Functional classification of genes induced (red) or repressed (green), while the unknown are not reported. The NaCl concentration is expressed in mM. Putative function Putative protein mM Ribosomal Protein 40S ribosomal protein S4 100 Ubiquitin 100 Proteasome subunit alpha type 6 100 Pathogenesis-related protein precursor 150 Non-specific lipid-transfer protein 4.1 precursor 150 Glutathione S-transferase 100 Cytochrome P 450 monooxygenase 150 L-ascorbate peroxidase 1 cytosolic 150 C3 photosynthesis RuBisCO small subunit 150 Protein transport Oligopeptide transporter 7 100 Cytocrome P 450 monooxygenase 150 Bowman-Birk type trypsin inhibitor 150 Putative Sip 1 protein 100 S-adenosylmethionine decarboxylase proenzyme 100 Putative protease inhibitor 100 Cellular cycle regulation Rae 1 like protein 100 Folding and stability FKBP12-interacting protein of 37 kDa 50 Proteolysis Defence related Detoxification Stress responses View publication stats View publication stats