Differential Gene Expression Induced by Cold Stress in Olea europaea L.
Investigations on the abiotic stresses in plants consist in the identification of genes involved in the defence response and analysis of the mechanisms regulating their activity. The aim of the present works was to study the differential gene expression induced by cold stress in olive (Olea europaea L.) plants. Cold tolerance in plants is not constitutive, but it is induced by a prolonged exposition at low temperatures. The choice of the biological material is due to the economic relevance that this plant occupies in the Mediterranean country. Since the best quality of olive oil is derived from plants growing in regions frequently subjected to hard-frost (for instance, in Tuscany), it was necessary to set up advanced methodologies for the selection of resistant cultivars. For this purpose, it is necessary to use molecular markers isolated from resistant plants for nursery selection. Molecular biology methodologies were used in order to isolate genes involved in cold-induced tolerance processes in Olea europaea cultivars. In order to systematically isolate and study the involved genes we used the PCR-based cDNA subtraction method, termed suppression subtractive hybridisation (SSH) for generation of subtracted cDNA libraries from tolerant plants, both in control and cold treated material. This powerful method can achieve greater than 1,000 time enrichment for differentially expressed cDNAs. Samples of Olea europaea L. cv. Leccino, tolerant to low temperatures, were used. This material was selected from plants survived in Tuscany after very low temperature exposition in the year 1985. The clones were cultured for two years in controlled conditions. Control samples were kept in a mean temperature of 25 °C; cold stressed samples were treated with decreasing temperatures till to -10 °C. RNAs were extracted from control (25 °C) and cold treated (-10 °C) plants and used for the construction of reverse and forward cDNA libraries, that allow the identification of genes that are switched on/off after the treatments. Forward and reverse libraries are constituted respectively of sequences expressed and suppressed or diminished after the stressing treatment. The system allowed the rapid identification of differentially expressed genes. Selected cDNA clones were sequenced and analysed using the FASTA, BLASTX and BLASTN programmes. The expression levels of the sequences corresponding to the isolated clones were evaluated through slot blot and Northern blot of the total RNA from treated and control materials and the subsequent hybridisation using as labelled probes the selected clones.
Titolo: Differential Gene Expression Induced by Cold Stress in Olea europaea L.
BERNARDI, RODOLFO
ADAMO S.
FONTANA F.
MANZO M.
SALVINI M.
PETRUCCELLI R.
BARTOLINI G. , DURANTE M.
Abstract: Investigations on the abiotic stresses in plants consist in the identification of genes involved in the defence response and analysis of the mechanisms regulating their activity. The aim of the present works was to study the differential gene expression induced by cold stress in olive (Olea europaea L.) plants. Cold tolerance in plants is not constitutive, but it is induced by a prolonged exposition at low temperatures. The choice of the biological material is due to the economic relevance that this plant occupies in the Mediterranean country. Since the best quality of olive oil is derived from plants growing in regions frequently subjected to hard-frost (for instance, in Tuscany), it was necessary to set up advanced methodologies for the selection of resistant cultivars. For this purpose, it is necessary to use molecular markers isolated from resistant plants for nursery selection. Molecular biology methodologies were used in order to isolate genes involved in cold-induced tolerance processes in Olea europaea cultivars. In order to systematically isolate and study the involved genes we used the PCR-based cDNA subtraction method, termed suppression subtractive hybridisation (SSH) for generation of subtracted cDNA libraries from tolerant plants, both in control and cold treated material. This powerful method can achieve greater than 1,000 time enrichment for differentially expressed cDNAs. Samples of Olea europaea L. cv. Leccino, tolerant to low temperatures, were used. This material was selected from plants survived in Tuscany after very low temperature exposition in the year 1985. The clones were cultured for two years in controlled conditions. Control samples were kept in a mean temperature of 25 °C; cold stressed samples were treated with decreasing temperatures till to -10 °C. RNAs were extracted from control (25 °C) and cold treated (-10 °C) plants and used for the construction of reverse and forward cDNA libraries, that allow the identification of genes that are switched on/off after the treatments. Forward and reverse libraries are constituted respectively of sequences expressed and suppressed or diminished after the stressing treatment. The system allowed the rapid identification of differentially expressed genes. Selected cDNA clones were sequenced and analysed using the FASTA, BLASTX and BLASTN programmes. The expression levels of the sequences corresponding to the isolated clones were evaluated through slot blot and Northern blot of the total RNA from treated and control materials and the subsequent hybridisation using as labelled probes the selected clones.