Proceedings
of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
ISBN 88-900622-1-5
Poster Abstract
SUPPRESSION SUBTRACTIVE HYBRIDISATION FOR COLD
STRESS INDUCED GENE ACTIVATION AND INACTIVATION IN OLEA EUROPAEA L.
BERNARDI R.*, ADAMO S.*, SALVINI M.*, MANZO M.*,
BARTOLINI G.**, DURANTE M.*
* Dipartimento di Biologia delle Piante Agrarie, Sez.
Genetica, Via Matteotti 1/B, 56124 Pisa
mdurante@agr.unipi.it; rbernard@agr.unipi.it
** Istituto Propagazione Specie Legnose, C.N.R. Via
Ponte di Formicola 76, Scandicci, (FI)
Olea europaea L., cold stress, suppression subtractive
hybridisation
Plant growth and
development are strictly related to environmental factors such as water,
nutrilites, quantity and quality of light, temperature. For this reason plants
developed the capability of adaptive responses, genetically induced, to the
environmental challenges. The genetic approach to the study of stress tolerance
in the plants consists in the identification of genes and regulatory mechanisms
involved in the defence responses.
In this
preliminary work we are trying to characterise the gene expression induced by
low temperatures in olive(Olea europaea L.) plants. 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.
In the present
work we have applied a new method, termed suppression subtractive hybridisation
(SSH) for the generation of subtracted cDNA libraries and for the
identification and isolation of differentially expressed transcripts after cold
treatments. The technique is base on the construction of reverse and forward
cDNA libraries that allow the identification of the genes that are switched on
/off after the treatments.
We demonstrated
the effectiveness of the SSH method by generating cold stress specific cDNA
libraries and characterising selected cDNA clones that were sequenced and
analysed using the FASTA, BLASTX and BLASTN programmes.
Till now have been
isolated the five clones SAG3, SA, SABS, SAAC and SABP. The SAG3 clone, coming
from the forward library, is constituted by a sequence homologous to open
reading frame ycf2 isolated from Spinacia oleracea cpDNA whose
putative product seems to be indispensable for cell survival. The SA clone
isolated from the reverse library shows a high identity with the ATAB5238
sequence from Arabidopsis thaliana: its function is
unknown. SABS clone codifies for bark storage proteins; SABP codifies for
chlorophyll A/B binding proteins: both are differentially modulated in control
and cold treated plants. SAAC codifies for cp carbonic anhydrase and is active
in the control plants.