Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
ISOLATION AND CHARACTERIZATION OF POTATO STRESS RESPONSIVE GENES BY A FUNCTIONAL APPROACH IN E. COLI
MASSARELLI I., CIOFFI R., GRILLO S., LEONE A.
Research Institute for Vegetable and Ornamental Plant Breeding (CNR-IMOF), Via Università 133, 80055 Portici, Italy
Tel. 081 7885411
potato cells, Solanum commersonii, gene isolation, osmotic stress
Drought and high salinity, are major abiotic stress-limiting factors for crop productivity. These two stress conditions, which share a common osmotic component, affects almost all plant functions, including growth and development and takes effect on microorganism vitality. Plants and microorganism respond to osmotic stresses by activating mechanisms of repair and protection of cellular metabolism as a result of the action of different genes, which operate in a complex and coordinate network.
In order to identify key gene functions involved in the potato cellular response to stress we adopted a functional approach. The methodology used led to the isolation of potato gene whose over-expression enables E. coli cells to become NaCl tolerant. A cDNA library from Solanum commersonii was used to generate a phagemid library by helper phage-mediated in vivo excision. After transformation of E.coli cells with the rescued library, the IPTG-induced bacterial cells were selected on plates supplemented with different concentration of NaCl (0,5-1,17 M). After repeated selection, 12 salt stress tolerant colonies (N1-N12), containing plasmids with 0.8 - 2.5 kb inserts, were obtained. The inserts were isolated, recloned and introduced into a different E.coli strain. All transformed cells exhibited tolerance in the range 0.5-1.17 M NaCl.
Eight of the cDNA clones were sequenced and partially characterized. Comparative sequence analyses showed that N1 and N8 have significant homology with known plant genes. N1 has high homology (66%) to dehydrin from different plant species, while N8 has 98% identity to Solanum tuberosum chaperonin 60 B subunit. The remaining clones, all characterized by sequence domains (myristilation, phosphorylation, Ca++ binding motifs) characteristic of genes involved in signalling cascades, do not display any significant homology with already described proteins. Homology search identified similar gene sequences of unknown function in the arabidopsis genome.
Preliminary Northern analysis revealed that the expression of N1, N2.1, N2.2 and N8 clones were rapidly induced in potato cells upon high salt ( NaCl 200mM) e water deficit (PEG 20%) conditions, thus suggesting a putative role of the genes in the potato cellular metabolism under stress conditions.
The present data show that the approach utilized is valuable for the identification of plant genes involved in response to stress signals. Further characterization of the corresponding full-length cDNAs will give information about the functional role of the isolated genes in the metabolic and molecular events occurring in the cellular response to environmental stress and in the adaptation process.