MOLECULAR CLONING OF Stcdpk1 GENE

GARGANTINI P; GIAMMARIA V; ULLOA, R M

Pinamar, Prov. de Buenos Aires

Congreso; XLI reunión de SAIB; 2005

Sociedad Argentina de Investigaciones Bioquímicas

In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are key intermediates in calcium-mediated signalling, that couple changes in Ca2+ levels to a specific response. These enzymes have a unique structure consisting of an aminoterminal catalytic domain fused to a carboxyterminal calmodulin-like domain (CLD) with four EF hands Ca2+ binding sites, and require only micromolar concentrations of free Ca2+ for their activity (Harmon et al., 1986; Harper et al., 1991).During potato tuberization, dormancy and sprouting of potato tubers, StCDPK1 (GenBank Database accession numbers AF115406) is differentially expressed .In order to clone the Stcdpk1 gene, a CLONTECH genomic library was screened using the full-length StCDPK1 cDNA as probe. One of the positive clones contained the 3 end of the gene. Based on this sequence and on StCDPK1 coding sequence a PCR approach was used to amplify the complete gene. Stcdpk1 spans a 6290 pb region from the ATG to the polyadenylation consensus. The gene comprises 8 exons and 7 introns. E1, E2 and E3 encode the protein kinase domain while E4 encodes the junction region and the CLD is encoded by 4 small exons. Most CDPKs from Arabidopsis share this structure but with smaller intronic regions. Mapping of Stcdpk1 gene in the potato genome is currently being performed by SSCP analysis.