MOLECULAR CLONING AND CHARACTERIZATION OF StCDPK3

GRANDELLIS CAROLINA; GIAMMARIA VERÓNICA; ULLOA RITA

Mar del Plata

Congreso; XLIII reunión de SAIB; 2007

SAIB

Ca2+ is a crucial signalling molecule in plants involved in the transduction of environmental, hormonal and developmental stimuli. Ca2+-dependent protein kinases (CDPKs) play distinct roles in mediating Ca2+ signalling. CDPKs are monomeric proteins, with an N-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). Different CDPK isoforms are expressed during stolon to tuber transition in potato plants: StCDPK3 in early stolons and StCDPK1 in induced stolons. StCDPK3 was isolated as a partial clone containing the autoinhibitory domain, the CaM-LD and part of the 3´UTR region; it is 98% homologous to LeCPK1 and NtCDPK1, two CDPK from tomato and tobacco. StCDPK3 was used to probe a genomic potato library prepared in Dr Glenn Bryan´s lab at SCRI. A BAC clone, B15, containing the StCDPK3 gene, was isolated. The 5´end of the gene was amplified using primers directed against the 5´UTR region of LeCPK1 and NtCDPK1, subcloned into pGEM-T and sequenced. StCDPK3 presents an N-terminal myristoylation and palmitoylation consensus. Expression of StCDPK3 was analysed in different tissues, the complete coding sequence was cloned and the recombinant StCDPK3-6xHis protein will be expressed in bacteria. The structure of the gene is being analysed. We expect to obtain promoter sequences to understand the transcriptional regulation of StCDPK3.