Cloning of StCDPK1 and StCDPK3 promoters using Genome Walker kit and fusion to a reporter gene

GRANDELLIS C; HANNAPEL D; ULLOA RM

Tucumán

Congreso; XLV reunión de SAIB; 2009

SAIB

Cloning of StCDPK1 and StCDPK3 promoter sequences using Genome Walker Kit and fusion to a reporter gene (GUS) Carolina Grandellis1, David Hannapel2, Rita M. Ulloa1 1INGEBI-CONICET-UBA, Buenos Aires, Argentina 2 Iowa State University, IA, USA  Transient changes in the cytosolic calcium concentration are sensed and decoded by calcium sensors, such as calcium-dependent protein kinases (CDPKs). Protein phosphorylation functions as a major mechanism involved in transducing external stimuli. Our group has cloned and characterized three CDPK isoforms, StCDPK1, StCDPK2 and StCDPK3, which are differentially expressed during tuber development and sprouting. To perform functional studies of these genes, the promoter sequences of StCDPK1 and StCDPK3 were amplified using Genome Walker Kit. Genomic DNA libraries were generated using genomic DNA from potato. Gene specific primers were designed for StCDPK1 and StCDPK3. Upstream sequences of both genes (StCDPK1, 2279 bp and StCDPK3,1841 bp) were amplified, cloned into pBI101 containing the reporter gene â-glucuronidase and transformed into Agrobacterium tumefaciens GV2206 strain. Two different potato subspecies (Solanum tuberosum ssp andigena and tuberosum var. Spunta) were transformed using leaf and tuber explants. In silico analysis using the PlantCARE was performed for both promoters. In the future, molecular characterization of the transgenic events and histochemical analysis will be performed and the pattern of tissue expression and distribution of StCDPK1 and StCDPK3 will be compared.