Expression and characterization of a calcium dependent protein kinase from wheat

CALÓ G; MARTÍNEZ-NOËL G; PONTIS H

Pinamar

Congreso; XLI Reunión Anual SAIB; 2005

SAIB

Calcium-dependent protein kinases (CDPKs) include an extensive group of signaling molecules that mediate many processes in plants, such as growth, development and responses to external stimuli. We have previously identified in the wheat genome an ORF encoding a putative CDPK (Tacdpk1) that is up-regulated by sucrose (Suc) and possibly involved in sugar signaling. The aim of this work was the expression of Tacdpk1 in an heterolog system (E. coli) and its characterization. For this purpose the full length encoding sequence (1828 bp) was obtained by RT-PCR from the RNA extracted from wheat leaves treated with Suc. The deduced protein had the typical structural features of the CDPK family and high similarity with other CDPKs (>70%). The Tacdpk1 was expressed in E. coli as a 6xHis fusion protein using the pRSETA vector. The recombinant protein was in the soluble fraction and had the expected size (/58 kDa). Immunoassays with antibodies against a conserved region of CDPKs confirmed the identity of the heterolog protein. Expression analyses of Tacdpk1 by semiquantitative RT-PCR were carried out in leaves and roots under cold, salt stress and hormones treatments. In conclusion, in this work we expressed the first CDPK from wheat and gave more evidences for its possible role on signaling. Supported by ANPCyT, CONICET, UNMdP and FIBA.