Biochemical characterization and expression analysis of StCDPK22, a potato CDPK isoform belonging to group III

SCIORRA, M; FANTINO, ELISA; SANTIN FRANCO; ULLOA RITA M

Foz de Iguazu

Congreso; XI International Plamt Molecular Biology Meeting; 2015

IPMB

Biochemical characterization and expression analysis of StCDPK19, a potato CDPK isoform belonging to group IIISCIORRA Marcelo1, FANTINO Elisa1; SANTIN Franco1; ULLOA Rita María1-2. e-mail: msciorra@dna.uba.ar1-Instituto de Investigaciones en Biología Molecular e Ingeniería Genética ?Dr. Héctor N. Torres? INGEBI-CONICET,Vuelta de Obligado 2490 CABA. Argentina 2-FCEyN-DQB-UBACalcium dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. A new potato isoform belonging to group III was cloned using RNA from stems. Isoform StCDPK19,encodes a 59.4 kDa protein (524 aa) withpI: 5.9. In silico analysis revealed that this isoform contains an N-terminal variable domain (NTV) spanning 54 aa, a typical serine/threonine kinase domain (KD) fused through a hinge region to a calmodulin like domain (CLD) presenting only three canonical calcium binding sites (EF hands). The coding sequence was cloned into pET-22b (+) expression vector in order to obtain the recombinant protein tagged with 6 His in its C-terminus. RNA seq data indicates that this isoform is ubiquitously expressed; however RT-PCR expression assays indicated that StCDPK19is mainly present in stems and leaves. The recombinant protein encodes an active kinase. Its biochemical parameters (Km and Vmax) will be determined using syntide-2 and ATP as acceptor and donor substrates respectively. The biochemical parameters of this isoform, its requirement for calcium and its capability to autophosphorylatewill be compared with that of isoform 7 (group I) and isoforms 1,2 and 3 (group II) in order to determine if the lack of one EF-hand affects the kinase activity. This work was supported by CONICET, UBA and ANPCyT.