Cloning and biochemical properties of CDPK gene OsCDPK14 from rice.
A rice CDPK gene, OsCDPK14 (AY144497), was cloned from developing caryopses of rice (Oryza sativa cv. Zhonghua 15). Its cDNA sequence (1922 bp) contains an ORF encoding a 514 amino acids protein (56.7kD, pl 5.18). OsCDPK14 shows the typical structural features of the CDPK family, including a conserved catalytic Ser/Thr kinase domain, an autoinhibitory domain and a CaM-like domain with four putative Ca2+-binding EF hands. Subcellular targeting indicated that OsCDPK14 was located in the cytoplasm, probably due to the absence of myristoylation and palmitoylation motifs. OsCDPK14 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using Syntide-2, a synthetic peptide. Kinase activity was shown to be Ca2+-dependent, and this activation was strongly enhanced by Mn2+ and inhibited by W7 in vitro. These results provide significant insights into the regulation and biochemical properties of OsCDPK14, suggesting OsCDPK14 may be a signal factor of cytoplasm in rice plant.
Zhang
Tiegang
T
Laboratory of Plant Development Physiology and Molecular Biology, College of Life Sciences, Beijing Normal University, PR China.
Wang
Qi
Q
Chen
Xing
X
Tian
Chunhua
C
Wang
Xinshen
X
Xing
Tim
T
Li
Yi
Y
Wang
Yingdian
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D017931:DNA Primers; D018744:DNA, Plant; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases