cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases.
Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library. We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings. These clones were classified into four groups based on their DNA cross-hybridization. Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized. The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively. The catalytic and regulatory domains were linked by a well-conserved junction domain. The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal. Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves. To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags. A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate.
Saijo
Y
Y
Laboratory of Applied Botany, Faculty of Agriculture, Kyoto University, Japan.
Hata
S
S
Sheen
J
J
Izui
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases