CDNA CLONING AND PROKARYOTIC EXPRESSION OF MAIZE CALCIUM-DEPENDENT PROTEIN-KINASES

Using degenerate oligonucleotide primers corresponding to conserved re gions of the calcium-dependent protein kinase (CDPK) family, we carrie d out a polymerase chain reaction and obtained four distinct partial-l ength cDNAs from a maize leaf library. We then used these clones as pr obes for conventional screening and isolated 19 longer clones from ano ther cDNA library of maize seedlings. These clones were classified int o four groups based on their DNA cross-hybridization, Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and character ized. The predicted protein of each clone was a typical CDPK with elev en canonical subdomains of protein kinases, and four EF-hand calcium-b inding motifs in its N-terminal and C-terminal halves, respectively. T he catalytic and regulatory domains were linked by a well-conserved ju nction domain. The N-terminus of the protein also contained a consensu s sequence for an N-myristoylation signal. Northern blot analysis show ed that the transcription level of each gene was higher in roots and e tiolated leaves than in green leaves. To confirm the calcium dependenc y of the maize enzymes, the entire coding region of ZmCDPK9 was subclo ned into an expression vector so that it was in frame with the vector- encoded peptide tags. A cell-free extract of Escherichia coli transfor med with the recombinant plasmid exhibited calcium-dependent phosphory lation activity, using casein as a substrate.