Molecular cloning and characterization of calcium-dependent protein kinase genes in Dendrobium officinale / 中国药学杂志

ABSTRACT

OBJECTIVE: To clone and characterize calcium-dependent protein kinases (CDPKs) genes in Dendrobium officinale Kimura et Migo. METHODS: Reverse transcription PCR(RT-PCR) and rapid-amplification of cDNA ends (RACE) methods were used to isolate CDPKs genes from the leaf cDNA of D. officinale. Characteristics including the molecular weight, theoretical pI (isoelectric point), conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced proteins were analyzed using serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed using DNASTAR and MEGA4. Tissue specific expression patterns were determined using real-time quantitative PCR (qPCR) analyses. RESULTS: Two full length genes DoCPK2 and DoCPK3 (GenBank accessions JX219469 and JX219470), 2119 and 2418 bp in length, respectively, were obtained. DoCPK2 was deduced to a 541 aa (amino acid) protein with a molecular weight of 60.46·103 and a pI of 6.14, while DoCPK3 encoded a 536 aa protein with a molecular weight of 60.30·103 and a pI of 6.32. The two deduced proteins, without signal peptide, both contained the conserved caniocal serine/threonine- protein kinase catalytic domain, Ca2+ binding EF hand motifs, and a 19 aa transmembrane structure at 248-266 and 243-261 aa position, respectively. They were highly homologues (67%-81%) to the plant CDPKs genes, and were mostly close to monocots CDPKs genes from wheat and rice. DoCPK2 and DoCPK3 were constitutively expressed among the five tissues. DoCPK2 transcripts were more abundant in the roots and PLBs (protocorm-like bodies), while the transcription levels of DoCPK3 were repressed in the seeds and stems. CONCLUSION: Two calcium-dependent kinases genes DoCPK2 and DoCPK3 are identified from the valuable herb D. officinale, which will be useful for further functional determination of the genes involving in the growth, development, biotic and abiotic responses in D. officinale.