Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinale

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.