Molecular characterization of a protein kinase gene regulated by wounding in tobacco (Nicotiana tabacum)

Abstract

Nine kinds of putative protein kinase gene fragments of approximately 210 to 300 bp were isolated from Nicotiana tabacum by RT-PCR using protein kinase degenerative primers. These DNA fragments could be divided into four groups according to their deduced amino acid homology. Theses groups represent the ABA-inducible protein kinase homologs, AGC group kinase homolog, S-receptor kinase homologs, and receptor-like protein kinase homologs. One of DNA fragments included in ABA-inducible protein kinase homologs was proved to be induced transcriptionally upon wounding through northern blot analysis. Corresponding cDNA, which may be involved in wound signal transduction pathway, was isolated. The cDNA, named WAPK (wound- and ABA-induced protein kinase), is 1,227 bp in length and contains an open reading frame of 1,017 bp. The open reading frame designates a polypeptide of 339 amino acids with a calculated molecular mass of 38,234 Da. Deduced amino acid sequence analysis shows that the N-terminal region of WAPK contains a catalytic region composed of twelve subdomains which are typically found in Ser/Thr protein kinase. This region shows 78-84% amino acid sequence identity with similar regions of abscisic acid-induced and external stimuli responsive protein kinases. However, the C-terminal region of WAPK shows little homology with similar regions of Ser/Thr protein kinases, except a 16-amino acid stretch near the end of the catalytic domain. Kinase assay using WAPK fusion protein expressed in E. coli revealed that WAPK autophosphorylates on serine residue(s). The WAPK gene was predominantly expressed in flowers, moderately in roots, and poorly in leaves. Transcripts were not detected in stems. The WAPK gene was induced by wounding (within 1.5hr), by abscisic acid (within 0.5hr), and by methyl jasmonate (within 2hr), but not by TMV infection, dehydration, and cold stress. The induction pattern of WAPK mRNA upon wounding was not affected by treatment with diethyldithioca...