Molecular characterisation of plant cDNAs BnMAP4Kα1 and BnMAP4Kα2 belonging to the GCK/SPS1 subfamily of MAP kinase kinase kinase kinase

Several yeast and mammal MAP kinase modules require, upstream of their MAP kinase kinase kinase (MAP3K), a MAP3K kinase (MAP4K). An Arabidopsis thaliana EST clone, sharing identity to MAP4Ks from yeast and mammals, has been used to isolate cDNA clones from a Brassica napus microspore-derived embryo cDNA library. The BnMAP4K alpha 1 and BnMAP4K-alpha 2 clones encode putative proteins possessing the 12 subdomains of the serine/threonine protein kinase catalytic domain. A detailed analysis showed that they belong to the GCK/SPS1 subfamily of MAP4K proteins which possess an amino terminal catalytic domain and a long carboxy terminal tail. A Southern blot analysis suggested that the two proteins are encoded by a small multigene family. Expression studies revealed the presence of BnMAP4K alpha 1 and -alpha 2 transcripts in all the tissues examined; however, they are most abundant in roots, siliques and flower buds. The expression of BnMAP4K alpha 1 and -alpha 2 at the three main developmental stages of microspore-derived embryos (i.e., globular/heart, torpedo and cotyledonary) was confirmed by northern blot and RT-PCR analysis. An expression analysis of the above genes using synchronised Arabidopsis thaliana cell suspensions showed that the homologues genes are cell cycle regulated. (C) 1999 Elsevier Science B.V. All rights reserved.