Cloning and homology modelling of a Pto-like protein kinase family of common bean (Phaseolus vulgaris L.)
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Degenerate primers, based on conserved subdomains of several plant serine/threonine kinases (STK) similar to the tomato Pto protein kinase, were designed to amplify similar regions from the common bean genome. Sequence analysis of the products defined five distinct classes sharing from 56.9 to 63.9% amino-acid identity with Pto. Inter-class identity ranged from 61.2 to 81.4%. Each of the five classes contain the conserved residues found in subdomains II through IX of most STKs. Multiple sequence and neighbor-joining tree analysis suggest the Pto and the cloned common bean sequences define a unique class of plant protein kinases. Southern hybridization to common bean DNA determined that the sequence classes represent low to moderate copy number families. Using PCR amplification with class-specific primers followed by restriction enzyme digestion of the products, these five classes were found to be essentially monomorphic among 20 divergent common bean genotypes. Each class was determined to be expressed in a leaf mRNA population. Further analysis of the Sg5 class using 3′-RACE (rapid amplification of cDNA ends) identified seven unique family members. All Sg5 3′-RACE products share a high degree of identity, but contain numerous differentiating features that demonstrate the presence of microheterogeneity within the Sg5 class. Three-dimensional homology modelling demonstrated that Pto and Sg5–3e contain nearly all of the structural features found in type α cyclic AMP-dependent protein kinase (cAPKα) except α-helices within subdomains II and XI. Based on these homology models and models of ten other plant kinases, two subfamilies of plant protein kinase sequence could be differentiated based on subdomain XI structure. Database searches revealed that subdomains VIa, VIb, VIII and IX of the Pto-like class are unique to plant species, whereas for a second subfamily of plant protein kinases (containing the common bean kinase PvPKI) these subdomains are also similar to those found in non-plant eukaryotic species.
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