Expression and autophosphorylation analysis of XA21 and PI-D2 protein kinases in Saccharomyces cerevisiae

Abstract:

Rice bacterial blight and blast are the most crucial rice disease. Xa21 confers resistance to bacterial blight，while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor kinase-like proteins. Biochemical properties of XA21 kinase expressed in bacterial were characterized in our previous report. In this study，both XA21 and PI-D2 kinase domain were PCR amplified and cloned into yeast expression vector pEGH via recombinational cloning strategy，kinase proteins expressed in eukaryotic yeast system was purified and autophosphorylation assay was carried out. The results indicated that XA21 and PI-D2 protein can be detected by SDS-PAGE and showed expected molecular weight. Autophosphorylation assay indicated that yeast expressed XA21 and PI-D2 were active when incubated with P32 labelled ATP. The experiment provided basic materials for biochemical prosperity analysis，protein-protein interaction and substrate screening research.