Expression optimization of rice bacterial blight resistance gene Xa21 and blast resistance gene Pi-d2 protein kinases in Pichia Pastoris

Abstract:

Abstract: [Objective] Bacterial blight and blast are the most severe rice diseases. Xa21 confers resistance to bacterial blight, while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor-like kinase proteins. The aim of this study was to express kinase domain of XA21 and PI-D2 proteins in Pichia pastoris yeast system. [Methods] We amplified the coding regions for the kinase domains of Xa21 and Pi-d2 and constructed recombinant plasmids pPICZαA-Xa21K and pPICZαA-Pi-d2K, respectively. Restriction enzyme digestion and sequence verified plasmids were linearized and transformed into yeast strains. We compared the expression of recombinant proteins in 3 Pichia pastoris strains (KM71,GS115 and X33), with various methanol concentration (1%, 2% and 3%), pH (pH5, pH6, pH7 and pH8) and induction time (24 h, 48 h and 72 h). [Results] The recombinant kinase domain of XA21 and PI-D2 proteins were expressed in yeast, however, they were detected only in pellet of yeast cells but not in the supernatant of medium. This indicated that the recombinant proteins were not secretive. Comparison results revealed that Pichia pastoris strains (KM71 and X33), 2% methanol, pH5 and 48 h or longer induction time were optimal for the expression of the two rice kinase domain proteins. Finally, we purified soluble recombinant proteins and detected them by SDS-PAGE. [Conclusion] We obtained purified domain of XA21 and PI-D2 proteins from Pichia pastoris expression strain, which will facilitate the investigations of their biochemical properties.

Key words:Keywords: rice; bacterial blight; rice blast; disease resistance gene; Pichia pastoris yeast; protein expression