Phenotype identification, cloning and expression of B55 subunit mutants of protein phosphatase 2A in Arabidopsis model plant

Abstract

Plant photoreceptors sense light and regulate certain downstream genes to alter morphological features. In Arabidopsis, these are three classes of photoreceptors: far/red light absorbing phyochromes, UV/blue light absorbing cryptochromes and phototropins. Reversible protein phosphorylation is a major signaling mechanism in eukaryotic cellular function. Recent research suggests that reversible phosphorylation of photoreceptors is a vital regulatory mechanism in light signal transduction pathway. Protein phosphatase has been proposed to play an important role in cellular function both plants and animals. The B subunits of PP2A play a crucial role in localization and substrate specificity of PP2A in mammals. It has also been proposed that B subunits have the same role in plants. The Arabidopsis seedlings were planted on half MS medium containing 1% sucrose and without sucrose in different light conditions to observe the phenotypic appearance. To investigate a possible dephosphorylation in the signaling pathways from phytochromes and cryptochromes by PP2A in the nucleus, phenotypic identification of the Arabidopsis single B55 mutant seedlings were first investigated in this study. Subcloning and expression of B55 and B`β recombinant proteins were then conducted to probe the target proteins with antibody by Western blot analysis. The primary purpose of this study is then to identify the phenotypic observations (particularly the hypocotyl elongation) of B55 subunit mutants in Arabidopsis seedlings and compare with that of wild type (Col). For further study of role of PP2A in de-etioltion, the genes for B55 subunit and B`β in Arabidopsis as a model organism had to be subcloned into expression plasmid vector, PGEX-6p-1. The expression plasmid vector was transformed into E. coli, DE3, for protein expression under IPTG induction. The proteins were co-purified with GST by using Gluthathione-agarose beads and eluted with elution buffer. The purified proteins were detected with secondary antibody conjugated with Horse Radish peroxidase (HRP). Some problems were encountered for optimization of the proteins during purification and Western blot detection.