Expression of a Wheat Endosperm 14-3-3 Protein and Its Interactions with Starch Biosynthetic Enzymes in Amyloplasts

Abstract

Wheat endosperm starch is the major determinant of grain yield and processing quality.The quality and quantity of starch are controlled by a number of starch biosynthetic enzymes.14-3-3 proteins,involved in many biological processes,are ubiquitous and important regulators in all eukaryotic cells from yeast to mammals and plants.Protein-protein interactions between a wheat endosperm 14-3-3 protein and starch biosynthetic enzymes from amyloplast were investigated in this study.A 14-3-3 gene was cloned from developing wheat endosperm and inserted into plasmid vectors pET29c and pET41c.The recombinant vectors were transformed into Escherichia coli strain BL21-CodonPlus(DE3)-RP and expressed at very high level.The fusion protein existed mainly as an insoluble inclusion body after extraction by BugBuster Protein Extraction Reagent.The soluble fusion protein was purified by bounding to S-protein agarose,while the inclusion body should be dissolved in 8 mol L- 1urea and refolded firstly.Sucrose synthase activity was observed to be inhibited by exogenous recombinant 14-3-3 protein in a dosage-dependent manner,which suggested the refolded protein was successfully activated and could be used in the following research.The purified recombinant 14-3-3 protein was bound to S-protein agarose as a biochemical bait,and then incubated with wheat amyloplast extract.Proteins interacting specifically with the 14-3-3 protein and remaining on the resin were analyzed by SDS-PAGE and Western blot.These assays showed that starch synthase I(SSI),starch synthase II(SSII),starch branching enzyme IIa(SBEIIa),starch branching enzyme IIb(SBEIIb),and ADP glucose pyrophosphorylase large subunit(SH2)interacted with 14-3-3 protein,whereasSBEI,starch phosphorylase(SP),D-enzyme(DE),and ADP glucose pyrophosphorylase small subunit(BT2)did not bind with the 14-3-3 protein.The results suggest a role for the wheat endosperm 14-3-3 protein in regulation of grain starch biosynthetic enzymes.