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

作者：Song Jian · 发表于：ACTA AGRONOMICA SINICA · 年份：2009 · 被引用次数：1 · 研究领域：14-3-3 protein interactions、Enzyme Production and Characterization、Microbial Metabolites in Food Biotechnology

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.Thes...