Optimization Of 2-DAE Conditions For Maize Endosperm Proteins And Separation & Characterization Of Proteins Involved In Maize Starch Metabolism

Starch is the main food source and important industrial material for human and it plays a very important role in human's life. Cereal starch metabolism is a very sophisticated system related with the activities of a range of enzymes. The fact that some enzymes forming much bigger protein complexes has made the research on the starch-biosynthetic enzymes more difficult. Traditionally, researchers use the mutations of some specific enzymes and reverse transformation to study the roles of different enzymes'functions, at the same time, some researchers use affinity electrophoresis to discuss the precise roles of enzymes. The technique, 2-DAE can separate native proteins from crude extracts based on their interactions with the substrates, compared with 2-DE, it can display the functions of proteins on the gel. In this study, a new technique-two dimensional affinity electrophoresis has been used to separate the enzymes participating in starch biosynthesis based on their interactions with the polysaccharide from the protein crude extract of maize endosperm in the stage of 7-37 days after pollination. In the first dimension, proteins are separated by native polyacrylamide gel electrophoresis (PAGE). In the second dimension we add a certain amount of substrates, designed amylopectin, beta-dextrin, glycogen and sucrose in the native polyacrylamide gel. The proteins interacting with the substrate during electrophoresis will be separated from the bulk of proteins showing obvious distarded mobility on the gel. In this study, in affinity gels containing four kinds of substrates, results show that there are no interacting proteins with the substrates in the protein extracts extracted at the stage of 7-9DAP. Three proteins show the interactions with the substrates in the protein samples extracted after 16DAP of maize endosperm development, to be more precise, in sucrose-containing affinity gel, there is one protein spot interacting with sucrose; in amylopectin-containing gel, there are two protein spots interacting with the substrate; in the affinity gel immobilized with glycogen, there are two protein spots interacting with the substrate between the phase of 16DAP and 28DAP, at the same time, three spots has been detected in the phase of 35 to 37 DAP. Confirmation by LC-MS/MS and zymogram analysis, they have been demonstrated sucrose synthase-SH1(SUS-SH1), starch phosphorylase, starch phosphorylase and pullulanase-type starch debranching enzyme. The 2-DAE coupled with LC-MS/MS described here is simple, highly effective method for separating interacting proteins based on their functions from crude protein extract. This is the first time in domestic using two dimensional affinity electrophoresis (2-DAE) coupled with LC-MS/MS for separation and characterization of protein involved in maize starch metabolism, and at the same time, this is the first example of founding the phenomenon that sucrose synthase- SH1 participating in the starch metabolism of maize endosperm and the changes of expression levels of key enzymes during the maize endosperm. This research has provided necessary experimental data for the further research on regulation of maize endosperm starch metabolism and laid the foundation for maize breeding.