【作者】 辛洪宝；

【导师】 郝东云；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 二维亲和电泳技术能够利用天然状态下蛋白与底物之间的相互作用而将其从蛋白粗提物中分离出来。与二维电泳相比,它能够将蛋白的功能在胶上展示出来。在本研究中,基于天然状态的酶与玉米胚乳淀粉代谢中间产物之间的相互作用,通过二维亲和电泳的方法从授粉后7～37天的玉米胚乳蛋白的粗提物中分离得到了参与玉米淀粉代谢的关键酶。通过LC-MS/MS鉴定以及酶谱的验证,四种蛋白分别为蔗糖合成酶-SH1、淀粉磷酸化酶前体、淀粉磷酸化酶和普鲁兰型淀粉脱支酶。经试验二维亲和电泳结合质谱技术是一种简单、高效的,基于蛋白功能从粗提物中分离和鉴定淀粉代谢相关蛋白的有效方法。本研究率先在国内利用二维亲和电泳来研究参与玉米胚乳淀粉代谢的关键酶,同时发现蔗糖合成酶SH-1与其他亚型的蔗糖合成酶相似,在玉米的淀粉代谢过程中同样发挥着重要的作用。更多还原

【Abstract】 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.更多还原