【目的】从蛋白质组学的层面探讨玉米籽粒发育过程中胁迫相关蛋白的表达特性,分析其功能,揭示籽粒自身防御系统的分子调控机理。【方法】大田条件下,以玉米品种登海661(DH661)为供试材料,67 500株/hm~2密度下种植,开花期人工饱和授粉后第3、5、10、15、20、30、40和50天(DAP)取果穗中部籽粒。TCA-丙酮沉淀法提取籽粒总蛋白,用同位素标记相对定量(i TRAQ)技术进行蛋白质组学分析。通过匹配Uniprot玉米蛋白数据库鉴定籽粒总蛋白,并且用基因本论(GO)注释按照生物过程、分子功能及细胞组件进行功能分类。分析鉴定籽粒发育过程中显著差异表达的胁迫相关蛋白,并且将其分层聚类以展示其在籽粒发育过程中的表达模式。【结果】通过匹配玉米蛋白数据库,籽粒中总计鉴定到4 751个蛋白,这些蛋白涉及多种生物过程与分子功能,其中代谢过程与分子过程是最主要的两个生物过程,而催化活性与绑定功能是最主要的两个分子功能类别,表明这些生物过程与分子功能对籽粒发育具有重要作用。定量分析检测到123个胁迫相关蛋白在玉米籽粒发育过程中显著差异表达,主要参与籽粒蛋白修饰(33个)、活性氧(ROS)体内平衡(31个)、贮藏物质保护(17个)、病虫害响应(8个)及其他胁迫响应过程(34个)。蛋白修饰相关蛋白主要包含一系列的热激蛋白、肽基脯氨酰顺反异构酶及蛋白二硫键异构酶,并且这些蛋白在籽粒不同发育阶段均显著积累,这对稳定籽粒中的蛋白结构具有重要作用。ROS相关蛋白包含不同的抗氧化酶系,并且主要在籽粒发育前、后期显著积累,维护了ROS的体内平衡。贮藏物质保护相关蛋白主要包含多种蛋白酶抑制剂、油脂体蛋白及油脂体固醇蛋白,并且这些蛋白随着籽粒发育不断上调表达,保护了贮藏物质的合成与积累。病虫害响应相关
【Objective】In order to understand the molecular regulation mechanism of defense system in maize grain,the expression characteristics of stress-related proteins during grain development were studied by using approach of plant proteomics.【Method】Denghai 661(DH661) was used as experimental material and planted at 67 500 plants/hm~2 in field.The middle grains were harvested after flowering artificial saturation pollination at 3,5,10,15,20,30,40,50 d,respectively.The total proteins were extracted by the TCA-acetone precipitation method and then were analyzed by isobaric tags for relative and absolute quantitation(i TRAQ) proteomics.The proteins in maize grain were identified by searching the Uniprot maize protein database and gene ontology(GO) annotation was used to classify the functions of these proteins according to the biological process,molecular function and cellular component.Quantitative analysis was applied to identify stress-related proteins that were significantly differentially expressed during grain development.Hierarchical cluster analysis was used to show the expression patterns of these stress-related proteins during grain development.【Result】A total of 4 751 proteins were identified in maize grain by matching the maize protein database,and these proteins were involved in diverse biological processes and molecular functions,of which the metabolic process and molecular processes were the main biological processes,and the catalytic activity and binding function were the main molecular categories,showing that these biological processes and molecular functions played important roles in maize grain development.Quantitative analysis detected 123 stress-related proteins were significantly differentially expressed during grain development,and these proteins were mainly involved in grain protein modification(33),reactive oxygen species(ROS) homeostasis(31),storage material protection(17),disease response(8) and other stress response process(34).The proteins relate