Abstract: 【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/hm2 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 (iTRAQ) 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 related to protein modification mainly included a series of heat shock protein, peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase, and these proteins significantly accumulated at different development stages, which played important roles in stability of protein structure. ROS related proteins contained a variety of antioxidants, and mainly significantly accumulated at both early and late development stages, which maintained the homeostasis of ROS. Storage material protection related proteins mainly contained a variety of protease inhibitors, oleosin and steroleosin, and the expression of these proteins were constantly raised with the grain development, which protected the synthesis and accumulation of storage material. The proteins involved in disease response also significantly accumulated at late development stage, which enhanced the grain resistance to biological stresses. Proteins involved in other stress response mainly included a series of late embryogenesis abundant protein (LEA), annexin, lipid transfer protein, nonspecific lipid transfer protein and lipoxygenase, of which all of the LEA significantly accumulated at late development stage, annexin and lipoxygenase significantly accumulated at early development stage, while lipid transfer protein and nonspecific lipid transfer proteins were accumulated at different development stages, showing that these proteins played important roles in different grain development stages.【Conclusion】Stress-related proteins were accumulated during maize grain different development stages, which constructed a harmonious, diverse and stable defense regulatory mechanism, and thus maintained the normal development of maize grain.

Key words: maize, grain development, iTRAQ proteomics, stress-related protein, protein function