Differential Proteomics Analysis Of Floret Intact Chloroplasts Proteins Of Physiological Male Sterile Line Induced By CHA-SQ-1 In Wheat

Chemical hybridization agents (CHA) break the three-line breeding and is widely used in the two-line breeding, SQ-1 is a new kind of CHA with self-knowledge property in China. It can induce 95%-100% male sterility through pharmacodynamic test in Northwest A& F University. Now CHA has been a sophisticated technology and applied to produce hybrid wheat seeds in a large area, such as Hybrid Wheat Xiza 1, Hybrid Wheat Xiza 5, etc In order to better understand the molecular mechanism on chloroplasts proteins level and find the crucial proteins which related to wheat male sterility. In this study, the floret of wheat XiNong1376 induced by SQ-1 and its untreated check were studied, results are as follows:1. To comparatively study on the two different sucrose density gradient isolation methods of intact chloroplasts from wheat floret at thinucleate stage, the microscope and SDS-PAGE were used. The loading quantity of sample and the pH range of IPG for the analysis of 2-DE system were also optimized in this study. The results showed that, it was an efficient method of isolating intact chloroplasts using 30%, 45%, 60% sucrose density gradient. The chloroplast proteins were extracted by the TCA/acetone method, about 150 protein spots could be detected on the map when 280μg proteins of chloroplast were loaded onto 17cm, pH 4 ~ 7 IPG strip for 2-DE followed by silver staining. The system was suitable for isolation of intact chloroplasts.2. In this study, cells of the wheat florets were broken using cell disruption by blender and filtered through four layers of muslin, the intact chloroplast proteins of floret were isolated by differential centrifugation and sucrose density gradient purification. The proteins of floret intact chloroplasts from wheat male sterile line induced by chemical hybridizing agent SQ-1 were separated by two-dimensional electrophoresis.3. The silver-stained protein spots were analyzed using PDQuest software, about 150 protein spots were detected reproducibly within pI 4～7 on each 2D-gel, 17 spots were found to be differentially expressed, 7 of them were identified by MALDI-TOF-MS analysis and Swiss-prot database searching, which are PAP-fibrillin, ATRABB1A, pleckstrin homology (PH) domain-containing protein/RhoGAP domain-containing protein, copper/zinc superoxide dismutase 1, R2R3-MYB transcriptional regulator and hypothetical protein. These proteins were involved in hormone modulation, translocation of protein, protein-protein interaction, active oxygen accumulation and anther development. The male sterility induced by CHA-SQ-1 might be related to these physiological processes.