| Maize is the first crop applied male sterile for hybrid seed production, it plays an active role in reduction of production costs and improving seed purity. However, the molecular mechanism of nuclear male sterility in maize is far from clear, which has hampered the extending of its use. In this study, using maize K305ms as material, the protein profile of fertile and sterile anthers with 3 different anther development periods were constructed by iTRAQ technology, and the differentially expressed proteins were compared. Differentially expressed fertility related proteins were screened and enriched GO?COG and Pathway analysis were conducted on differentially expressed proteins and related pathways. We explored the K305ms abortion mechanism to lay a foundation for further research by binding protein immunoassay, fluorescent quantitative techniques and gas-mass spectrometry technology. The main results are as follows:1.27607 peptides and 6606 proteins were identified by using iTRAQ technology. GO analysis showed that these proteins are mainly distributed in the cells and organelles and mainly are binding proteins and catalytically proteins, and mainly involved in cellular metabolism and cellular processes; the functional classification of COG showed that in addition to general function, most proteins are involved post-translational modification, molecular chaperone, protein turnover, translation, ribosomal structure and protein biosynthesis; annotation of pathway found 304 metabolic pathways, mainly in carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, membrane transport and signal transduction and cellular material of secondary metabolites metabolic pathways. Cluster analysis showed that sterile and fertile anther protein expression pattern was similar in the early and late anther development, and was different with medium-term, which implied medium-term(meiosis stage) may be the key period of abortion.2.175,215 and 426 proteins in the 3 periods of sterile and fertile anther developments respectively, and a total of 721 significant differentially expressed proteins were screened. These proteins in the early period fertile anthers than sterile up-regulated 23,down-regulated 24;in the medium-term up-regulated 16,down-regulated 49;late period up-regulated 59,down-regulated 12.GO enrichment analysis indicated that the main activity including transport and ecetron carrier protein activity, mainly involved in cell metabolism, growth and signal transduction.COG functional categories showed the most functions are post-translational modification, protein chaperones and protein turnover. Pathway annotation showed that the differentially expressed proteins are involved in 198 pathways. According the enrichment of related proteins and continuous differentially expressed, we suggested that the carbohydrate and energy metabolism, fatty acid metabolism and fatty acid chain elongation may be one of pathways caused abortion.3. Western blot immune expression analysis was performed in which Hsp70,ADGP and ATP ? were screened as antibody, and ?-Actin as internal antibody. The results showed that Hsp70 expression differences during anther development was not significant, ADGP difference expression in the late anther development was significant, and ATP ? difference expression in the medium-term anther development was significant. The results were consistent with iTRAQ results. ATP ? plays an important role in energy metabolism, there was almost a trace amount in sterile anthers of medium-term.ATP? content in sterile anthers was less than fertile anthers of medium-term, which may lead to insufficient energy supply in medium-term, and affect normal meiosis.4.7 differentially expressed proteins were selected and specific primers were designed according the corresponding genes. Through the qRT-PCR reaction to detect the fertile and sterile anther in 3 periods, the levels of transcription expression results showed that transcript levels are basically the same with the corresponding protein expression levels. Metabolic pathways analysis found that main function of these proteins is to play an important role in lipid metabolism and fatty acid biosynthesis pathway, by influencing fatty acid chain extension and degradation of lipid synthesis. Reducing lipids may cause degradation of the outer wall, unable to form pollen grains and causes abortion.5. GC-MS were performed to analysis the contents of wax and cutin of fertile and sterile anthers in 3 stages. We found wax contents of sterile anthers was slightly lower than that of fertile anthers during the 3 stages, and the differences was not significant; Cutin contents of sterile anthers was lower than that of fertile anthers in early and late stages, but that of fertile anthers was lower than that of sterile anthers in the medium-term, and the differences reach extremely significant level. The contents of part long-chain cutin monomer of sterile anthers were extremely significant lower than that of fertile anthers. These results further validated that abnormity of the fatty acid chain elongation and lipid metabolism may lead to male sterility in maize.6. We speculate that maize K305ms abortion may be due to lack of carbohydrates and energy supply in early and medium-term periods, leading to related gene transcription, translation and ribosome synthesis was inhibited, resulting in abnormal meiosis; related metabolic pathways blocked in late period, especially fatty acid synthesis and lipid metabolism in the fatty acid chain extended cannot be normal, resulting in anther tapetum dysplasia; tapetum abnormalities lead to degradation of anthers, meiotic abnormalities lead to the functional pollen can't be formed, eventually. |
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