To investigate the CMS mechanism of wheat on proteomic level and find the crucial proteins which related to fertility,mitochondria was isolated from young spike of wheat by differential centrifugation and Percoll density-gradient methods.Determined by marker enzyme assays and chlorophyll content,the mainly contaminants in the spike mitochondrial fraction were caused by peroxisomes,plastids and chloroplasts after the first discontinuous Percoll density gradient centrifugation.In order to improve the purity of spike mitochondria,a second 28% Percoll self-forming density gradient centrifugation was further carried out,the result showed that the contaminants were decreased to negligible amount,meanwhile the integrity of mitochondria （88%） was improved to 90%.The spike mitochondria proteins extracted from uninucleate stage of （S）-1376A and （A）-1376B were separated by two-dimensional electrophoresis （2-DE）,and the silver stained gels were analyzed by PDQuest 2-DE software,about 326 protein spots could be visualized on the 2-DE maps,and also revealed a similar pattern between the male sterile line and its maintainer line,except 11 spots were differentially expressed.A total of five differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）,three of them were identified as manganese superoxide dismutase and T5E216 following NCBInr database by the Mascot software.These results may contribute to further understanding of the mechanisms of CMS in wheat.
