Mitochondrial Proteomic Analysis of Cytoplasmic Male Sterility Line and Its Maintainer in Wheat (Triticum aestivum L.)
Mitochondrial Proteomic Analysis of Cytoplasmic Male Sterility Line and Its Maintainer in Wheat (Triticum aestivum L.)
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DOI：
10.1016/s1671-2927(09)60154-1
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发表时间：
2010-06-20
影响因子：
--
通讯作者：
Ye Jing-xiu
中科院分区：
文献类型：
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作者：
Chen Rui-hong;Liu Wei;Ye Jing-xiu
为了从蛋白质组水平上研究小麦细胞质雄性不育的发生机制，寻找与育性相关的关键蛋白质，采用差速离心法和Percoll密度梯度法从小麦幼穗中分离线粒体。第一次不连续Percoll密度梯度离心后的质体和叶绿体为了提高穗粒线体的纯度，进一步进行第二次28%Percoll自生密度梯度离心，结果表明，外源污染物减少到可以忽略不计的程度，同时线粒体的完整性（88%）提高到90%。提取（S）-1376 A和（A）-1376 B单核期的纤突线粒体蛋白，采用双向电泳（2-DE）进行分离，银染凝胶用PDQuest 2-DE软件进行分析，在2-DE图谱上共检测到326个蛋白质点，除11个蛋白质点存在差异表达外，不育系与保持系的蛋白质图谱基本一致。通过MALDI-TOF-MS分析，利用Mascot软件对其中3个突变体进行NCBInr数据库检索，鉴定为锰超氧化物歧化酶（MnSOD）和T5 E216。
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 live 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