温敏核不育水稻花药蛋白质组初步分析

Preliminary Proteomic Analysis of the Proteins of Thermo-sensitive Genetic Sterile Rice Anther

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,通过银染显色 ,获得了分辨率和重复性较好的双向电泳图谱 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和

The proteins of thermo-sensitive genetic sterile rice anther were separated by two-dimensional electrophoresis with immobilized pH(3-10) gradients as the first dimension and SDS-PAGE as the second.Mean standard deviations of about 1.45±0.23 mm for IPG-IEF and 1615±0.17 mm for SDS-PAGE were obtained.The silver-stained protein spots were analyzed and identified by employing an improved procedure,including (1) in-gel reduction,alkylation and enzymatic digestion;(2) extraction and desalting by using the Ziptip TM;(3)direct MALDI-TOF mass spectrometry analysis and protein database searching.60 protein spots out of about 950 detectable spots on the 2D-gels were identified.The 2D protein maps of the rice anther during the sterile and fertile stages were compared.While the anther changed from sterile to fertile stages,about 20 protein sports showed variations that were significant and reproducible.Five up-regulated proteins and three down-regulated proteins were identified.