分析接种小麦白粉菌E09后Pm21近等基因系及其亲本叶片的总蛋白质差异,从蛋白质组调控角度揭示Pm21基因抗白粉病的抗病机理。以Pm21近等基因系及其亲本百农3217为试材,显微观察接种后5 d内的叶片表面细胞与白粉菌孢子表型差异;采用非离子去污剂提取总蛋白质,运用双向电泳、质谱分析与数据库检索技术,比较Pm21近等基因系及其亲本接菌第4天后的叶片总蛋白质差异。超显微和显微观察结果表明,12 h后百农3217叶细胞表面与Pm21近等基因系相比褶皱明显;在接菌第2天后,在Pm21近等基因系白粉菌孢子生长明显慢于其亲本表面孢子,且孢子侵入个数较少,不萌发现象较多;在第4天后Pm21近等基因系被侵染叶片细胞出现过敏性反应;通过MALDI-TOF-MS/MS质谱测序和MASCOT软件序列分析,注释了15个表达上调蛋白点;功能分类表明,差异蛋白质分别参与叶绿体光合呼吸作用(40%) 、放氧作用(13%) 、代谢物合成(20%) 、抗病、动力蛋白、运输等生理功能。蛋白质组水平发现,光合呼吸蛋白和叶谷氨酰胺合成酶在Pm21近等基因系感病3～4 h后就大量表达,可能是导致叶片明显变绿原因;叶谷氨酰胺合成酶的增加与光合呼吸蛋白的增加密不可分;光合呼吸蛋白和抗坏血酸过氧化物酶的上调表达为植物体在高H2O2浓度下正常生长提供了保障。另外,β-1,3-葡聚糖苷酶的过量表达也是Pm21基因前期抵御白粉菌侵染有效物质。钾离子通道蛋白可能在抗病防御过程中起到了信号转导的作用。
The purpose of this project was to study inoculated by E09 in wheat leaves to confer durable and broad spectrum resistance to wheat powdery mildew through proteomic analysis. To carry out the cytological observations of Wheat(Triticum aestivum) cultivars Bainong 3217 and near-isogenic Bainong 3217/Pm21(BC7F6) plants were inoculated with E09 powdery mildew after 5 days. Total proteins were extracted by nonionic detergent, were separated by two-dimensional gel electrophoresis(2-DE) 3 replicates and analysed through MALDI-TOF-MS/MS mass spectrometry. Microscopic observation results showed that Bainong 3217 leaves cell surface folds after 12hr, and spores growing significantly inhibited on leave surface of near-isogenic Bainong 3217/Pm21(BC7F6) after 2 days inoculation. Besides, hypersensitive reaction was found at 4th day post inoculation. And 15 of up-regulated proteins were identified homologous to known proteins in various databases by MALDI-TOF-MS/MS and MASCOT analysis. The predicted functions of the 5 proteins were related to Chloroplasts photosynthesis(40%), oxygen releasing(13%), Metabolites synthesis(20%), resistance, transcription. One result of leaves deepening green may cause by Enzyme Rubisco and Glutamine synthetase leaf isozyme proteins increased. These proteins increased after about 3 or 4 hours infection. Enzyme Rubisco and Ascorbate peroxidase expressed were up-regulated to protect Calvin cycle in high concentration of H2O2. In addition, g lucan endo-1, 3-beta-D-glucosidase precursor induced were up-regulated to suppress further development of haustoria. The potassium channel protein may play an important role in early disease-resistance signal transduction in Pm21.
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