【作者】 刘静；

【导师】 赵奎军；

【作者基本信息】 东北农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 蛋白质组学是生命科学研究的热点和前沿。蛋白质组学的发展是受技术限制的,也是受技术推动的。目前小麦叶片蛋白质组研究主要采用双向电泳(2-DE)分离和肽质量指纹(PMF)分析技术。探索新的分离、分析方法,可为更深入全面研究小麦叶片蛋白质组提供技术保证。二维液相色谱(2D-LC)与2-DE技术具有互补性,本文探讨了利用2D-LC和纳升级液相色谱串联质谱(Nano LC-MS/MS)技术分离鉴定小麦叶片蛋白质组的新方法。小麦叶片蛋白经提取、脱盐后,进行第一维阴离子交换分离;收集洗脱组分进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,并对非高丰度蛋白组分进行第二维反相液相色谱分离;随机选择含较低吸收峰的部分组分经胰蛋白酶水解后进行Nano LC-MS/MS分析;将串联质谱数据通过MASCOT搜索NCBInr和EST数据库,并对二级质谱获得的蛋白序列进行MS BLAST分析。结果表明,经第一维阴离子交换色谱分离,收集得到15个组分,其中第15组分包含小麦叶片丰度最高的蛋白——1,5-二磷酸核酮糖羧化酶/加氧酶(RuBisCO);其余14个组分经反相液相色谱分离,共收集1551个组分,获得1867个色谱峰;随机对其中6个含较低吸收峰的收集组分酶解和Nano LC-MS/MS检测,9种蛋白得到鉴定。小麦条锈病(Puccinia striiformis f.sp.tritici)一直是我国小麦的主要病害,成灾频率高、流行范围广。利用抗病品种是防治小麦条锈病最有效、经济和安全的途径,选育和推广抗病品种成为控制病害流行的首选措施。但由于病原菌生理小种变异快,品种抗性频繁丧失,抗病育种始终处于被动应付状态。本文从蛋白质组学角度出发深入研究小麦对条锈病抗性的分子机理,揭示寄主小麦与病菌之间的互作机制,在小麦抗病品种的培育和利用上具有重要理论和实际意义。由中国农科院植保所培育的小麦抗条锈病品系Taichung29*6/Yr5目前在我国尚未发现对其致病的条锈菌生理小种。本试验联合应用双向电泳、二维液相色谱和质谱技术,以接种当前条锈菌主要流行小种条中32号(CY32)的小麦抗条锈病品系Taichung29*6/Yr5为材料,研究其叶片差异表达蛋白质组,以期发现特异的抗病相关蛋白。经双向电泳分析,初步确定19个差异表达蛋白质点。其中,接种后诱导表达的蛋白质点有8个;上调表达的蛋白质点有10个;下调表达的蛋白质点有1个;未发现表达被抑制的蛋白质点。经二维液相色谱分析,初步确定17个差异色谱峰。其中,接种后新出现的色谱峰有1个;峰面积增加的色谱峰有6个;峰面积减少的色谱峰有9个;缺失的色谱峰有1个。所有差异蛋白组分均进行了质谱分析与数据库检索,11个蛋白质获得鉴定结果,包含8个与植物源蛋白相匹配的蛋白、2个与真菌蛋白高度同源的蛋白和1个与假设蛋白高度同源的蛋白。鉴定蛋白的功能主要涉及植物防卫和植物光合作用。试验结果表明利用2D-LC和Nano LC-MS/MS技术可有效地分离和鉴定小麦叶片蛋白质组;同时采用双向电泳和二维液相色谱技术分析小麦抗条锈病品系Taichung29*6/Yr5接种条锈菌CY32的差异表达蛋白质组,能够更全面的获取差异表达蛋白质信息;本文利用小麦抗条锈病品系Taichung29*6/Yr5开展的比较蛋白质组研究工作,为深入理解小麦与条锈病菌的互作机制提供了重要的基础。更多还原

【Abstract】 Proteomics is one of the hot spots in life science and proteomics technologies have been applied in almost all biology research. Two-dimensional gel electrophoresis(2-DE) and peptide mass fingerprinting(PMF)have been the main techniques for the wheat leaf proteome analysis. Two-dimensional liquid chromatography(2D-LC) can be used as a complementary approach to protein separation with 2-DE. The purpose of the study is to establish a protocol to separate and identify wheat leaf proteome by combination of 2D-LC and nanoflow liquid chromatography tandem mass spectrometry(Nano LC-MS/MS). The soluble protein extracted from wheat leaf were desalted by gel filter chromatography and separated by strong anion exchange chromatography (SAX) in the first dimension. The elution fractions were subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis(SDS-PAGE)and the fractions without the most abundant protein(s) were further separated by reversed phase liquid chromatography (RPLC) in the second dimension. To test the effectiveness of the separation method, some of 2D-LC fractions including lower intensity peaks were digested and analyzed by Nano LC-MS/MS. The MS/MS data was used to search against NCBInr and EST database using MASCOT search engine. Meanwhile, de novo sequencing was performed manually and the data was used to search against nrdb95 database using MS BLAST search engine. A total of 15 collections were obtained through the first dimensional separation and the most abundant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in plant leaf, could only be observed in the 15th collection. After the second dimensional separation, 1551 collections were collected with 1867 resolved protein peaks from the other 14 fractions without RuBisCO. Nine proteins were identified from six 2D-LC collections with lower intensity peaks selected randomly.Wheat stripe rust (Puccinia striiformis f.sp.tritici) is one of the most destructive diseases to wheat in China .Use of resistant cultivars has become the major means of controlling stripe rust and resistant cultivars utilization is the most effective,economical and safe way.Because of development of the new physiologic races, wheat stripe rust resistance genes frequently became susceptible and thus breeding for disease-resistance is passive usually. Taichung29*6/Yr5 resistant to wheat stripe rust was established by institute of plant protection of Chinese Academy of Agricultural Sciences and no pathogenic races are found up to the present in our country. So, We chose Taichung29*6/Yr5 as the experimental material.Through proteomic analysis of the changes of proteins in Taichung29*6/Yr5 inoculated by the epidemic race CY32,we wanted to find some specific resistance-related proteins. As a result, 19 protein spots resolved on two-dimensional gels were found to be differentially expressed after analyzing Coomassie-blue stained gels of the soluble protein samples from the control and disease leaves 8 days post inoculation.8 of those were induced,10 of those were up-regulated,1 of those was down-regulated and no protein was repressed. 17 protein peaks resolved by the 2D-LC were found to be differentially expressed after analyzing two dimensional chromatograms of the soluble protein samples from the control and disease leaves 8 days post inoculation. 1 of those was induced,6 of those were up-regulated, 9 of those were down-regulated and 1 protein peak was repressed. 11 proteins were identified at last by MS or MS/MS analysis and data searching. Of these, 8 matched plant proteins,2 were highly homologous with fungal proteins and 1 was highly homologous with hypothetical protein from Oryza sativa Indica Group. Functions of identified proteins were mainly involved in plant defense responses and plant photosynthesis.Based on the experiment setup and results, we tentatively concluded that the combination of 2D-LC and Nano LC-MS/MS could be an effective method in future wheat leaf proteomics analysis; utilization of both 2-DE and 2D-LC could acquire more differentially expressed proteins; differential proteomic analysis of Taichung29*6/Yr5 helped us to elucidate the resistant mechanisms and provide theoretical principle and resources for molecular breeding of wheat resistance to puccinia striiformis.更多还原