Proteome Analysis Of Taichung29~*6/Yr5 Resistant To Wheat Stripe Rust

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.