Differential Proteomic Analysis Of Wheat Powdery Mildew Resistance Near Isogenic Line

Wheat powder mildew, caused by the fungus Blumeria graminis f.sp.Tritici (Bgt), is one of the most important wheat diseases worldwide. It can lead to nearly 40 percent of yield loss in some fields in epidemic years. Xiao Baidong is north of ancient local varieties of winter in Xinjiang, distributed mainly in Xinjiang Urumqi and Jinghua etc, and its powdery mildew resistance ability is outstanding, and is the special local resistant varieties. Xiao Baidong contained a resistant genes named xbd, and located in chromosome 7B with Pm5 closely, retained powdery mildew resistance capacity and low toxicity frequency up to now, is resistant to many wheat powder mildew bacteria (E01, E03, E05, E09, E15, E16, E18, E20, E26, E30, E31).This study chooses two near isogenic line as research materials, Bainong3217(susceptibility to powdery mildew) and xbd/Bainong 3217 (BC7F8)(resistant to powdery mildew). We studied wheat powdery mildew resistance on physiological and biochemical and proteomic method aiming at finding the differences between the two near isogenic line, and revealed: (1) After inoculated by Blumeria graminis, the wheat leaves were detected on concentrations of hydrogen peroxideanalysis (H2O2) and biological activities of relevant kinds of metabolic enzymes during 0-6 days. (2) Differential proteomic of the two near isogenic line, after seedling inoculation with four days later. Aimed at finding the interaction mechanisms between the two near isogenic line, and gaining valuable specific resistant proteins.The main results as follows:1. H2O2 content in the susceptible and resistant lines were significantly decreased and increased after inoculation with powdery mildew, respectively. H2O2 might play an important role in the wheat responses, several metabolic mechanisms might also involve in the responses, and H2O2 content could be influenced by these various enzymes.2. Metabolic enzymes activities related H2O2 in the leaves of susceptible line increased significantly during post-inoculation, and were higher than those in the resistant lines. After inoculated with bacteria in 5d, GPX, GR, SOD, APX, GST enzyme activity significantly increased in susceptible line, and this enzyme activity no significant changes in the resistant lines. Physiological indexes have changed significantly during the pathogen infection period, explain to interested disease strains infect bacteria is a kind of disease stress to adjust after a response to stress.3. Optimized the 2D technology including protein extraction, separation and staining techniques in leaves of wheat seedling, obtained a map which has clear separation and numbers of proteins. Add 10% PVP when grinding leaves, increase CHAPS and Thiourea content moderatly, and increased ultrasonic processing steps during protein extraction; Add 4% DTT in IEF hydration solution and add one step to remove the ions, enhancing the voltages to 500V and enlonging the time to 5h, isoelectric focusing voltage is 8000V, the time to 13h; Adjust SDS concentration to 0.1% and agarose concentration of fixative to 0.5% in SDS gel electrophoresis buffer solution. Thus, we detected 2500 protein spots by using pH 3-10, 24cm strip and 2D, silver staining and PDQuest software analysis.4. After 2D separation, we detected significantly different protein spots 230 between the susceptible and resistant lines, and obtained 19 proteins with clear features through MS and database searches. These proteins include: resistance-related protein, photosynthetic and electron transport proteins, metabolism-related protein, and other functional proteins. Resistance related proteins includ pathogenesis-related protein in systemic acquired resistance(SAR), such as beta-1,3-glucosidase(spot 2), beta-glucosidase(spot 6), 60 kDa jasmonate-induced protein(spot 7), and induction of defense proteins in SAR, such as O-methyltransferase (spot 1), TIR-NBS-LRR class disease resistance protein(spot 8); photosynthetic and electron transport proteins: optical system II protein(spot 15), ribulose-1,5-bisphosphate carboxylase(spot 11, 12, 13, 16), cytochrome b6(spot 4), oxygen-evolving complex precursor(spot 14); Metabolism-related protein: malate dehydrogenase(spot 19), lipase I(spot 21), plastidic glutamine synthetase(spot 10); And other functional proteins, such as ribosomal protein L11(spot 3), protein synthesis related, Adenosyl-homocysteinase(spot 20), nucleotide transporter related, potassium channel protein(spot 9) belongs to membrane transportproteins, beta tubulin (spot 17) belongs to structural protein.