The Optimizing Of Protein Sample Preparation From Crops And The Proteome Analysis Of The Wheat Isogenic Line Tyr5

The wheat, rice, corn and soybean are the main four major food crops in China and playing the vital role in China’s food security. It is so important to carry out the studies of the chill and salt resistance as well as anti-waterlogging of the crops to guarantee the production in harsh environments and make the crops plantable. At present accumulating achievement of the four major food crops in insect resistance, disease resistance, salt or drought tolerance etc. has carried out extensive research. The function proteins, the product of gene, are the key to understand the resistant genes how to exert its effect. So the comparative study of the proteins specific in stress has important significance for the exploration of crop resistance mechanisms.In this thesis the yellow rust resistance wheat Yr5 and its isogenic line Taichung29* 6 /Yr5 (TYr5), back spike rice mutant plants/wild type, soybean short petioles plant/long petiole and the AA/aa mutant strains of corn provided by the Institute of Plant Protection, the Chinese Academy of Agricultural Sciences are used as materials. The protein isolation methods were comparatively analyzed with those crops and then a primary proteomics of yellow rust Yr5 isogenic was formulated. The efficient plant leaf protein sample preparation methodology was first set with the four kinds of crops. Then the TYr5 that resistance to wheat rust proteome is obtained with TripleTOF5600 mass spectrometry (AB SCIEX) by the mass spectrometry analysis and qualitative analysis. A large number of peptides that may be associated with the disease resistance were deduced. The main results were as follows:(1) To establish and optimize the wheat, rice, soybeans, corn leaf total protein separation method, the 1 DE electrophoresis and in gel protein digestion method was set for four crops. The protein extraction and separation of the rapid identification of protein, compared the different treatment on the premise of different protein extraction buffer extraction method and the differences between the protein enzyme digestion method. The proposed protein of different buffer of peptides and protein enzyme extraction efficiency were analyzed. The result indicates that urea thiourea extraction buffer extracted wheat, corn and soy protein showed better in bands number, good stability, high extraction yield and the TCA acetone after processing of rice leaf protein extraction phenol formulation of higher purity clear sample. But the traditional enzyme digestion can get higher peptide recovery rate relative to the microwave-assisted enzyme digestion method.(2) The results of TripleTOF5600 mass spectrometry (AB SCIEX) cascade with liquid chromatography tandem were subject to qualitative analysis. Some 46108 of the 9001 segments of peptides and proteins are showed up in the two samples of yellow rust resistance wheat Yr5 and its isogenic line, Taichung29* 6/Yr5 (TYr5). We get 2254 proteins with accurate quantitative information and 78 proteins differences in protein content change is greater than 2 times by NCBI blast. We run the Gene Ontology (GO) of 78 differentially expressed proteins in the functional annotation and analysis. It was found that most of the proteins located in cell matrix and ribosome organelles function in combination, catalytic, and other functions mainly involved in metabolism, cell metabolic process, biological process and stress reaction.