Study On The Proteomic Difference Of Wheat Leaf Under Salt Stress

Secondary salinization of soil is an important abiotic factor limiting the crop yields worldwide. Due to different degrees salt injuries, large proportion of crop species are difficult to exert great potentiality of high production and good quality. According to statistics, drought and salinization caused by more than 40% reduction in the impact of crop yields in the various environmental factors. How to improve salt tolerance of plants has become an increasingly imminent and needs to be resolved. If we want to select high-salt-tolerance plant varieties, we must study the mechanism of salt-tolerant plants depthly. Traditional researches focused on a small number of genes and expression field, it is difficult to reveal a comprehensive mechanism of salt-tolerant plants. Protein is the implementation of the various physiological functions, and is a direct manifestation of the phenomenon of life. So the study of the protein structure and function of plants can directly state the mechanism of salt stress condition changes.Two-dimesional gel electrophoresis(2-DE) is one of the most useful methods in proteins research, which has been widely used in biology and biochemistry. At the same time, it still had many deficiencies, such as poor reproducibility, relatively small size sample loading and difficult preparation, and low abundance protein can easily be overshadowed by the High, a number of high molecular weight or very acid and very alkaline proteins could not be separated. Wheat is the world's largest crop planting acreage, There are 1/3 percent of the population eat wheat as the main food. The development of wheat production is importance to meet the food needs of the people and improve living standards of urban and rural residents and ensure national security and social stability in the new circumstances. So the study on salt tolerance of wheat has attracted extensive attention.In this study, wheat leaves were used to study the proteomic difference habitats of salt stress. But in view of its rich colors, polyphenols, polysaccharides, organic acids and other secondary metabolites, the sample preparation becomed more difficult. Firstly, a system of 2-DE suitable for wheat leaf was established.Then the protein in leaves of wheat which was planted in salt stress conditions were compared with those planted in normal conditions. The main results are summarized as belows:(1). The whole protein of leaves under salt stress were extracted and analysed through 2-DE methods, respectively. The TCA/acetone precipitation method could identified more bands than other methods.While using 2-DE methods with the phenol extraction, and analyzed by ImageMaster 2D Platinum 6.0 software, 486 proteins spots on 2-DE gels were identified, which was 17 proteins spots more than lysis-buffer method and far more than urea/thiourea method and KCl-methanol/ammonium-acetate method. The proteins which uniformly spread on the gel were round or oval shape, regularly, clear and good repeatability. This method could not effectively remove the phenol and quinone-type material (the upper part of the high abundance of impurities difficult to remove), but tests showed that the reproducibility of this method compared with other methods are high.(2). Based on the traditional TCA/acetone method, prolong centrifugal force and centrifugation time after the sample dissolved by the lysis buffer. This step could effectively remove the polyphenol compounds and quinones and access to gain shape, regular, clear, less interference with vertical stripes protein spots. This method could meet the needs of follow-up mass spectrometry sequencing well.(3). This research showed that most of the wheat proteins are acid proteins and most of them centered largely on the acidic side of the scope of pH4-7. Using ImageMaster 2D Platinum 6.0 software for analysis of 2-D map patterns showed that the conditions of salt stress proteins and the control pattern have similar patterns, but there were also quite difference. Under in the salt stress, some new proteins expressed increasingly and some were difference in expressing content.(4). The protein in the leaves of wheat leaf which was planted in 24 h, 7 d salt stress conditions were compared with the control which planted in normal conditions. The 24 h salt stress condition had 12 protein spots were down-regulated, 34 protein spots were up-regulated and 4 new protein sports were appeared in the protein maps, but was not existed incontrol. The results aslo showed that there were 13 protein spots significantly difference using the Image Master 2D Platinum 6.0 software. These would have important functions of wheat response to salt stress.(5). This study identified eight protein spots successfully. They represented five types protein: ribulose-1,5-bisphosphate carboxylase/oxygenase large, photosystem I reaction center subunit, oxygen-evolving complex precursor, oxygen evolving complex 33 kDa photosystem II, 2-Cys peroxiredoxin BAS1. Campared with the control they were all up-regulated. Among them only the 2-Cys peroxiredoxin BAS1 content was up-regulated with the treatment time prolonged. Others were up-regulated in the 24h salt stress condition, but down-regulated in the 7 d. This study speculated that these differentially expressed proteins are connect intensivly with plant physiological and bio-chemical activity metabolism, photosynthesis and degradation of synthetic substances.