| Soil salinization is one of the world’s resource and ecological problems, according to incomplete statistics, there are about 99.13 millions hectare of saline-alkali soil in China. Alkali stress seriously restricted the agricultural production of our country. The development and utilization of saline-alkali soil is a major problem urgently to be solved to ensure the efficient and sustainable development of agriculture and food security. Glycine soja, the wild relatives of the cultivated soybean, has excellent resistance to salt, alkali, cold and disease. It not only has high resistance to the alkali(can survive at soil p H>10), but also can reduce the content of the water soluble ions in the saline-alkali soil. Therefore, the study of alkali resistance mechanism of Glycine soja, has an important role to develop alkali resistant crops, control and use of saline alkali soil effective. Protein is the direct execution of plant physiological activities, therefore, it is important establishment the protein profile of Glycine soja under alkali stress by two-dimensional electrophoresis technology, detect and analyze of the expression patterns of differentially expressed proteins for study the resistant mechanism of Glycine soja ender alkali stress.In this study, we use the alkali resistant Glycine soja G07256 as experiment material, analyze7 physiological indexes related with alkali stress; established two-dimensional electrophoresis system of Glycine soja leaves proteomics; and established the profiles of different expression proteins in Glycine soja leaves under alkaline stress. Analyzed preliminarily of the isoelectric point, molecular weight distribution and expression pattern of differential expression protein. This study laid a good foundation to obtain the response protein under alkali stress. The main results are as follows:1. Analysis of physiological index of Glycine soja under alkali stressTo understand the alkali resistance characteristics of Glycine soja. Treated the seedlings at the age of 21 days with 50mmol·L-1 Na HCO3. Detected the content and activity of MDA, proline,soluble sugar, SOD, POD, CAT and ROS, 7 physiological indexes of alkali stress treatment 0h, 12 h,6h, 24 h and 48 h. The MDA, proline and soluble sugar reached maximum accumulation in treatment of 48h; SOD, POD and CAT respectively reached the highest activity in 6h, 24 h and 48 h,at the same time, ROS reached the maximum accumulation at 12 h and decreased to normal level at48 h. This shows that the Glycine soja resist the stress by accumulating of proline and soluble sugar and expression SOD, POD, CAT in turn.2. The establishment of two dimensional electrophoresis systemTo establish a two-dimensional electrophoresis(2-DE) system with high resolution and good repeatability. We extracted the total proteins of Glycine soja leaves through Tris-phenol method,Tris-HCl method and TCA-acetone method, compared the protein quantity and yield, and compared the 2-DE images obtained by using different protein loading amounts, isoelectric focusing(IEF) parameters, and gel concentrations. Our results identified a suitable protocol for2-DE analysis, using the Tris-phenol method for protein extraction, 2 000 μg protein loading amount, 90 000 V·h IEF condition, 10% gel concentration. Under this condition, we obtained good2-DE images with much clearer background, more protein spots, and better reproducibility.3. Analysis of differentially expressed protome under alkali stressIn order to obtain proteins response to alkali stress. Using the 2-DE system we estabished,extract proteins and two-dimensional electrophoresis of Glycine soja leaves treatment by Na HCO3 stress 0 h, 6 h, 12 h, 24 h and 48 h, in order to ensure the repeatability of 2-DE, at least three times repeat to each treatment. Using PDquest software analysis the gels, compared every two treatment gels, select differentially expressed proteins with a threshold fold > 1.5, P < 0.05, received 101 protein points significant change. The molecular weight of the protein points differentially expressed mainly distributed between 20 to 60 kD, and the isoelectric point mainly distributed between 5.6 to 6.0. Among the different treatments, detect most different protein points in 24 h and48 h, show that treatment maybe the most active period of the stress response system. 101 protein points in different treatment period, have a total down-regulate expression 127 times and up-regulate expression 52 times, number of down-regulate account for 71% of the total number.Illustrate the Glycine soja may resistance to alkali stress by lowering metabolism and reduce energy consumption. |
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