Proteome And Its Experimental System From Leaves Of Populus Cathayana Under Salt Stress By Two-dimensional Gel Electrophoresis

Salt stress is one of the major abiotic stressed conditions in natural habitats, which decreasing plant growth, influence plant population and community stucture, regulating the biodiversity and stability in ecosystem. On the basis of this, most of researchs were foucsed on the relationship between plants and salt soil. One of the hotspots is developing plant breeding, which indicate to improve the ability of plant species living in salt soil. In order to fit this demands, proteins should be study further for these plants, which was the most important contents in organism cells.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, there are many problems about two-dimesional gel electrophoresis that still have not discovered, such as lower repeatability, little quantitative loading of sample, unsuitable for alkaline proteins and acidic proteins. Furthermore, many plant tissue contains a lot of secondary metaolites, such as pigments, phenol, polysacchavide. In this study, we carried out the experiments to study the protein in the leaves of Populus cathayana. Firstly, a system of 2-DE suitable for P. cathayana was established. Then the protein in leaves of P. cathayana which was planted in salt stress conditions were compared with those planted in normal conditions. The main results are below:1) The whole protein of leaves under salt stress were extracted and analysed through SDS-PAGE methods and 2-DE methods, respectively. The phenol extraction methods could identified more bands than the improved acetone precipitation methods and the thichloroacetic acid acetone methods. But this methods still could not gave enough imformation about the differential proteins. While using 2-DE methods with the phenol extraction, and analyzed by ImageMaster 2D Platinum 6.0 software, 721 proteins spots on 2-DE gels were identified, which is 118 proteins spots more than the improved acetone precipitation methods, and 284 protein spots more than the TCA-acetone precipitation methods, respectively.2) When the quantitative loading of samples were 100/IPG gel, results showed that the protein spots were few. While the quantitative loading of samples were 300/IPG gel, more than 600 protin spots was detected. The results showed that 300/IPG gel was the suitable treatment. When the quantitative loading of samples were 600/IPG gel, there were many horizontal lines appeared on the 2-DE gels maps, means that the samples were too much.3) It has been demonstrated that prolonging the time of IEF (isoelectric focusing) under low voltage could make the final voltage reached 8 000V. The results showed that when the time of IEF was 90 000 Vh, the protein spots were clearness and unifornly separate on the 2-DE gels maps. While the time of IEF was 40 000 Vh, there were many protein spots could not move to their isoelectyic points. The 2-DE gels had many horizontal lines of proteins. When the time of IEF reached 150 000 Vh, the cathode drift was appearance.4) The results showed that while the equilibrium time was 10 min, the protein spots had verical streaks. When equilibrium time was 20 min, the protein spots were clearness and unifornly separate on the 2-DE gels maps, means that 20 min was the best equilibrium time of the IPG gel. When equilibrium time was 30 min, some of the protein spots were lost.5) The protein in the leaves of P. cathayana which was planted in salt stress conditions were compared with those planted in normal conditions. The results showed that there were 46 protein spots significantly difference using the Image Master 2D Platinum 6.0 software. Thereinto, 36 protein spots were up-regulated and 10 protein spots were down-regulated. Four protein spots were new appeared in the protein maps of the leaves salt stress, but was not existed in CK. Those protein may be relationship with the signal trancduction, photosynthesis, energy metabolism, cell growth /division and eytoskeleton stability. These may be have important functions of P. cathayana response to salt stress.