Proteomic Analysis Of Sickle Alfalfa (Medicago Falcata L.) Proteins Under Cold Stress

Plants suffer from various of biotic and abiotic stress during the grow process. Cold is one of the severe phenomena. Almost all plants undergo chilly stress. It restricts severely the distribution of the plants and affects yield. Every year, crop losing attains to hundreds of billion because of low temperature in the world. In China, the area that endures low temperature reaches to two million km2 each year. So it is crucial and valuable to improve the quality of cold resistance of the plants. In the end of the 18th century, many scholars had studied the theory of cold resistance. However, the early researches focused on cell modality changes and the transformation of biophysics and biochemistry after the cold stress condition. With the prompt development of the moleculer biology technology, the researches about cold stress has attained to molecular level. It accelerates the development of the plants gene engineering to cold resistance.The sickle alfalfa has been used as tested materials to study on the discrepancy expression of proteome by the method of two-dimensional gel electrophoresis. Some important steps, such as sample preparation, the selection of lysis solution, the choice of stress time, the definition of protein quantity, isoelectric focusing, balancing and staining, were discussed and improved. The conclusion is as follow: comparing the three different methods of proteins extraction, including TCA-acetone precipitation, phenol extraction methanol /ammonium acetate precipitation and trizol precipitation, we have concluded that the method of trizol precipitation is most suitable for the extraction of sickle alfalfa proteins. It has high efficiency, and favourable compatibility to the next steps. In the lysis solution, we selected 8M urea, 2%CHAPS and 1%DTT. It is suitable for dissolving of sickle alfalfa proteins. Comparing with 6h, 12h, 24h, 12h are the best stress time. For 11cm strip with a linear gradient of pH4-7, the protein quantity for silver staining method is 50-80μg, and for CBB staining method is about 300μg. For 24cm strip with a linear gradient of pH4-7, the protein quantity for silver staining method is 100-120μg, and for CBB staining method is 500-600μg.Adopting this protein quantity, we can get 2-DE gels with fine focusing effect and limpid background. Silver staining method is sensitive and propitious to gels analysis. The method of Coomassie Brilliant Blue staining can obtain clear backdrop, and it is compatible with Mass spectrometry. Therefor we establish a technique method of 2-DE which is applied in sickle alfalfa. Profiling the discrepancy expression of sickle alfalfa proteins under cold-stress condition with the improved method of two-dimensional gel electrophoresis, the result indicated 84 spots showed a 2-fold change under cold stress condition. 58 proteins were up-regulated, and 26 proteins were down-regulated. These protein spots were analyzed by ImageMaster 2D platinum 5.0 software. We get the identification of molecular weights and pI of these proteins. This study gives us new insights into cold stress response in sickle alfalfa and demonstrates the power of the proteomic approaches in forage biology studies.