Seperation And Analysis Of Proteins Extracted From Intercellular Washing Fluid Of Wheat Leaf

Intercellular washing fluid (IWF) protein in plant cells plays an important role in various biological processes in plant such as plant growth and development,disease and stress resistance, and so forth. As the content of IWF protein in plants is low, and the cell membrane is easily damaged, it is difficult to extract and separate IWF proteins effectively. This paper aims to establish an effective and suitable extraction method for wheat leaf IWF protein, and apply various research methods of proteomics to separate the wheat leaf IWF protein. It provides a new way to conduct a research on IWF proteins in wheat growth and development, disease and stress resistance and so on. It also laid a solid foundation for the reveal of the biological functions of IWF proteins.Firstly, we optimized an extraction method that is more suitable for wheat leaf IWF protein. According to our results, the optimized method can be simply stated as follows:wheat leaves were completely soaked in 50 mM NaAC as the extraction buffer,then vacuumed for 20 min, and the IWF was collected by centrifugation at 2 000×g for 15 min after the extraction buffer was removed by centrifugation at 40×g for 5 min. IWF were collected and freeze-dried, then separate the IWF proteins using SDS-PAGE.44 small bands separated by SDS-PAGE were cut and digested respectively, and then analysis by MALDI-TOF/TOF tandem mass spectrometry. 27 proteins were identified in all. Some of these proteins are reported as intercellular located. When using two-dimensional electrophoresis (2-DE), a total of 1247 and 522 protein spots can be detected in the 2-DE image of wheat leaf total protein and IWF protein. When total proteins were separated by 2D-DIGE technology,1376 protein spots were detected. It can improve the quantitative accuracy and is able to reveal more protein spots.High performance liquid chromatography (HPLC) can make up for deficiencies in SDS-PAGE and 2-DE technology. For separate and analysis IWF proteins at the maximum extent, we explored the HPLC method for the separation of wheat leaf protein in this paper. We compared the effect of different extraction buffer and separation buffer in HPLC separation. The results shown that, extraction buffer 1# (1M KH2PO4+K2HPO4) and 3# (50 mM Tris-HCl 10 mM MgSO4, 0.1%β-mercaptoethanol,2 mM PMSF) have their own advantages. When the two buffers were mixed in equal volumes, most fractionations can be separated. And proteins of wheat leaves can get the best separation with 0.1% TFA aqueous solution as separation buffer.