| Sonchus brachyotus DC. is perennial herbal member of sonchus Compositae family, one of the"three weeds"in Northeast Soybean, caused great loss to agricultural production. Existing herbicides only have effect on the ground part of Sonchus, but powerless to the underground part. Root cause is herbicide have poor conduction in the underground rhizomes, and the underground part of Sonchus are able to sleep through the cold winter, the following year continue to harm. This study detect the dynamic changes of proteins in the course of Sonchus rhizome dormancy by two-dimensional electrophoresis and mass spectrometry to find and identify the different expressed proteins to find proteins associated with dormancy in order to explore their dormancy mechanism and provide new ideas for control of Sonchus. Currently, no such studies were reported at home and abroad.This study selected perennial weeds Sonchus underground rhizomes as experimental material, the main study for the following two aspects. First screening total protein extraction method for Sonchus rhizome and establish the two-dimensional electrophoresis analysis system suitable for Sonchus, then sampling and analysis different time points in the course of dormancy of Sonchus by two-dimensional electrophoresis, find differential protein spots and mass spectrometry analysis these protein spots. The main results are as follows:(1) Determine Trizol precipitation method for total protein extraction method of Sonchus brachyotus DC rhizome.The purity of the protein and SDS-PAGE electrophoresis separation as an index to compare the total protein extracted by TCA-acetone precipitation method, phenol method, Tris-Hcl method, Tris-acetone-phenol method, Trizol precipitation method, phenol-TCA- acetone method and double ethyl method seven methods, The results showed that Trizol precipitation method have the highest purity of protein and the largest, most clear band of SDS-PAGE electrophoresis, most protein spots and clear map of two-dimensional electrophoresis, without vertical and horizontal stripes, is the best total protein extraction method of Sonchus brachyotus DC rhizome.(2) Established SDS-PAGE and two-dimensional electrophoresis system suitable for Sonchus brachyotus DC rhizomeSDS-PAGE: With 5% stacking gel and 12% separating gel, the sample volume was 15μg, when sample in stacking gel, the current is 10mA; arrived separating gel, the current is 40mA, when the bromophenol blue to the bottom of the gel, end the electrophoresis. Two-dimensional electrophoresis: The isoelectric focusing use 24cm linear IPG strips (PH=4-7), taking 450μL hydration solution containing about 1200μg protein, passive hydration, hydration 14 h. Isoelectric focusing were carried out at 20℃, 50 V, 30 min; 100 V, 1 h; 300 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 2000 V, 1 h; 5000 V, 1 h; 8500 V, 80000Vhr; 500 V, 1 h. Electrophoresis followed after completion of gel balance, the strips in turn in the balance fluid containing DTT and iodoacetamide each 15 min. The second electrophoresis by 12.5% SDS-polyacrylamide gel, pre-electrophoresis 45 min, 2w/gel; official electrophoresis 5-6 h, 15w/gel. Gels were stained by thermal CBB staining.(3) Mass spectrometry analysis 46 differential protein spots, obtained 45 statistically significant protein spots, including 16 proteins related to energy metabolism, 9 proteins associated with plant adversity physiological, 4 proteins associated with plants antioxidation, 3 proteins related to cell wall synthesis, 3 proteins related to protein hydrolysis, 2 proteins associated with signal transduction and 8 unknown proteins and hypothetical proteins. These results showed that multiple physiological processes changed in the course of dormancy of Sonchus brachyotus DC rhizome. |
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