Proteomic Analysis For Root Secretion And Leaf Intercellular Fluid From Brassica Napus

Plant roots secrete a variety of compounds called root secretion which is the important medium that has been shown to be involved in the processes of nutrient acquisition, energy and media communication between plant and soil microenvironment. The root secretory proteins are thought to play important roles in the regulation of soil chemical properties and soil microorganisms. To analysis the root secretory proteins of Brassica napus may help to understand the mechanism of plant adaptation to growth environment.Intercellular-fluid which has important significance for plant growth and metabolism refers to liquid of cell apoplast, serving as a bridge that perceives and conducts signals between symplast and environment. Proteins secreted into apoplast reflect its functional diversity. Studying on the intercellular-fluid proteins revealed new sights into plant responses to stress.In this work, Brassica napus “Dingyou-4” was chosen as experimental material. The method for protein extraction from root secretion and leaf intercellular fluid from Brassica napus was established and optimized. The platform for root secretion and leaf intercellular-fluid proteomics of Brassica napus research was established by two-dimensional electrophoresis(2-DE) and mass spectrometry(MS). The main results are as follows:1. The sterile hydro culture and microbial detection system was established for Brassica napus rhizosecretion proteomic research. Based on the traditional sterile hydro culture system, microbial detection system was introduced and result that sterility testing and seedling cultivation were processed simultaneously. Thus the operation steps were simplified, the experiment cycle was shorted and the pollution rate was reduced.2. The process of protein extraction from root secretion was optimized. The optimized TCA-acetone process was assisted by N-Lauroylsarcosine sodium salt which is a protein detergent. Better result was obtained from the optimized method.3. The root secreted proteins from Brassica napus was analyzed using the platformestablished by 2-DE and MS. Based on 2-DE, a separated gel map with clear background of root secreted proteins was obtained. 11 proteins were identified successfully by MALDI-TOF-MS/MS and bioinformatics technology. Some related literatures and protein database suggested that most were secondary metabolism and4. A method was established for leaf intercellular-fluid collection of Brassica napus. Based on the infiltration-centrifugation method, double centrifuge tubes were used for leaf intercellular-fluid collection. The concentration of Chlorophyll in the collected intercellular fluid was less 3% than the control show that the collected intercellular-fluid could use for the lasting experiment.5. The leaf intercellular-fluid proteomics was separated by 2-DE and the separation gel maps were obtained. Two maps with clear background were chosen for under low phosphorus stress analysis using image software. The results revealed that 5 proteins were significantly up-regulated and 4 proteins down-regulated.6. For MALDI- TOF- MS/MS analysis, 7 proteins were identified successfully. Related literature and protein database indicated that these proteins involved in stress defense process. Germin-like protein 1 was a typical defense-related intercellular-fluid protein and protein aspartic protease was found that it could induce defense response in the wild Rice and Arabidopsis.