| Oil body is a subcellular structure of oil storage in plants. Its central part is thetranslucent matrix composed of triacylglycerol. The outer layer is wrapped by amonolayer of phospholipids in which there are all kinds of binding proteins. Oil bodybinding proteins play a very important role in many processes,for instance, theoccurrence and enrichment of oil bodies, the stability and degradation of oil bodies,signal transduction, the transport of proteins and lipids. Previous researches aremainly confined to the static research of oil bodies and oil body proteins in plants,however, the dynamic process of the degradation of oil bodies is seldomstudied.Therefore, using the relevant techniques of proteomics to study thecomparative proteomics of oil bodies in the germination period helps to further statechange rules of the components and modified states of oil body proteins in thedegradation process of oil bodies, which has important significance in gaining insightinto the molecular mechanism of oil bodies’degradation as well as exploring thestructures and functions of proteins-associated oil bodies.In this study, Brassica napus L.(Ding you za4) was used as the experiment materialsto study the changes of oil bodies’ shape and physiological indexes in the germinationperiod of rape seeds.The two-dimensional electrophoresis was used to analyze theexpression and change rules of oil body proteins in the germination period. This willlay the foundation for mass spectrometry identification of oil body proteins havingthe stable differential expression patterns in the germination period of rape seeds infollow-on experiments and the study of the molecular mechanism in the the oilbodies’ degradation.The main results are summarized as follows:1. The fluorescence microscopic observation method of oil bodies in the rape seedswas established. the change rules of oil bodies’ shape in the seeds’ germinationperiod were explored and the sampling time of two-dimensional electrophoresis tostudy oil body proteins was determined.The method of paraffin section was used toobserve oil bodies’ shape in the seeds’ germination period combined with the nile red dyeing.The results showed that the degradation of oil bodies mainly occurs after24hours, in addition, the degradation of oil bodies in very big extent between24hoursand36hours.The physiological researches and change laws of oil bodies morphologyin the rape seeds’ germination period were evaluated.On this basis, the sampling timeof two-dimensional electrophoresis research on oil body proteins was identified as0h,24h,36h and48h.2. The method of extracting oil bodies was optimized and obtained high purity of oilbodies.In addition, the effect of environmental factors on the oil bodies’ stability wereexplored.The results showed that pH and ion concentration have a greater influenceon the oil bodies’ stability.however, the temperature has a little influence on the oilbodies’ stability.On the basis of the original method of extracting oil bodies, the highpurity of oil bodies were obtained by increasing two steps-elution through9M ureaand extraction through n-hexane.Microscopy results showed that the averagediameter of oil bodies is2.19m. The effects of three environmental factors-temperature, pH and ion concentration on the oil bodies’ stability weresynthesized.The results showed that the oil bodies’ stability decreases obviouslywhen pH is between4and6.However, the diameter of oil bodies increasesobviously,besides,the diameter of oil body reaches maximum5.5m when pH is5,which is1.28times more than the diameter of the control group.The oil bodies’stability decreases and the diameter of oil body continues to increase with theincrease of ion concentration.The diameter of oil body reaches5.62m while NaClconcentration increases to250mM,which is1.57times more than the diameter of thecontrol group. The temperature almost has no effects on the oil bodies’ stability andthe average diameter of oil body is2.33m at different temperatures.3. The two-dimensional electrophoresis method based tube gels for the separation ofoil body protein in Brassica napus seed was established and optimized. The oil bodyproteins during germination period were extracted by purifying oil body and on thebasis of protein extraction method established in the laboratory. The60mM Tris lysisbuffer (7M Urea,2M thiourea,4%CHAPS,2%ampholyte pH3-10,2%DTT,60mMTris) was used to cleave the protein powder and the silver nitrate staining method thatis suited for two-dimensional electrophoresis gels of oil body proteins was assuresed as the best staining method.4.The oil body proteins were extracted from rapeseeds during germination period andseparated by2D-PAGE. After silver staining, the proteinic image patterns wereanalyzed by PDQuest8.0.1software. Patterns analysis showed that: different proteinswere stably expressed. The study has a better repeatability of the patterns, the overallcorrelation coefficient of the oil body protein profiles during germination is0.706021.117proteins were obtained after overall match of the four maps.47proteins showeddiffenently expressed between0h and24h,49proteins showed diffenently expressedbetween24h and36h.61proteins showed diffenently expressed between36h and48h.19proteins showed diffenently expressed between0h and48h. The study finallyassured that there are7proteins which are persistently up-regulated and there are10proteins which are persistently down-regulated by further analysis through PDQuestsoftware combined with manual correction. |
