| Xanthium spp. is a widely distributed wild weeds which contains abundant disease resistance substances, including plenty of small molecular substances and protein substances. In this study, the method of confront culture (plate growth rate method)were adopted. Fusarium oxysporum f.sp.Perniciosum., Valsasordida Nits., Botryosphaeria dothidea (Moug. ex Fr.) Ces. et de Not., Myrothecium rorideum Tode ex Fr., Sphaceloma euonymi- japonici Kurosawa and Colletortrichun graminicola (Ces.)Wilson were tested. We studied the inhibitory effect of 4 solvent extracts of X.sibiricum and extracting solution of proteins in different parts to the stains and determined their inhibitory effects of the different extacts to TMV by the method of lesion host. Afterwards, we isolated and purificated the proteins with strong resistance from seedlings of X. sibiricum by using the way of tracking activity.The results showed that the inhibitory effects of different solvent extracts of X. sibiricum to the stains had significant differences, and ethyl acetate extract had the highest inhibitory effect. When concentration of ethyl acetate extract solution was 1g/ml, the inhibitory rate of extracts to the mycelium growth of C.graminicola was 81.25%. When we put N. glutinosa as lesion host, the inactivation effect of acetone extract to TMV was 78.20%, the next was ethyl acetate extract, and the effect of ethanol and chloroform extracts to the fungi were not stable and obvious to TMV.The extracting solution of proteins in different parts of X. sibiricum to the stains had different inhibitory effect, and the inhibitory rate to the spores was invisible. Crude protein solution from seeds and seedlings of X. sibiricum had significant inhibitory effect, it was high when the fungus grew fast, the opposite was low when the fungus grew slowly. The most obviously TMV-resistance activity in different parts of X. sibiricum was 0-40% extract protein salted out from seeds, the next was 0-40% extract protein salted out from seedlings. In conclusion, the antipathogen activity of 0-40% and 40%-80% extract proteins salted out from seedlings were relatively remarkable, so we mainly used seedlings as experiment material in the follow-up study of DEAE- Sephoraes Fast Flow elution curve. The results of chromatographic separation were different by adjusting different loading volumes, elution flow rate, concentration of sodium chloride and the final elution volume when we studied the chromatographic behavior about 0-60% extract proteins salted out from seedlings by DEAE-Sepharose Fast Flow column. The results showed that the more the loading volume, the worse separation effect. The higher the flow rate, the thinner the elution peak. The more miscellaneous peak, the stabler the chromatographic effects not. The higher the concentration of sodium chloride, the thicker the elution peak, the more seriously the tailing peak. The bigger the total elution volume, the wider the each elution peak, the better the separation effect. In summary, the optimum conditions of 0-60% extract proteins salted out from seedlings by DEAE-Sepharose Fast Flow column were that the loading volume for each time was 10ml, the elution rate was 0.8ml/min, the concentration of sodium chloride was 2mol/L, the total elution volume was 200ml.In the 0-60% extract proteins from seedlings purification process, a elution peak could be obtained by CM-Sepharose Fast Flow ion-exchange chromatography. According to experiment analysis, this component contained 4 proteins, 6 relative independent peaks were found by DEAE-Sepharose Fast Flow anion exchange chromatography. |
PDF Full Text Request