Isolation, Purification And Characterization Of An Antifungal Protein To Verticillium Dahliae Of Cotton

Cotton is one of the most important economic crop in our country. However, Ver -ticillium wilt of cotton, which is one of the most dangerous fascicular disease caused by Vertidllium dahliae Kleb., is the major diseases which seriously affect the yield and quality of cotton in our country. Although much progress has been made in prevention and control of Vertidllium wilt of cotton, the progess is slow. Because Vertidllium dahliae is soil-borned fungal pathogen, the effect of prevention and control is very little using the physical and chemical method.It is strongly consided that developing resistant and tolerant cultivar is the most economic and effective ways to resolve this problem. But the basic research about the pathogenic mechanism of Vertidllium dahliae shows that developing resistant and tolerant cultivar by conventional breeding is far from enough because lack of resistant resources. Objectives of this study were to isolate and purify the antifungal protein excreted by antifungal microbe in Microsclerotiniasphere of cotton field infected by Vertidllium dahliae to learn the mechanism of the antifungal protein, and to do the basic work on the wilt resistant gene engineering of cotton.The antifungal microbe separated from cotton field infected by Vertidllium dahliae were cultivated and the rough proteins of them were isolated by ammonium sulfate from 40% to 60%. The condition of the purification of the rough proteins were determined by HiTrap CM Sepharose Fast Flow column (1ml) and DEAE Sepharose Fast Flow( 1ml). DEAE week anion-exchange chromatogram is used and the best con dition is MES buffer at pH6.0, and 0-lmol/L NaCL is used to wash the column. The proteins were collected at the peak curve and the antifungal activities were tested.EacAbstract-h peak with antiftmgal activities was collected and then molecular sievechromatogra -phy were used. A purified protein was gained,it was test by SDS-PAGE which shows that it has only one ingredient.Through tests of stabilization of the rough protein done by protein enzyme and by high temperature,we can see that the purified proteins were sensitived to them. The test of stablization of the purified antifungal protein at different pH show that it was steady from pHS.O to pH 10.0. The shower test of fungal lawn, the burgeon of the spo -re and the effect to hypha of Verticillium dahliae showed that the mechanism of anti -fungal proteins had 3 facets: first, the spore become abnormality and then dissolved; second,the cellular wall was split and dissolved; third, the burgeon of Microsclerotinia were resisted.Until now, not a report was seen about the isolation and purification of antifungal protein from antifungal microbe in the Microsclerotiniasphere of cotton fie -Id. This test quested for the method of purification of the antifungal protein, and purif -ied one ingredient with its character was analysed. This research may use for referenc -e on the mechanism of antifungal proteins of the antifungal proteins of the antifungal mirobe in the Microsclerotiniasphere of cotton field and on the the wilt resistant gene engineering of cotton.