Purification And Characterization Of Proteins From Plants With Activity Against Fungi And Study Of Antifungal Mechanism

In the long-term evolution,plants can produce some peptides and proteins to resist the invasion of pathogens.These substances are referred to as plant antimicrobial peptides or proteins depending on the molecular weight.China is a country with an abundance of plant resources.The intent of this project was to screen and purify antimicrobial peptides or proteins to facilitate the future development of new antibiotics.In this study,after eighty-one times of preliminary screening tests,the seeds of JingShuiCai(Brassica juncea var.multisecta L.H.Bailey)and ZiCaiTai(Brassica campestris L)were shortlisted since they contain such proteins.Ion-exchange chromatography on SP-Sepharose and fast protein liquid chromatography on Mono S and Superdex 75 were used to isolate the antifungal proteins from their seed extracts.Their molecular weights were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)to be 30 kDa and 13 kDa,respectively.Then thermal stability and pH stability of the two proteins and their stability in presence of metal ions were tested.It was discovered that theactivity of the two proteinswas fully preserved in the pH range 1-13 and in the temperature range 40-80℃.The antifungal activity of JingShuiCai antifungal protein toward Fusarium oxysporum was fully preserved in presence of Al3+、Zn2+、Cu2+、Fe3+、Pb2+、Mg2+、K+、Cr3+and Mn2+ions but augmented by Ca2+ions.The activity of ZiCaiTai antifungal protein toward Colletotrichum gloeosporioides was maintained in the presence of Al3+、Zn2+、Cu2+、Fe3+、Pb2+、Mg2+、K+and Cr3+ions but augmented by Mn2+、Ca2+ions.The antifungal activity of ZiCaiTai antifungal protein toward Colletotrichum gloeosporioides was retained despite the presence of surface active agents and organic solvents.It was discovered that theactivity of the ZiCaiTai antifungal protein toward Colletotrichum gloeosporioides was fully preserved in presence of surface active agents and organic solvents.Then the mechanism of antifungal action of JingShuiCai antifungal protein toward Fusarium oxysporum was discussed.With the use of EDTA,it was revealed that the antifungal activity of JingShuiCai antifungal protein was dependent on Cr3+ ions.With the use of SYTOX Green、Rh-123、and H2DCFDA,it wasdisclosed that JingShuiCai antifungal protein destroyed the integrity of the fungal cell membrane,elicited changes in the mitochondrial membrane potential,and brought about accumulation ofreactive oxygen species(ROS)in fungal cell.DAPI dyes was employed to stain the nuclei compared to extracted DNA,It was found that theJingShuiCai antifungal protein was able to decompose DNA both intracellularly and extracellularly.