Purification And Characterization Of An Anti-fungal Peptide From Braassica Campesttris L.ssp.Chinesis(L.) Makino Var.Rosularis Tsen Et Lee

Fungal diseases generate new challenges for agriculture and cause economic loses.The traditional chemical pesticides have several drawbacks:high toxicity,poor target selectivity,polluting the environment and inducing resistance of insects.All of these problems prompted researchers to focus on environmental friendly pesticides and antimicrobial peptides represented by biological.Pesticides have become a trend.Antimicrobial peptides exist extensively in plants.Antimicrobial peptides have many advantages:broad-spectrum antimicrobial activity,stability,reverses the bacteria's resistance,safety and efficient.Antimicrobial peptides have a broad application prospect in industry,agriculture and animal husbandry.Wuta-tsai is a variety of Brassica campestris ssp.chinensis var.communis and has nearly one thousand years planting history.The study of the Antimicrobial peptides from Wuta-tsai has not been reported.The intent of this research proposal is to isolate and characterize proteins with antifungal potential from seeds of Wuta-tsai(Brassica campestris L.ssp.Cinensis(L.)Makino var.rosularis Tsen et Lee).A 5.7 kDa antifungal peptide(SUP-2 peptide)was purified from the seeds of Wuta-tsai.The purification procedure entailed affinity chromatography on Affi-gel blue gel,ion exchange chromatography on SP-Sepharose Fast Flow,FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex-75.It inhibited mycelial growth in fourteen kinds of phytopathogenic fungi including Mycosphaerella arachidicola(IC50=1.46 ?M),Exserohilum monoceras(IC50=2.91 ?M),Fusarium olanif.sp.glycines(IC50=3.52 ?M),Valsa mali(IC50=3.98?M),Fusarium oxysporum(IC50=4.82 ?M),Colletotrichum micotianae(IC50=5.24 ?M),Alternaria longipes(IC50=6.51 ?M),Fusarium graminearum(IC50=8.17 ?M),Helminthosporium maydis(IC50=11.21 ?M),Botrytis cinerea(IC50=19.38 ?M),Colletotrichum higginsianum(IC50=26.17 ?M),Magnaporthe oryzae(IC50=50.29 ?M),Colletotrichum gloeosporioides(IC50=73.68 ?M)and Gliocladium catenulatum(IC50=183.49 ?M).Spore germination in Colletotrichum micotianae,Magnaporthe oryzae,Helminthosporium maydis,Colletotrichum higginsianum and Exserohilum monoceras was inhibited by 500?M SUP-2 peptide by 15.73%,59.49%,87.63%,88.05%,94.10%and the EC50 toward Exserohilum monoceras was 134.06 ?M.The peptide had no inhibitory effect toward bacteria and hepatoma cells.In conclusion,the above results evince that SUP-2 peptide from seeds of Wuta-tsai have broad-spectrum resistance to fungi and is a good candidate for the exploration of anti-fungal biopesticides in agriculture.Further research on its efficacy in agriculture as biopesticide is warranted.