Recombinant Expression And Antifungal Mechanism Of Permatin

Thaumatin-like proteins(TLPs)are the family 5 of pathogenesis related proteins(PR-5),sharing significant amino acid sequence homologeous with the sweet tasting protein thaumatin,which is present in the fruits of the tropical plant thaumatococcus daniellii.At present,TLPs are commonly found in insect,fungus and plants.TLPs are a group of cysteine-rich proteins,in which the disulfide bridges of TLPs help stabilize the protein.Recently,many members of TLPs are studied because of their significant antifungal activity and the similar functions with adiponectin.The permatin protein isolated from naked oat(Avena nuda)seeds belongs to the PR-5.In order to recombinant exression of permatin gene from naked oat seeds by E.coil.,permatin gene were tested by fused with soluble tag protein or periplasmic express signal peptide.The results showed that only NusA-Permain protein was expressed solubly.The antifungal activity against Trichoderma spp,Fusarium oxysporum and Saccharomyces cerevisiae of permatin was tested.Firstly,we determined the half-inhibitory concentration of permatin against Trichoderma spp.and Fusarium oxysporum using radial growth assays;Next,the effect of permatin on spore germination and mycelium growth of Trichoderma spp.was observed by Delta Vision,whilst inhibition rate of spore germination was counted;Further more,the effect of permatin on fungal hyphae and spores permeability was examined by fluorescent probe propidium iodide(PI).The results indicated that permatin showed remarkable antifungal activity against Trichoderma spp.and Fusarium oxysporum and the IC50 values were determined to be 0.73 ?M,21.42 ?M,respectively.And permatin could significantly inhibit germination of the Trichoderma spp.spore,as well as the growth of hyphae.In addition,permatin was capable of causing membrane permeabilization in the fungi Trichoderma spp.and Fusarium oxysporum.ROS of Fusarium oxysporum treated with permatin was assemblaging.On the other hand,the effect of CaCl2 on the antifungal activity of permatin was determined with Saccharomyces cerevisiae.Liquid culture method was carried out and the growth curves of differently treated groups were drawn.Besides,morphologic and ROS change were detected using DCFH-DA(2',7'-dichloro fluorescein diacetate salt)by fluorescence microscope observation.The results suggested that CaCl2 could enhance the antifungal activity of permatin,promote the disruption of permatin against fungal membrane and raise ROS level.