Preliminary Study On The Mechanism Of Sanxiapeptin Against Citrus Decaying Fungi Based On Cell Surface Target

Citrus is one of the most important economic crops in the world and is favored by consumers because of its good taste and rich nutrition.However,in the process of storage and transportation of citrus,the spoilage caused by postharvest fungus caused huge economic losses.Among them,green mold attributed to Penicillium digitatum is one of the main diseases of citrus after harvest.Although traditional chemical antifungal agents(such as Bellkute,Prochloraz,etc.)have a good inhibitory effect on P.digitatum,they have a certain degree of harm to citrus fruit quality,ecological environment even human health.Therefore,the development of a green and efficient biological antifungal agent is of great significance and economic value for the postharvest storage of citrus.In the early stage,we isolated a unparalleled linear pentapeptide from the secondary metabolite of Penicillium oxalicum with the structure O-Me-Baba-N-Me-L-Thr-D-Thr-N-MeL-Val-L-Ser,named " Sanxiapeptin ",which has a strong inhibitory ability on P.digitatum.To study the potential target of Sanxiapeptin acting on P.digitatum,this subject is based on the morphological observation and the experiment of the antifungal effect of the protective agent,the method of transcriptomics was used to analyze and verify the differentially expressed genes only caused by Sanxiapeptin.and provide a theoretical basis for popularization and application.The main results of the current research are as follows:1.The morphological changes of P.digitatum were observed by microscopic technique.After treatment with Sanxiapeptin,the cell surface morphological changes of P.digitatum were significantly changed,which was significantly different from the control group.The effects of the cell wall protective agent sorbitol and the cell membrane protective agent ergosterol in the fungal inhibition process of Sanxiapeptin were analyzed.Different from the positive control membrane target fungicide,ergosterol,the cell membrane protective agent ergosterol can significantly relieve the inhibitory effect of Sanxiapeptin,while the cell wall protective agent sorbitol will promote its antifungal ability.It follows that both the cell wall and the cell membrane are involved in the process of Sanxiapeptin inhibiting P.digitatum.Since the GPI(Glycosylphosphatidylinositol)-anchored protein of fungal cells is distributed in both the cell wall and the cell membrane,it is preliminarily speculated that the action site of Sanxiapeptin is located on the cell surface,and the antifungal effect can be achieved by affecting the function of GPI lipid anchor or GPI-anchored protein.2.Through the BGISEQ-500 research platform,the transcriptome of P.digitatum treated with Sanxiapeptin was sequenced,and the 6.38 Gb sequence data generated by the sequencing was analyzed without parameters.The mutual comparison rate of transcriptome sequences was93.66%,showing high sequencing quality.After P.digitatum was treated with Sanxiapeptin,more than 2,000 genes were changed in expression,mainly involving pathways such as cell membrane,cell wall components,autophagy,and glucose metabolism.Given the different action pathways of Sanxiapeptin and Bellkute,530 differentially expressed genes caused by Sanxiapeptin alone were selected for analysis,and it was found that these genes were mainly related to hydrophobin,ergosterol synthesis,glycan synthesis,and GPI metabolism.3.Among the 17 candidate genes,the RT-PCR semi-quantitative expression detection of8 genes was completely consistent with the transcriptome sequencing results.As follows:CL254.Contig3,CL279.Contig24,CL1025.Contig2,CL2611.Contig2,CL3.Contig4,CL2032.Contig3,CL2193.Contig3,CL277.Contig8.Using Hyg R as the marker gene,the overexpression vectors of CL3.Contig4 and CL2193.Contig3 were successfully constructed and transformed into P.digitatum protoplasts.4.The ability of Sanxiapeptin to germination of spores has a more obvious inhibitory effect compared with that of Bellkute and Prochloraz.It is speculated that the antifungal effect of Sanxiapeptin begins in the spore germination stage of P.digitatum.The antifungal process may also be related to cell wall growth and elongation,transport and delivery of components,and remodeling.16 candidate genes were screened,and the results of RT-PCR expression verification of 2 genes,CL2443.Contig1、Unigene1157,were consistent with the transcriptome sequencing data.