Integrated Transcriptomics And Proteomics Analysis For The Mechanism Of Bacillus Pumilus HN-10 Antifungal Peptide P-1 Against Trichothecium Roseum

Trichothecium roseum is pathogens that infect a variety of fruits and vegetables,causing cephalothecium roseum,and easily accumulating toxins causing poisoning for humans and animals.The Bacillus pumilus HN-10 antifungal peptide P-1 is a novel peptide,which had strong activity against T.roseum.But it's mechanism remains unclear.In this experiment,the effects of antifungal peptide P-1 on the membrane permeability of T.roseum were studied by using atomic absorption spectrometry and spectrophotometry methods.SDS-PAGE and agarose gel electrophoresis were used to study the changes of proteins and nucleic acids in the cells.Based on transcriptomics and i TRAQ proteomics techniques,the intracellular mechanism of antifungal peptide P-1 against T.roseum was analyzed.It would provide theoretical basis and reference for fruits and vegetables post-harvest disease control and development of new food preservatives.The main results of the study are as follows:1.The effects of antifungal peptide P-1 on membrane permeability of T.roseum was ascertained.Studies showed that after treatment of T.roseum with antifungal peptide P-1,the proteins and nucleic acid,conductivity,K~+and Na~+release of T.roseum increased compared with the control group,meant membrane permeability increased.2.The effects of antifungal peptide P-1 on intracellular proteins,nucleic acids and enzymes of T.roseum was clarified.The results of SDS-PAGE test showed that the antifungal peptide P-1 inhibited the protein synthesis of T.roseum,especially the protein with molecular weight of 43.0KDa-97.4KDa.In the aspect of nucleic acid synthesis,the gel retardation of T.roseum DN A and competitive binding with EB were determined.The result showed that the antifugal peptide P-1 had not a significant gel retardation effect on T.roseum DNA,but it could compete with EB for DNA binding,and nucleic acid synthesis was inhibited.Meanwhile,the effect on the enzymes,such as the ATPase and TC A cycle key enzyme activities were reduced.3.Based on transcriptomics sequencing analysis,180 significantly Differential express genes(DEGs)were obtained.A total of 18,830 coding genes were detected in the control group,and 1,903 coding genes were detected in the treatment group.A total of 180 DEGs were obtained statistically significant(C hange Fold>2,p<0.05),which of 60 were up-regulated and 120 were down-regulated.4.The effects of the antifungal peptide P-1 for DEGs on the transcriptional level of the T.roseum were clarified.The DEGs were analyzed by bioinformatics.The results of GO function analysis indicated that the DEGs were mainly enriched in signal organization process,metabolic process,cell membrane and cell membrane composition,and transport activity.KOG analysis showed that DEGs were mainly enriched in amino acid and inorganic ion transport and metabolism,secondary metabolite transport and synthesis.K EGG pathway analysis revealed that DEGs were mainly in amino acid biosynthesis,carbohydrate metabolism,glycine serine and proline metabolism and ABC-binding cassette transporter(ABC)aspects.5.The effects of the antifungal peptide P-1 for Differential express proteins(DEPs)on the translational level of T.roseum was obtained.Based on i TRAQ proteomics,2780 proteins were identified,and 321 DEPs were obtained with statistical significance,there were 123up-regulated and 198 down-regulated proteins.GO function analysis indicated that DEPs mainly enriched in metabolic processes,signal organization processes,cell membrane functions,and catalytic activities.The results of K EGG pathway analysis indicated that the DEPs mainly enriched in endocytosis,amino sugar and nucleoside sugar metabolism,glycerophospholipid metabolism,and glycosaminoglycan and glycosaminoglycan degradation.In summary,based on the transcriptomic and proteomic data,combined with the physiological and biochemical indexes of membrane permeability and intracellular action,the antifungal mechanism of antifungal peptide P-1 against T.roseum was obtained from the following aspects:firstly,antifungal peptide P-1 increased the membrane permeability of T.roseum,and the down-regulation of related transporters proteins;secondly,resulted in the disorder of enzymes in T.roseum,it's growth was inhibited;thirdly,the protein synthesis of T.roseum was inhibited and the metabolism of T.roseum was disordered,thereby inhibiting T.roseum growth.