| Lanzhou lily is a medicinal and edible sweet lily,which has rich nutritional value and is one of the characteristic crops in Lanzhou.However,with the rapid development of lily industry,fungal diseases have become the main bottleneck restricting the fine processing of Lanzhou lily.The researchers found that the main spoilage pathogens were Aspergillus flavus and Rhizopus oryzae by analyzing the diversity of microorganisms in the bulbs of Lanzhou lily,and some reports showed that Rhizopus oryzae was also the main spoilage bacteria causing soft rot of Longya lily bulbs.Phenyllactic acid is a newly discovered antibacterial agent,which can not only inhibit the growth of bacteria but also inhibit the growth of fungi,and the antibacterial effect is obvious.The preliminary experiments showed that phenyllactic acid had a good inhibitory effect on Rhizopus oryzae.However,it was found in the literature that there was no report on the antibacterial mechanism of phenyllactic acid against Rhizopus oryzae.Therefore,in this study,we first used Rhizopus oryzae to infect Lanzhou lily bulbs and determined the pathogenicity of Rhizopus oryzae to Lanzhou lily bulbs.Then,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of phenyllactic acid against Rhizopus oryzae were determined by tube dilution method.The effects of different concentrations of phenyllactic acid on the growth of Rhizopus oryzae were measured by dry weight weighing method.The activities of enzymes related to metabolism of Rhizopus oryzae treated with phenyllactic acid were determined.The effects of phenyllactic acid on the morphology and structure of Rhizopus oryzae cells were observed by electron microscopy.Finally,we used proteomics and metabolomics techniques to analyze the types and changes of proteins and central carbon metabolites in Rhizopus oryzae,and used bioinformatics methods to analyze the metabolic pathways involved in differential molecules,and analyzed the correlation between proteomics and metabolomics data.The main research results are as follows :1.Study on the pathogenicity of Rhizopus oryzae to Lanzhou lily bulbs :Rhizopus oryzae increased the apparent color of Lanzhou lily bulbs,increased the relative permeability of cell membrane and MDA content,increased the activities of catalase and peroxidase,and decreased the activity of superoxide dismutase,which resulted in the gradual increase of browning degree and irreversible damage to cells.2.Antibacterial effect of phenyllactic acid against Rhizopus oryzae : The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of phenyllactic acid against Rhizopus oryzae determined by tube dilution method were 8mg/m L.The growth of Rhizopus oryzae under different concentrations of phenyllactic acid was measured by dry weight method.The results showed that the inhibitory effect on Rhizopus oryzae increased with the increase of phenyllactic acid concentration.The determination results of metabolic-related enzyme activity,pyruvate content and glucose content in Rhizopus oryzae showed that the activities of hexose kinase(HK),pyruvate kinase(PK),?-ketoglutarate dehydrogenase(?-KGDH)and pyruvate content in Rhizopus oryzae mycelia decreased with the increase of phenyllactic acid concentration after 24 h treatment with different concentrations(0,2 and 4 m /m L)of phenyllactic acid.Scanning electron microscopy and transmission electron microscopy were used to observe the morphological and structural changes of Rhizopus oryzae cultured for 24 h in the control group(0mg/m L)and the treatment group(4mg/m L).We found that phenyllactic acid treatment mainly damaged the cytoplasm and mitochondria of Rhizopus oryzae and affected the growth of Rhizopus oryzae.3.Proteomics study on the antibacterial effect of phenyllactic acid on Rhizopus oryzae : TMT marker quantitative proteomics study showed that 485 differentially expressed proteins were screened in PLA vs CK group,of which 205 were up-regulated and 280 were down-regulated.After functional classification of the screened differentially expressed proteins,the results of COG analysis showed that the significant classification of the annotated differentially expressed proteins mainly included translation,ribosome structure and biogenesis,routine functional detection and post-translational modification,protein update,and molecular chaperone.GO enrichment analysis showed that after the treatment of Rhizopus oryzae with phenyllactic acid,the expression of proteins involved in nitrogen-containing compound metabolism,biosynthesis and binding function in the cytoplasm of Rhizopus oryzae was different.Pathway enrichment analysis using KEGG database found that differentially expressed proteins involved in tryptophan metabolism,thiamine metabolism,cysteine and methionine metabolism,RNA transport and purine metabolism and other metabolic pathways.4.Metabonomics study on the bacteriostatic effect of phenyllactic acid on Rhizopus oryzae: through the study of high-throughput target metabonomics,it was found that a total of 51 central carbon differential metabolites were screened in PLA vs CK group,of which 22 were up-regulated and 29 were down-regulated.Using KEGG database to enrich metabolic pathways,the metabolic pathways of significant enrichment of differential metabolites were glycolysis,carbon sequestration of photosynthetic organisms,TCA cycle,pentose phosphate pathway,pyruvate metabolism and so on.5.Correlation analysis between proteomics and metabolomics of phenyllactic acid against Rhizopus oryzae: through the correlation analysis of proteomics and metabolomics of PLA vs CK,it was found that the main effect of phenyllactic acid on Rhizopus oryzae was to reduce the activity of key enzymes in the glycolysis pathway of Rhizopus oryzae,resulting in the down-regulation of the expression of pyruvate,the final product,and then affected the metabolism of TCA cycle.The down-regulation of the expression of various metabolites in the TCA cycle led to inhibite the growth of Rhizopus oryzae. |
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