| In this work,Arthrobotrys oligospora was used as the research material and treated by two representative inhibitory compounds in soil ammonia and benzaldehyde.We collected spore samples treated and untreated by these two compounds after 24 h of the germination.The ubiquitinated modified protein group of spores was determined by using a non-labeled quantitative proteomics(Label-free)technology.The ubiquitinated up-regulated and down-regulated proteins were screened at 2 and 0.5 fold,and it was found that under the bacteriostatic action of ammonia and benzaldehyde,the ubiquitination level of hundreds of lysine sites was up-regulated by at least 2 times.the results of the expression pattern cluster analysis also showed that the bacteriostasis of ammonia and benzaldehyde significantly changed the ubiquitination level of spore proteins.We also performed COG functional classification,KEGG metabolic pathway enrichment analysis,protein interaction analysis,and motif analysis of ubiquitin modification sites on these ubiquitin-modified differential proteins.We also analysed the correlations of the spore proteome and transcriptome data that collected by our group in the same condition and the ubiquitination modification data.The results revealed that increased ubiquitination level was only found among a small portion of down-regulated proteins in the presence of ammonia or benzaldehyde.The transcription of these down-regulated proteins are also decresed.The ubiquitination level of the majority of the down-regulated proteins are not increased and the transcription level remains almost unchanged.These results suggested that down-regulation of a large number of protein expression levels is not associated with elevated levels of ubiquitination and decreased levels of gene transcription under ammoxidation conditions.Increased levels of ubiquitin modification and decreased gene transcription levels in ammonia and benzaldehyde bacteriostatic conditions are not the main reasons for the decline in protein expression levels.We selected some differential proteins according to the omics data and knocked out the coding genes of these proteins(AOL_s00076g167,AOL_s00007g395,AOL_s00215g687,AOL_s00076g699,AOL_s00112g95,AOL_s00004g254,AOL_s00078g131,AOL_s00004g335)by homologous recombination.Finally,we studied these genes in the aspect of growth morphology,growth rate,stress resistance,the amount of spores and germination rate of the spores in the stress environment.Some the results indicated that Some strains were significantly different from the wild type in these phenotypes(1)AOL_s00076g699 gene encodes protein MAPK kinase MKK2/SSP33 the knockout strain did not sporulate and the morphology,growth rate and the density of itsmycelium were significantly different from those in the wild type.,and the growth rates in three basic mediums of PDA,TG and TYGA were decrased by 60.78%,83.24%,and 80.35%.(2)The protein encoded by the AOL_s00004g335 gene is lactyl glutathione lyase,which is a detoxifying enzyme for methylglyoxal in cells.This expression level was up-regulated under ammonia and benzaldehyde bacteriostatic conditions.Under the antibacterial action of benzaldehyde and methylglyoxal,the germination rate of the spores was significantly lower than that of the wild type,and the ability to resist the inhibition of benzaldehyde and methylglyoxal was greatly weakened.(3)Glucose can significantly relieve the inhibition of spore germination by ammonia gas;for the bacteriostasis of benzaldehyde,low concentration of glucose can significantly relieve the inhibition of spore germination.the high concentration of glucose stengthens the inhibition of spore germination by benzaldehyde.After knocking out the gene encoding of glucose-6-phosphate dehydrogenase(AOL_s00078g131),the germination rate of the spore inhibited by ammonia was not different from that of the wild type,and the germination rate of the spore inhibited by benzaldehyde decreased significantly.Glucose can still relieve the inhibition of the spore germination of the knockout strain by ammonia.These results shown that the ED metabolic pathway of the glucose played an important role in the antibacterial action of the spores of Arthrobotrys oligospora against benzaldehyde.This study found that the ammonia and benzaldehyde led to a significant increased in the level of ubiquitination of hundreds of lysine sites.Combining the data analysis of transcriptomics,proteomics and ubiquitin-modified transcriptomics,it was found that the expression level of hundreds of proteins decreased significantly under the bacteriostasis of these two compounds.Among these down-regulated proteins,the ubiquitination level of a small portion of down-regulated protein increased,the transcription level of a small portion of down-regulated proteins decreased,and the level of ubiquitination of most down-regulated proteins did not increase,and the level of gene transcription did not decrease.These results indicate that elevated levels of ubiquitin modification and decreased gene transcription levels under ammonia and benzaldehyde bacteriostatic conditions are not the main reasons for the decrease in protein expression levels.We screened several important proteins from the omics data,knocked out the coding genes of these proteins,and showed a series of phenotypic results on the mutants.The results showed that the genes AOL_s00076g699,AOL_s00004g335 and AOL_s00078g131 play a role in the antibacterial action of ammonia-benzaldehyde. |
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