| Copper(Cu)is known as one of the trace nutrient metal,Which is essential at low concentration but toxic at high concentrations.Research on copper resistance mechanism in microorganisms mostly focuses on bacteria and yeast,and the relevant mechanism has been studied clearly.Filamentous fungi have high resistance to heavy metals and are closely related to industrial and agricultural production and human health.The resistance mechanism of copper has great practical significance for the prevention and control of this fungi.At present,the mechanism of copper resistance to filamentous fungi remains unknown,and there are few reports about the mechanism of copper resistance of filamentous fungi both in domestic and abroad.A study on the mechanisms copper of filamentous has a great significance,so it is necessary to research on it in depth.In this study,a series of physiological and biochemical experiments were performed on Penicillium janthinellum strain GXCR(wild-type stain,WT)and two mutants with reduced copper resistance EC-6 and UC-8.The analysis of the results showed that the ability of resistance to copper was classified by size in the order of WT>UC-8>EC-6.On the PDA and TYA medium without copper,all the three strains could grow normally and sporulate quickly.After adding copper,the three strains'colony diameters drastically decreased and the sporulation rate reduced.Under a certain concentration of copper stress,the two mutants did not form pseudo root(vegetative mycelium),and WT could still grow on high-concentration copper medium.At this time,only very few pseudo roots were formed.The ability of WT to absorb nutrients is stronger than mutants.The TYA medium has fewer nutrients.Under the condition of adding copper,the aerial hyphae of the three strains has started to exhibit copper precipitation.In addition,the addition of manganese can increase the copper resistance of strains.Exogenous proline can enhance the ability of the strain to resist copper,indicating that proline plays a key role in the copper-resistant mechanism of Penicillium janthinellum,and the determination of proline content in the three strains of the three strains also verified this inference.The addition of exogenous ATP can enhance the growth ability of the strain under copper stress,while the ATPase inhibitor sodium orthovanadate decreases the copper resistance of the strain.Under copper stress,the biomass of WT was more than 2 mutants.In the experiment of spore germination rate,different heavy metal had different effects on spore germination of the strains.The germination ability of the WT was stronger than that of the two mutants.Active oxygen in the strain will increase under adverse conditions,and its content is closely related to the activity of antioxidant enzymes SOD,POD,CAT and intracellular MDA content,and it changes regularly with the increase in copper concentration.The detection of intracellular heavy metal content of the strains under stress of heavy metal found that under copper stress,the intracellular copper ion content of WT was lower than 2 mutants.Under the combined stress of copper and manganese,the intracellular copper ion of the three strains decreased significantly,and the manganese ion content decreased with the increase of the concentration.Under the cadmium stress,the intracellular cadmium content of the three strains increased with the increase of the concentration,The highest subsequent decrease was achieved at 0.03 mM Mn2+.In this process,WT intracellular cadmium content was less than two mutants.Under chromium stress,the intracellular chromium content of the three strains increased with increasing concentrations.Intracellular chromium content of WT was greater than 2 mutants at 3,4,and 5 mM Cr6+.In contrast,under lead stress,the intracellular lead content decreased with increasing concentration.Based on the results of physiological and biochemical experiments,six samples of WT and EC-6 in TYB supplemented with 0,0.5 and 3 mM Cu2+ were used for transcriptome sequencing.47407 transcripts with a length greater than 100 bp were obtained and 32585 Unigenes were finally obtained after analysis.Through data screening:1.Under non-copper stress treatment,a total of 13802 DETs,of which 6853 were down-regulated and 6949 were up-regulated.2.Under 0.5 mM Cu2+treatment,a total of 15359 DETs,of which 7099 were down-regulated,8260 Up-regulated.3.3 mM Cu2+ treatment,a total of 23634 DETs,of which 8211 were down-regulated,15423 up-regulated.To further ensure the reliability of the transcriptome sequencing data,38 copper-resistance-related genes were selected and verified using real-time fluorescence quantitative PCR,which was in line with the expected results.The transcriptome data was further analyzed by the weighted gene co-expression network analysis.Gene clusters were divided into 10 modules with 10 similar expression patterns.10 traits were associated and the four most relevant modules were selected for constructing the network map to find 116 hub genes after integrating physiological and biochemical experimental results.Finally,taken all the results of characteristic copper resistance of Penicillium janthinellum WT and 2 mutants,RNA-SEQ data,WGCNA data together,Roles of enzymatic and non-enzymatic antioxidants in resistance to low and high Cu were defined.The backbone paths,signaling systems,and determinants that involve resistance of filamentous fungi to high Cu were determined,discussed and outlined in a model. |
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