| Arthrobotrys oligospora is a filamentous fungus found all over the world,which can capture nematodes by producing adhesive networks.Peroxisomes are cellular organelles that are ubiquitous in eukaryotic cells,and multiple proteins involve in the synthesis of peroxisomes.In this study,three PEX genes were characterized in A.oligospora.The ?PEX1,?PEX6,and ?PEX14/17 mutants were obtained by homologous recombination method.Through phenotypic comparison and transcriptomic analysis,the functions of three PEX genes were investigated in A.oligospora.The main results are described as follows.(1)The physicochemical properties prediction of Pex proteins(Pexp).The isoelectric points(p I)of Pex6 p and Pex14/17 p are relatively close,4.74 and 4.78 respectively,while the p I of Pex1 p is 8.45.Pex14/17 p has a molecular weight of 49.27 KDa,while Pex1 p and Pex6 p have molecular weights of 127.88 and 126.95 KDa,respectively.Functional domain analysis shows that both Pex1 p and Pex6 p contain AAA ATPase domain(IPR003593),and Pex14 N domain(IPR006785)is found in Pex14/17 p.Three Pexp from different fungi were divided in three branches,and the homologous Pexp of nematode-trapping fungi clustered together.(2)Deletion of three PEX genes and phenotypic analysis.Three PEX genes were disrupted by homologous recombination technique.Deletion of three PEX genes caused a significantly reduction in mycelia growth,and the aerial hyphae of them were basically disappeared.Meanwhile,CFW staining revealed that the hyphae of three mutants were enlarged and deformed and the branches were complicated to form a network.?PEX1 and ?PEX6 mutants can not produce traps and conidia.The?PEX14/17 mutant can produce unmatured traps,and its conidial yield was decreased to 1% compared to the WT strain.In addition,the mutants became more sensitive to the cell wall systhesis-disturbed reagents,whereas their resistance to oxidative stress became weakened.(3)Comparison of fatty acid utilization between WT and mutant strains.The utilization of fatty acid was decreased in the ?PEX1,?PEX6 and ?PEX14/17 mutants by measuring the utilization capacity of different fatty acids.Also,it was found by BODIPY staining that the volume of lipid droplet was increased in the mutants.(4)Comparison of cell structure between WT and mutant strains.Transmission electron microscopy showed that the peroxisomes were absent in the ?PEX1,?PEX6and ?PEX14/17 mutants,and the woronin bodies near the septa were disappeared,too.At the same time,MDC staining revealed that the number of autophagosomes was reduced but the volume became larger in the ?PEX1 and ?PEX6 mutants,while there was no significant difference in the ?PEX14/17 mutant.(5)Transcriptomic data analysis of ?PEX1 and ?PEX6 mutant strains.Samples of WT and mutants were collected at 3 d and 5 d,and the sequencing data was analyzed by Venn,GO,and KEGG enrichment.The upregulated DEGs were enriched in proteasome,membrane,ribosome,and cell cycle,and the downregulated DEGs were enriched in the MAPK signaling pathway-yeast and nitrogen metabolism.Innovations of this paper:(1)The PEX1,PEX6 and PEX14/17 genes were knocked out in the A.oligospora,and the functions of three PEX genes in the mycelial growth and morphology,the utilization of fatty acid,stress resistance and pathogenicity were investigated.(2)It was speculated by analyzing transcriptomic data that PEX1 and PEX6 genes regulate multiple biological processes such as proteasome,membrane,ribosome,cell cycle and nitrogen metabolism. |
PDF Full Text Request