Deletion Of Lgk1 Gene And Preliminary Characterization On Its Function In Aspergillus Fumigatus

Aspergillus fumigatus infects patients with low immunity and leads to invasive aspergillosis(IA),which is one of the major causes of death of critically ill patients.To date,however,the pathogenesis and virulence factor of A.fumigatus remain unclear.The levoglucosan kinase is a newly found fungal kinase which is likely involved in the metabolism of cell wall.Therefore,to investigate the function of lgk of A.fumigatus is of great significance.Bioinformatics analysis showed that A.fumigatus has three lgk genes(lgk1,lgk2,lgk3),while the weak pathogenic Aspergilli such as Aspergillus flavus,Aspergillus niger,Aspergillus terreus and Aspergillus nidulans have one or two lgk genes,respectively.Gene detection indicated that A.fumigatus lgk expresses constitutively,while,apart from A.nidulans,the lgks of other Aspergilli are inductive.During the stage of spore germination,the lgk1 of A.fumigatus constitutively expresses,while lgk2 and lgk3 weakly.During the stage of mycelium growth,lgk1 and lgk2 express strongly,while lgk3 weakly.Using the traditional homologous recombination(THR)technology and the latest CRISPR/Cas9 gene-editing technology,we constructed lgk1-knockout strain of A.fumigatus.PCR method is used to check the validity of lgk1-deficient strain ZS1 and ZS2,constructed by THR and CRISPR,respectively.Sequencing the lgk1 of ZS2 showed that lgk1 is deleted completely.Gene detection approved that lgk1 does not express.Phenotype analysis revealed that ZS2 cannot grow in minimal medium containing levoglucosan as the sole carbon and energy source,while ZS1 grows faintly.The detection of spore germination rate showed that ZS1 decreases 14.50%,ZS2 decreases 23.32%.The detection of dry weight in the mycelium growth stage showed that ZS1 decreases 6.62%,ZS2 decreases 5.44%.In this study,we have successfully constructed lgk1-knockout strain of A.fumigates.Our preliminary works revealed that A.fumigatus lgk1 is involved in the spore germination and mycelium growth and is potential virulence factor.