Study of the role of bem46 gene of Aspergillus fumigatus in polar growth by constructing an overexpression strain
Objective To construct an overexpression strain of bem46 gene of A.fumigatus,and explore the polar growth of A.fumigatus and its interaction with other polar genes under the condition of bem46 gene overexpression.Methods The overexpression strain of bem46 gene was constructed by protoplast method.The green fluorescence localization of A.fu-migatus bem46 was observed by laser confocal microscope.The germination rates of A.fumigatus Ku80,Δbem46 and over-expressed strains were observed and recorded.The radial growth of A.fumigatus Ku80,Δbem46 and over-expressed strains was recorded.The growth of over-expressed colonies of A.fumigatus bem46 stained with lactic acid phenol cotton blue was observed under optical microscope.Real-time fluorescence quantitative PCR was used to determine the relative ex-pression of genes related to polar growth in A.fumigatus Ku80,Δbem46 and overexpressed strains.Results The overex-pression strain of A.fumigatus bem46 was successfully constructed by protoplast method.Laser confocal microscope ob-served that when bem46 was overexpressed,a large amount of green fluorescence gathered in the spore head,but there was almost no fluorescence in the mycelium.Compared with the germination of Ku80,the germination time of the overexpressed strain of bem46 gene was earlier than that of Δbem46 strain.There was no significant difference in radial growth among Ku80 strain,Δbem46 strain and overexpressed strain.After dyeing and observation under microscope,it was found that the spore heads of Ku80 strain,Δbem46 strain and overexpressed strain were obviously different,and the overexpressed strain could obviously stimulate the spore heads to expand.By measuring the relative expression levels of polar growth genes in A.fu-migatus Δbem46 and the overexpression strains,it was found that the relative expression levels of cdc24,bem1,bem3,bud5,rsr1 and rasb genes in A.fumigatus were significantly reduced,and the expression level of cdc24 was not changed.Conclusion The bem46 gene can promote the spore germination of A.fumigatus and affect the spore and mycelium mor-phology during the polar growth of A.fumigatus.In the signal transduction pathway of related genes,bem46 gene may participate in the polar growth signal transduction pathway related to cdc24.
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