Construction Of Bem46 Knockout Strain And Preliminary Study Of The Function Of Bem46 Gene In Polarized Growth Of Aspergillus Fumigatus

Objective:Polarized cell growth involves asymmetric growth from one region of a cell to form particular cell structures or shapes.The specialized structures,which are critical for the function of particular cell types,maybe mediate cellular interactions during development.Filamentous fungi are extremely polarized organisms,exhibiting continuous growth at their hyphae tips.This study will construct the Bem46 knockout strain of Aspergillus fumigatus,and we will observe the growth difference between the control strain and the Bem46 knockout strain on the medium added different antifungal drugs.We expect to find the new antifungal drug targets.We will construct the Bem46 localization strain and observe the cellular localization of Bem46 in the hyphae.We will investigate the germination rate between the control strain and knockout strain,then observe the expression of bem1 gene and bud5 gene which are involved in the polarized growth of Aspergillus fumigatus.Methods:The Bem46 gene was identified by sequence alignment in A.fumigatus genome database.The Bem46 knockout strain(?Bem46)was construction by the protoplast.The growth was observed and compared between the control strain and?Bem46 on basal medium and different culture medium containing antifungal drugs,such as amphotericin B,caspofungin,itraconazole,voriconazole and tacrolimus.The growth diameter was measured under 37? at 72 hours.The experiment repeated three times.The Bem46 fluorescence localization strain was constructed with the enhanced green fluorescent protein(eGFP).The fluorescence was cbserved under the laser scanning confocal microscope.The germination rate was observed and compared by microscope.RNA was extraction from mycelia.The transcription of Bem1 and Bud5 which were related to the signal transduction of the polarity growth was detected by Real-time fluorescent quantitative PCR(RT-qPCR).Results:The results showed that the Bem46 gene,Afu7g04660,contains 1116 bp bases pair and encoding 311 amino acids.Six transformans was obtained by protoplast method and only one was confirmed by PCR and Southernblot.There were no visible difference between control strain and?Bem46 on the basal medium and culture medium containing antifungal drugs.Bem46 fluorescence localization strain was obtained by protoplast and confirmed by PCR.The distribution of the fluorescence was location non-specificity.The germination rate of the?Bem46 was retarded than the control strain by the microscope in the GMM liquid medium.Compared with control strain,the expression of Bem1 and Bud5 in knockout strain was lower than the expression of the control strain by RT-qPCR.Conclusion:The Bem46 gene of A.fumigatus maybe not the drug target of amphotericin B,caspofungin,itraconazole,voriconazole and tacrolimus.The Bem46 gene does not take part in polarized growth related structures directly.But indeed,the Bem46 gene is involved in polarized growth process.The Bem46 gene has a positive effect on spore germination.And the deletion of Bem46 leads to low expression of two genes related to the polarity growth.The results shown the Bem46 gene could involve in conduction pathway of polarity growth,and provides new directions to study the growth mechanism of A.fumigatus.