The Genetic Transformation Of ZafA Gene Of Trichophyton Mentagrophytes Mediated By Agrobacterium Tumefaciens

Trichophyton mentagrophytes is an important zoonotic dermatophytes,mainly infected human and animal skin stratum corneum,nails or hair and other horny tissue.Zinc is an important micronutrient,the second largest transition metal in the cell,which plays an important role in the growth,proliferation and pathogenicity of pathogenic microorganisms.A variety of genes have been found to be involved in regulating the zinc homeostasis of fungal cells,ZafA protein and its homologous protein have been shown to play an important role in the regulation of zinc homeostasis and the pathogenicity of pathogenic fungi.The ZafA gene was found in Trichophyton mentagrophytes by transcriptome sequencing,but the gene and its expressed protein function are still unclear.Agrobacterium tumefaciens-mediated genetic transformation(ATMT)is one of the common methods of fungal gene knockout and has been successfully applied to genetic transformation of many filamentous fungi.In this paper,the ZafA gene of Trichophyton mentagrophytes was genetically transformed by ATMT method to construct ZafA gene transformants,which laid the foundation for the study of ZafA function.While optimizing trichophyton mentagrophytes ATMT transduction system and improve the transformation efficiency of Trichophyton mentagrophytes.The results were as follows:1.The constructed binary vector pDHt/Zaf A-ⅠⅡ::hph containing the ZafA gene homologous fragment and the hygromycin resistance marker gene was transformed into agrobacterium tumefaciens.PCR was used to confirm successful conversion.After transformation,agrobacterium tumefaciens-mediated transformation was used to transform the conidia.PCR results showed that the ZafA gene of trichophyton mentagrophytes was knocked out by homologous recombination.2.To obtain the optimal ATMT conversion conditions,we used different solid phase carriers(Hybond N + nylon membrane,PVDF membrane,filter paper),different co-culture time(36 h,48 h,60 h),AS in pre-culture or not.The transformation of trichophyton mentagrophytes was carried out at 28 ℃ co-culture temperature.It was found that the number of transformants obtained by adding AS in pre-culture was significantly higher than that without AS group,Hybond N+ nylon membrane and filter paper as solid carrier can be usedfor trichophyton mentagrophytes ATMT transformation,with no statistically significant difference.Under co-culture conditions at 28 ℃,the co-culture time was extended to 60 h,which could significantly improve the conversion efficiency.In summary,the ZafA gene of trichophyton mentagrophytes can be knocked out through the ATMT system and obtain genetically stable ZafA gene knockout strain.Agrobacterium tumefaciens was cultured in AS-induced culture and mixed with trichophyton mentagrophytes conidia(1×107CFU / mL)by 1: 1,coated in co-culture medium covering nylon membrane or filter paper,28 ℃ co-culture 60 h to obtain a higher number of transformants.