Transcriptome Sequencing Analysis Of Microsporum Canis And Construction Of Zrta Gene Transformants

Microsporum Canis is a common zoonotic dermatophyte that mainly infects skin,hair and nails,causing superficial mycosis.In the process of fungal growth and reproduction,zinc is an essential trace element that participates in many biological processes and plays an important role in the growth and metabolism of fungi.During the interaction between pathogenic fungi and host,the host's nutrient immunity limits the uptake of zinc by pathogenic fungi,and in order to survive and proliferate under zinc-limited conditions,pathogenic fungi have evolved a special zinc homeostasis regulation mechanism.Zinc-responsive activator protein?Zapl?and its homologs are the major transcription factors regulating zinc homeostasis genes and have been confirmed in fungi such as Saccharomyces cerevisiae,Aspergillus fumigatus and Candida.However,at present,the effects of zinc deficiency on gene expression of M.canis are still unclear.The function of zinc-responsive activator of M.canis and the biological processes involved are also unknown.To determine the effect of zinc deficiency on canine microspores and the role of zinc-responsive activator?Zrta?in the regulation of zinc homeostasis in M.canis is important for the study of the pathogenicity of M.canis.In this experiment,transcriptome sequencing was performed on M.canis strains grown under normal conditions?NORM?and under zinc-limited conditions?Zn200 and Zn800?.The effects of zinc deficiency on M.canis were determined by the raw data quality control,alignment analysis,expression quantitative analysis,differential expression analysis,functional enrichment and annotation of differentially expressed genes.The Agrobacterium tumefaciens-mediated transformation?ATMT?was used to construct the Zrta gene deletion strain,which laid a foundation for the functional analysis of the Zrta gene.The results are as follows:1.A large number of genetic data files were obtained,and the number of differentially expressed genes showed that compared with the NORM group,Zn200 group and Zn 800 group up-regulated genes was 798 and 764 respectively,down-regulated genes was 585 and 631 respectively.Among them,MCYG₀4486 and MCYG₀6235 were significantly up-regulated in the zinc-restricted group compared with the normal group,MCYG₀2504 is significantly up-regulated in Zn200;in the Zn800 group compared with the Zn200 group,the number of up-regulated and down-regulated genes was 3 and 8 respectively.Functional annotation analysis of differentially expressed genes indicated that MCYG₀6235?zinc-responsiveness transcriptional activator?may be an important zinc transport factor.2.High fidelity amplified the zinc-responsive activator gene Zrta of M.canis,and constructed the binary vector pDHt/Zrta?,?::hph,which contains the homologous fragment of the Zrta gene and the hygromycin resistance gene?hph?.The digestion and PCR verification confirmed that the binary vector was successfully constructed.3.The successful constructed binary vector was transformed into Agrobacterium tumefaciens.After PCR verification,the M.canis was transformed with Agrobacterium tumefaciens with binary vector,and the transformants were identified by PCR and Southern blot.The strain with homologous recombination of the microspore Zrta gene and the hygromycin resistance gene,the Zrta gene deletion strain was stably subcultured for 3 times,and the hereditary trait was stable.This study clarified the effects of zinc deficiency on gene expression and biological metabolism of M.canis,and successfully constructed the binary vectors pDHt/Zrta???::hph for transformation,and successfully obtained the Zrta gene deletion of M.canis by ATMT.The strain lays the foundation for the functional verification of the pathogenicity of Zrta gene.