| Fungi are important members of the natural, They are present in awide variety of species.Of the more than1.5million fungal species only about150–300are pathogenic for humans. Sporothrix schenckii is the dimorphic fungusthat causes sporotrichosis in humans and animals. In recent years, due to the growingnumber of people with immunosuppression, the number of patients at risk ofsystemic sporotrichosis has rising,a serious threat to human health.In this study, several methods have been used to optimize A.tumefaciens-mediated transformation system of S. schenckii. The molecular analysisof transformants were performed by PCR, TAIL-PCR, to determine the T-DNAinsertion site flanking sequences. utants’ phenotypes and genetic traits wereanalysed by reverse genetics and forward genetic strategy. Dimorphic related geneswere screened and the functions were revealed. At the same time, the establishmentof high-throughput method of mutants improves our ability to isolate mutantsintargeted genes, to reveal themechanism and provide for drug targets of S.schenckii.1. The establishment of A. tumefaciens-mediated transformation system ofS. schenckiiThis is the first time to establish the A.grobacterium-mediated transformation(ATMT) system of S. schenckii isolate IFM41598. The transformation systemincludes the using of S. schenckii conidia as starting material, A. tumefaciens strainAGL-1harboring binary vector pBHt1as DNA donor was pre-cultured in IM with200μ AS. A. tumefaciens cell were mixed with the same volume of conidiasuspension and pipetted onto HybondN+filters. Afterco-cultivation for48h at25°C in the dark, transformants were selected on S containing150μg/ml hygromycinBand200μ cefotaxime for5d at25°C. The transformation efficiency reached morethan600transformants per2×106conidia. The highly efficient transformationenabled us to obtain a large number of S.schenckii T-DNA insertion mutagenesiswithin a short experimental period and these mutants mitotic stability. A small scaleT-DNA tagged mutant library of S. schenckii including3000mutants wereestablished and some growth, development and metabolism phenotypic changesmutants were obtained which would provid materials for reveal the molecularmechanisms of S.schenckii.2. The Screening and Identification of dimorphic related genesA Plate method were used to screen dimorphic related mutants.PCR,sequencing, bioinformatics analysis and TAIL-PCR were performed to analysis S.schenckii T-DNA insertion mutants. A total of18mutants were obtained and6related genes were identified including two serine/threonine protein kinase genes,two mating genes, hexose transporter gene and natural resistance-associatedmacrophage protein gene.3. Tageted disruption of dimorphic related genesMolecular clone,overlap PCR and PEG-mediated transformation of protoplastswere used to obtain the knockout strains of one serine/threonine protein kinase geneand hexose transporter gene. Split-Marker recombinant and PEG-mediatedprotoplast transformation were used to obtain the knockout strains of two matin gtype genes. ATMT was used to obtain the knockout strains of the other serine/threonine protein kinase gene and natural resistance-associated macrophage proteingene. Pyrithiamine was used as screening tag to obtain revertants.4. Study on the functions of dimorphic related genesThe changes of morphology, drug sensitivity, cell composition and virulence ofknockout strains were studied. The main changes of morphology were myceliumconcentration, spores decreased and dimorphic disorder. Drug sensitivity hasdeclined. Chitin showed no significant changes, but glucan and ergosterol wereIV increasedand virulence also changed.In summary, for the first time, an optimizated ATMT system of S. schenckiiisolate IFM41598for insertional mutagenesis was established; small-scale S.schenckii T-DNA tagged mutants library and DNA libraries were constructed; Thedimorphic related genes were analysed; the method of high-throughput screeningmutants’ for the reverse genetics was established which would help to investigate thepathogenic mechanism and develop drug targets of pathogenic fungi. This studyshould be possible to adapt to other clinically important pathogenic fungi. |
PDF Full Text Request