The Study Of Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis In Sporothrix Schenckii

Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Recent years, with the growing number of people with impaired immune, the incidence of sporotrichosis has rising, a serious threat to human health. Although the studies of genotyping, rapid identification and immune response had been carried out, however, the growth, metabolism, development, disease, dimorphic conversion mechanisms details that underlie S. schenckii remain uncharacterized owing to the lack of efficient genetic methods that can generate mutants.Mutants obtained by molecular tags are the most effective means of cloning genes and genetic analysis. Agrobacterium tumefaciens is a Gram-negative soil bacteria which has the ability to transfer its T-DNA (transfer DNA), located on a tumor inducing plasmid, to its host genome at random sites. A. tumefaciens-mediated T-DNA insertional mutagenesis is used widely owing to receptors types, high efficiency, stable transformants, easy to operation and so on.In this study, an optimized A. tumefaciens-mediated transformation system of S. schenckii was established. The molecular analysis of transformants were performed by PCR, Southern blotting, TAIL-PCR, to determine the T-DNA insertion copy numbers and insertion site flanking sequences. Mutants'phenotypes and genetic traits were analysis by reverse genetics and forward genetic strategy to reveal the growth and metabolism molecular mechanism and provide for drug targets of S. schenckii. At the same time, the establishment of high-throughput screening method of mutants improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of S. schenckii.1. The establishment of A. tumefaciens-mediated transformation system of S. schenckiiThis is the first time to establishment of the Agrobacterium-mediated transformation (ATMT) system and an investigation into the important factors affecting the transformation frequency of S. schenckii. The transformation system includes the using of S. schenckii conidia as starting material, A. tumefaciens strain AGL-1 harboring binary vector pBHt1 as DNA donor was pre-cultured in IM with 200μM AS. A. tumefaciens cell were mixed with the same volume of conidia suspension and pipetted onto HybondN+ filters. After co-cultivation for 48h at 25°C in the dark, transformants were selected on SM containing 100μg/ml hygromycinB and 200μM cefotaxime for 5d at 25°C. The transformation efficiency reached more than 600 transformants per 10~6 conidia. The highly efficient transformation enabled us to obtain a large number of S.schenckii T-DNA insertion mutagenesis within a short experimental period and these mutants mitotic stability. A small scale T-DNA tagged mutant library of S. schenckii including 2130 mutants were established and some growth, development and metabolism phenotypic changes mutants were obtained which would provid materials for reveal the molecular mechanisms of S.schenckii.2. The molecular analysis of S. schenckii T-DNA insertion mutantsPCR, Southern blotting, TAIL-PCR were performed to analysis S. schenckii T-DNA insertion mutants. Hygromycin phosphotransferase gene (hph) were amplified by PCR in 15 ATMT transformants selected randomly of S. schenckii, 0.8kb target bands were obtained, indicating that the T-DNA had been inserted into the S. schenckii genomes. Southern blotting results show that 87% mutants were T-DNA insertion single copy, 13% mutants were T-DNA insertion more copies in randomly selected mutants. TAIL-PCR amplified T-DNA insertion site flanking sequences analysis showed that 14 mutants had obtained sequences flanking from the left arm, 13 mutants had obtained flanking sequences from right arm in the 15 randomly selected mutants. Combined with PCR amplification results from complete T-DNA suggested there was missing borders in the T-DNA transfer process. These results indicated that ATMT was an effective insertion mutation strategy and provide a powerful tool to S. schenckii functional genomics.3. The analysis of pigment defects mutant of S. schenckii JLCC32757-M2013By screening T-DNA insertion mutants of S. schenckii, pigment defects mutant strains JLCC32757-M2013 was obtained. Compared with the wild type strains, this strain growth rate was normal, the colony was white, lost the ability to produce pigment, and its hyphae, conidia morphological were changed obviously. Molecular analysis showed that this mutant were T-DNA insertion single copy into wild type S. schenckii genomics. TAIL-PCR cloned T-DNA insertion site flanking sequence of JLCC32757-M2013. The NCBI database showed that T-DNA inserted into the ubiquitin conjugating enzyme E2 catalytic domain gene of S. schenckii, this gene was named SsUBCc. Designed specific primers of SsUBCc gene and pigment synthesis related genes exons for Real-time PCR, results showed that SsUBCc gene and pigment synthesis related genes expression at transcription level were lower than the wild-type strain. Animal experiments showed that the virulence of mutant strain was significantly lower than the wild type strain. Speculated that the insertion of T-DNA destructed the expression of ubiquitin conjugating enzyme E2 catalytic domain gene which affected parts of proteins post-translational modification and degradation, resulting in the loss of pigment production capacity and virulence decreased significantly. Speculating preliminary that SsUBCc was associated with pigment synthesis and pathogenic.4. The establishment of high-throughput screening T-DNA insertion mutants for reverse geneticsSsUBCc gene of JLCC32757-M2013 strain as a reference, the minimum concentration required template DNA of PCR amplification was detected, the results showed that clear PCR amplification products still could be obtained when 50ng/μl template DNA was diluted to1600 times. So mutants'pools were constructed per 100, 200, 400 mutants and the corresponding mutants DNA pools. PCR amplified the SsUBCc gene from mutants DNA pools results showed that DNA pools were sufficient to ensure that each strain template DNA could be amplified in the mutants'pools which containing 400 mutants. To ensure the reliability and facilitate to isolate specific mutant from the mutants pools by PCR and 96-well plates in the future, per 100 mutants as unit to construct 21 mutants pools and DNA pools in 2100 mutants for high-throughput screening of mutants. The establishment of high-throughput screening mutants'method could improve our ability to isolate mutants in targeted genes from mutants'library and facilitating the molecular genetic analysis of S. Schenckii.In summary, for the first time, an optimizated ATMT system of S. schenckii for insertional mutagenesis was established; small-scale S. schenckii T-DNA tagged mutants library and DNA libraries were constructed; the mutant that T-DNA inserted into the ubiquitin-like conjugating enzyme E2 gene was analyses; the method of high-throughput screening mutants'for the reverse genetics was established which would help to investigate the pathogenic mechanism and develop drug targets of pathogenic fungi. This study should be possible to adapt to other clinically important pathogenic fungi.