| Sporotrichosis is a kind of skin disease caused by Sporothrix schenckii, andoccassionally caused systemic disseminated infections which spreaded to internal organs. Inorder to prevent and treat with Sporotrichosis effectively, it is important to obtain the data ofepidemiology and pathology characteristics. Sporotrichosis often occurred in the Northeastof China, and it was a higher incidence in Jilin province. In recent years, the incidence offungi skin disease had a trend to upward. If we had knowed the data of epidemiology ofSporotrichosis in Jilin province, it was important to prevent and treat with the disease. Fromthe molecular level studying gene function of Sporothrix schenckii, it would help to revealthe etiology characteristics and pathogenic mechanisms. Mutants obtained by molecular tagswere the most effective means of cloning genes. The technology of Agrobacteriumtumefaciens-mediated T-DNA insertional mutagenesis (ATMT) was widely used owing toreceptors types, high efficiency, stable transformants, easy to operation and so on.ATMT technology was applied to the study of Sporothrix schenckii. This studyoptimized many factors that affected transformation efficiency such as the concentration ofspore suspension, the induction time of the Agrobacterium and co-culture medium, improvedthe conversion efficiency of the T-DNA, and established T-DNA insertion mutant library ofSporothrix schenckii. This study obtained a number of significant changes in phenotypemutants by screening, and obtained T-DNA insertion flanking sequences by TAIL-PCR,positioned the insertion site, analysed the function of the mutant gene. Among them, themutant841was a copper transporter gene which was the interrupted mutant. This studyinferred that the copper transporter gene was involved in the regulation of growth andanti-oxidative stress of Sporothrix schenckii, and associated with the pathogenicity ofSporothrix schenckii.1. The epidemiological analysis of sporotrichosisIn order to investigate the epidemicological characteristics of sporotrichosis of JilinProvince, this thesis maked a retrospective study of208cases of sporotrichosis. The resultsof this study showed higher incidence of female, male and female incidence ratio was1:1.51.The age of sporotrichosis patients was from3years old to87years old with the average ageof47years old. The morbidity of youth and prime people were67.3%, which was the highest, followed by the elderly, and the mobidity of minor people was the lowest, whichwas12.5%. The mobidity of occupation for farmers were higher than others, which was68.6%, and most of them had a history of trauma. Sporotrichosis of fixed type wassignificantly more than the lymphatic type. There was a patient with the disseminatedsporotrichosis, who is a farmer. The lesion on face and upper limbs were more common, andthe fixed type was more than lymphatic type. The mobidity of lymphatic type of upper limbswere the highest. The incidence in winter was higher than other seasons, which was37.5%.All of these showed that sporotrichosis had a significant seasonal incidence.2. Optimization of Agrobacterium tumefaciens-mediated transformation system andestablish of the mutant libraryThis research had established a stable and efficient of Agrobacteriumtumefaciens-mediated transformation system by optimizing five key factors:(1) inducingconditions: the initial concentration of Agrobacterium tumefaciens was0.3in OD600nm andthe inducing time was6hours; the concentration of acetosyringone was200μM;(2)Co-cultivated medium and condition: Agrobacterium tumefaciens which cultured to6hoursin induced medium with200μM mixed with same volume Sporothrix schenckii conidiasuspension which concentration was106condia per milliliter,spreading100μl mixture on themicroporous membrane medium,incubating48h at25℃;(3) Screening of culture conditions:incubating4days at25℃. Under these conditions, the transformation efficiency of ATMT inSporothrix schenckii was1000-1200mutants per106conidia. This study preliminaryconstructed a T-DNA insertional mutant liarary of Sporothrix schenckii with4500mutants.The quality of the mutant library was evaluated.All the randomly selected mutants wereproved to be stable through mitotic cell division. From the mutants library, we screenedseven mutants of obviously different phenotypic that the T-DNA insertion flankingsequences were successfully amplified, and conducted a preliminary analysis of genefunction by using the bioinformatics.3. Gene functional study of the copper transporter protein in Sporothrix schenckiiThe trace elements copper is one of the indispensable conditions for micro-organisms tomaintain their own grown and development.Copper often plays an important role in someenzymes when they play function. In this study, we screened a mutant that the coppertransporter gene was interrupted which named mutant841,and successfully coloned the genesequences about1300bp. By bioinformatics analysis, we find that the gene was highlyhomologous with the copper transporter protein of Magnaporthe grisea, Penicillium marneffei, Aspergillus niger, Paracoccidiodes brasiliensis, and then named the gene for CTPgene. Under the microscope of the mutant841, we finded that the morphogenesis hadlargerly changed: the mycelium arranged in haphazard, the number of the conidia werereduced which scattered around of the mycelium. Compared with wild type, the growth rateof the mutant didn’t change, but the conversion ability of yeast phase was weakened, and themutant was more to sensitive to1M hydrogen peroxide which showed poor ability toresistant to oxidative stress. In the systemic infection mouse model, the mutant exhibitedlower virulence. Thus, these results suggest that copper transporter protein was required forspore germination and the protection of Sporothrix schenckii against oxidative stresses, andfor the full virulence of the organism to be expressed. |
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