Microbiol Characteristics And Pathogenicity Of Cladosporium Cladosporioides Under Microwave And Nitroso-guanidin

Objective:Relatively little information is available on the microbiolcharacteristics of C. Cladosporioides. Because of the increasing number of immunocompromised patients, the number of fungi causing subcutaneous phaeohyphomycosis has been increasing because of the opportunistic nature of its etiologic agents and the increase in organ transplantation, widely used of immunosuppressive agents, chemotherapeutics, etc. This study was to further explore the molecular pathogenesis of skin disease of the giant panda and human skin disease caused by C.Cladosporioides,by SSH.Provide more research data for gene polymorphism, diagnosis and treatment of C. Cladosporioides.Method:In this study, C. Cladosporioides separated from the giant panda as original strain, with different concentrations of NTG and microwave mutagenesis get the mutant strain whose phenotype and virulence have undergone significant changes, and then using suppression subtractive hybridization technique to build subtracted cDNA library of differentially expressed genes of C.Cladosporioides, differential expression gene fragments obtained, sequencing and bioinformatics analysis.Results:1. The optimal conditions of C Cladosporioides for mutation was microwave mutagenesis 60s with 2mg/mL NTG 30min. Screening a strains by mutation which colony color is significantly lighter, less sporulation, sporulation slow, easy aging.Determination of pathogenicity and infection experiments in mice,the results show that the wild and mutant strains to have the opportunity to infect mouse skin,to wild strains of mice infection rate is up to 99%,mutant is 40%.2. Succeed in gaining total RNA both in wild type and Mutant of C. cladosporioides and isolating their mRNA. Two differential cDNA libraries had been found with C. Cladosporioides in wild type and its Mutant.3. Five positive clones acquired by subtraction in positive direction. Two positive clones were acquired by subtraction in reverse direction. Seven fragments were compared in GenBank by BLAST. All of them have more or less homologous with protein or gene from different database. The result is as follow: ①Fragments homologize to biochemical metabolic enzyme:FH1-3,FH1-4,FH1-5;②Fragments homologize to cell structure:FH1-1, FH2-1; ③Others:FH1-2.Seven fragments were analyzed by ORF finder, Predictprotein and Omiga2.0.Open reading frameworks and fungi motifs have been found existing in the fragments and may encoding protein in C. cladosporioides.Conclusions:The mutaenesis conditions of C. cladosporioides were obtained.Get the expected mutant.Mutant phenotype and virulence changes,suggesting that some genes may be related to differences in expression. Succeed in founding two differential cDNA library with C. cladosporioides in wild type and Mutant by suppressive subtractive hybridization techniques. Part of fragments were screend which may be contained gene related to virulence of C.cladosporioides. The fragments have more or less homologous with cell structure,biochemical metabolic enzyme and fungal virulence.