Function Analysis Of Sir2 Gene Of Sporothrix Globosa

Sporothrix schenckii complex is widely distributed around the world and has been found to contain seven genotypes,S.schenckii sensu stricto,S.globosa,S.brasiliensis,S.mexicana,S.pallida,S.luriei,S.chilensis.The endemic strains in China are mainly S.globosa and S.schenckii sensu stricto,and S.globosa is the only genotype found in northeast China.At present,the pathogenesis of Sporotrichosis is not completely clear,especially after the difference of genotype is confirmed,there are few studies on S.globosa.In response to changes in environmental pressure,S.globosa produced two kinds of morphological patterns.As a human opportunistic pathogen,the pathogenicity of Sporotrichosis depends on its ability to survive the cytotoxic process of the host immune system and to grow in host macrophages.The mechanisms that allow Sporotrichosis to survive in macrophages remain unclear.In this study,we investigated the role of histone deacetylase silencing information regulation factor 2(sir2)gene in growth,morphogenesis,cell wall integrity and pressure sensitivity from S.globosa.The results are as follows:1.By referring to relevant papers,we learned that sir2 gene was up-regulated in the yeast phase of S.schenckii.We hypothesized that sir2gene may play an important role in dimorphic transformation and pathogenicity of S.globosa.Firstly,I identified the genotypes of Sporotrichosis that were available in the lab,and the results showed that they were all S.globosa.Moreover,sir2 gene could be cloned from S.globosa.The knockout plasmid was synthesized by overlapping extension PCR technology,digested by enzyme,connected to p Fast Bac ^TMDual plasmid,and transformed into TOP10 competent cells.The knockout plasmid was successfully constructed by PCR verification,enzyme digestion verification and sequencing verification.I prepared protoplasts of S.globosa and transformed the knockout plasmid into protoplasts through PEG-CaCl₂mediated protoplast transformation.Then,protoplasts were regenerated,further screened and identified to obtain knockout strains.2.The differences in morphogenesis,conidia production,cell wall integrity and resistance to various stressors between the wild-type strain and(?)sir2 strain were compared.The results showed that the deletion of sir2 gene resulted in a slower growth rate of S.globosa in its mycelium phase,and the colony size was significantly smaller than that of the wild type.Conidia production was significantly reduced,and the conidia matured slowly and was not easy to shed from mycelia.The hyphae appeared twining and abnormal branching.There was no significant difference in cell wall integrity and sensitivity to salt stress and osmotic stress,but the sensitivity of(?)sir2 strain to oxidative stress was significantly increased.During the transformation from mycelium phase to yeast phase,more mycelia of(?)sir2 strain did not transform into yeast phase.In the morphogenesis of yeast phase,the growth of(?)sir2 strain was slower,but there was no significant difference in the early stage,and significant difference appeared in the late stage.There were still more mycelia of(?)sir2 strain with extended phase transition time.These results indicated that sir2 gene was involved in mycelial radial growth,conidial production,mycelial morphogenesis,resistance to oxidative stress,dimorphic transformation,and yeast phase morphogenesis.This is the first study to investigate the role of sir2 gene in morphology and pathogenicity of dimorphic fungal pathogen S.globosa.