| BackgroundPenicillium marneffei is a temperature-dependent dimorphic opportunity pathogenic fungi that mainly infects immunocompromised population in Southeast Asia and southern China. Recently,because of the increasing number of population with human immunodeficiency virus (HIV),the number of patients at risk of systemic Penicilliosis marneffei(PSM) has rising.It is a serious threat to human health. And its pathogenic molecular mechanism remains unclear.To explore the molecular mechanisms, quantitative real time PCR(qRT-PCR) has been one of the most important approaches to perform gene expression analyses. However, more suitable reference genes for qRT-PCR data normalization need to be developed. In this study, we selected 4 most stably expressed genes (Pfp;Rpl23;FacpA; DigA) during dimorphic phase transition from our previous RNA-seq dataset and 3 traditional reference genes(18SrRNA; Actl;Btu) as candidate reference genes.Two analytical software packages (BestKeeper and geNorm) were used to assess the stability of gene expression.Recently, it is believed that the molecular mechanism of pathogenicity of Penicillium marneffei is related with its dimorphic transition is closely. In this study,we employed high-throughput ssRNA-seq to get the transcriptional profiles of 4 different kinds of growth stages of P. marneffei and find out the transcription factor genes related to the dimorphic transition,such as AmyR and ctf1β.According to the annotation of the high-throughput ssRNA-seq, we found that AmyR and ctf1β are the C6 transcription factor genes, which regulate the process for the hydro lysis of starch and degradation of cutin. Through the study of the function of these two transcription factor genes, it may contribute to the excavation of pathogenic molecular mechanism of Penicillium marneffei.Objective:1 After screening out the suitable reference gene, apply the accurate analysis of qRT-PCR on dimorphic transition and pathogenic molecular mechanism research; 2 Through the preliminary functional analysis of transcription factor genes, AmyR and ctf1β, it may provide clues to reveal the molecular mechanisms.Methods:1 After selecting most stably expressed genes during dimorphic phase transition from our previous RNA-seq dataset and traditional reference genes as candidate reference genes, we use two software packages to analysis the Ct value to determine the ideal reference genes; 2 We use Split-Marker recombinant method to establish mutant strains and analyze the function of genes comparatively.Results:1 FacpA and DigA are the optimum genes at all dimorphic phase transition stages and can be used as reference genes for analysis of the qRT-PCR in Penicillium marneffei;2 The AmyR and ctf1β mutants were successfully constructed.In cell experiment, drug sensitivity test and stress test,we found some obvious differences among control strain and mutants.Morover,AmyR did participate in the function of hydrolysis of starch.Discussion:Quantitative real time PCR(qRT-PCR) has been one of the most important approaches to perform gene expression analyses.In different laboratories, researchers will choose different reference genes as a yardstick.Screening more ideal reference genes for qRT-PCR analysis in Penicillium mameffei is necessary.In this study,we found FacpA and DigA genes are more suitable and can be used as reference genes for qRT-PCR analysis in Penicillium marneffei. A preliminary study on the transcription factor genes mainly includes:the expression difference among related genes; the morphological changes of knockout strains; macrophage phagocytosis and the growth morphology changes in macrophages; changes in drug sensitivity and the AAmyR strains ability of hydrolyzed starch were studied. These results will contribute to the preliminary analysis of the transcription factor genes,which will lay the foundation for the future study of the morphological transformation and molecular pathogenesis in the dimorphic fungus. |
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