Research On The Function Of Atf21 And Phx1 Gene In PenicilLium Marneffei And The Expression Of Cutinase Gene In Prokaryote

Penicillium marneffei(Renamed as Talaromyces marneffei)is a temperature-dependent dimorphic opportunistic fungal pathogen of Penicilliosis marneffei(PSM).At 25 ℃,this fungus grows as mycelia,but at 37 ℃ in vitro culture or in the host grows as a pathogenic yeast form;Penicillium marneffei is endemic inthe tropical Southeast Asia area.It can cause fatal systemic infections in immunocompromised individuals.The molecular mechanism of dimorphic transition and the pathogenesis is still obscure.To response the upstream signal,transcriptional factors are the key molecules to regulate gene expression.The transcriptional factor genes are regulated by the upstream signal and these gene could be related to the dimorphic transition.Research on the function oftranscriptional factor genes is hope to clarify the molecular mechanism of the phase transition and pathogenesis in P.marneffei and to provide the basic knowledge to develop novel strategies for therapy or prevention of Penicilliosis marneffei.Purpose: 1、we found that the Atf21 gene encoded a b ZIP transcription factor and the Phx1 gene encoded a homeobox transcription factor were differentially expressed in the process of dimorphic transition in Penicillium marneffei by high-throughput ssRNA-seq.In this study,we construct the gene encoded transcription factor,Atf21 and Phx1 gene deletion mutants to understand the functions of these genes in P.marneffei.Research on the function of transcriptional factor genes is hope to clarify the molecular mechanism of the phase transition and pathogenesis in P.marneffei and to provide the basic knowledge to develop novel strategies for therapy or prevention of Penicilliosis marneffei.2 、 Our previous studies have found that the gene expression of Ctf1β gene,encoded cutinase transcription factor β subunit,and the gene encoded cutinase were increased significantly in yeast phase.In this study,we aim to clone the cutinase gene in Penicillium marneffei and express the recombinant cutinase in prokaryote.Methods: 1、Using real-time fluorescent quantitative PCR to analyze the feature of Atf21 and Phx1 gene expression in the process of temperature-dependent dimorphic transition in Penicillium marneffei strain GXFF.2、To analyze the gene function by constuction gene deletion mutants based on homologous recombination.3 、 PCR was used to clone cutinase gene in Penicillium marneffei,Obtain the recombinant cutinase by construction cutinase gene expression vector and the recombinant plasmid was transfered into E.coli.Identification of recombinant plasmid by PCR and sequencing;Determination of enzyme activity by alkali titration method.Results: 1、Atf21and Phx1 gene expression in the mycelia growth condition is significantly higher than in the yeast phase growth state in Penicillium marneffei strain GXFF by RT-PCR analysis,and the same result is got in Penicillium marneffei strain FRR2161 by ssRNA-seq.Atf21 gene expression changes obviously in the early stage of the process of dimorphic transition in Penicillium marneffei strain GXFF Phx1 gene expression changes obviously at the late stage of the process of dimorphic transition in Penicillium marneffei strain GXFF.2、Successfully constructed Atf21 and Phx1 gene knockout mutants,Compare to the wild type strain Spm4,Atf21 mutants is more sensitive to Cu2+.3、Successfully cloned cutinase gene in Penicillium marneffei,construct the cutinase gene expression vector and the cutinase gene expression vector was transfered into E.coli.Enzyme activity of cutinase in recombinant strains was about 1.7 times higher than control strain,enzyme activity of crude enzyme liquid can reach to21.8 U/mL.Conclusion: 1、Atf21and Phx1 gene expression in the mycelia growth condition is significantly higher than in the yeast phase growth state in Penicillium marneffei.Atf21 gene expression changes obviously in the early stage of the process of dimorphic transition in Penicillium marneffei strain GXFF,Phx1 gene expression changes obviously at the late stage of the process of dimorphic transition in Penicillium marneffei strain GXFF.2 、 Successfully constructed Atf21 and Phx1 knockout strains,innovative to find Penicillium marneffei Atf21 transcription factor genes regulate copper ion metabolism.3、Successfully cloned cutinase gene in Penicillium marneffei,construct the cutinase gene expressionvector and the cutinase gene expression vector was transfered into E.coli.