Screening And Functional Analysis Of Genes Involved In Growth And Pathogenicity In Aspergillus Fumigatus

Aspergillus fumigatus is an opportunistic pathogen common to the environment and infected by air.Clinically,it mainly infects the lungs of patients with low immunity and may invade other organs and tissues in the body.In recent years,with the increasing number of aging populations in the country and the abuse of various antifungal drugs,the number of people with low immunity has increased sharply.As a result,the number of patients with deep infections caused by Aspergillus fumigatus clinically has ranked the first in the number of deep fungal infections.Two.Threats to human health are gradually increasing.In this study,the mature Aspergillus fumigatus ATMT transformation system in the previous laboratory was used to successfully transform Aspergillus fumigatus using ATMT technology,and 1513 mutants of Aspergillus fumigatus were obtained.1513 mutants were screened for morphology,including changes in factors such as mycelium,spore production,pigment,and albino,to find mutants with significant changes,and further morphological and molecular biological verification to find their mutation sites and mutations The relationship between locus-associated genes and morphological changes has laid a foundation for studying the mechanisms related to Aspergillus fumigatus growth and development or pathogenicity.Aspergillus fumigatus is a high temperature resistant strain.In the experiment,1513 strains of Aspergillus fumigatus mutants were cultured in 25 degree(ambient temperature),37 degree(human temperature)and 48 degree(high temperature)incubators,and the phenotype was screened to obtain A total of 8 mutants,including AFM70808 strains,with large morphological changes,successfully mapped the mutation sites of these 8 strains,and specific analysis of their mutation sites revealed that 5 of them had mutation sites in In one gene,the mutation sites of three strains were between two or more genes.Nematode pathogenicity experiments wereperformed on 8 mutant strains to verify the pathogenicity changes of the strains.It was found that the strain AFM6284 had the lowest pathogenicity based on phenotypic changes.Combined with a lot of literature review,the study of the mutant gene of the strain AFM6284 mutant was the most significant,and it was selected as the main research object in the later period.Gene analysis of the AFM6284 knockout mutation site revealed that the gene is Phosducin family protein(Pho),and relatively few studies have been conducted on the function of Pho protein in fungi.It is known that its homologs are mainly in model animals.Binding to the ? protein subunit of G protein is involved in mediating signal conditioning of rod cells or binding to ?? subunit in the manner of a chaperone molecule is involved in the regulation of the G protein coupling-cAMP-PKA pathway.Other features are reported relatively rarely.In this study,the CRISPR / Cas9 system was used to perform gene knockout,the Pho gene was constructed by CRISPR / Cas9 knockout plasmid,and the Pho gene was successfully deleted.The phenotypic change of the ?Pho knockout strain was consistent with that of AFM6284 at three different temperatures,and the nematode model confirmed its pathogenicity.In order to further determine the gene pathway in which the gene is located,it was found through light experiments and cAMP concentration detection that the gene did not play a significant role in the light-transmitting gene pathway,and the deletion of the Pho gene significantly reduced the cAMP concentration,thus finding that the Pho gene was mainly involved in the G protein Coupling-cAMP-PKA pathway.To sum up,the AFM6284 strain and the ?Pho knockout strain have a slow growth and reduced pathogenicity due to the deletion of the Pho gene.Further verification found that the Pho protein encoded by the Pho gene mainly participates in the G protein-coupling-cAMP-PKA pathway in Aspergillus fumigatus,and the deletion of the Pho protein inhibits the production of cAMP,and regulates the concentration of cAMP in a positive manner,thereby regulating cAMP.Downstream pathway.This study lays the foundation for further exploring the role of the Pho gene in the gene pathway and further elucidating its pathogenic mechanism,and providestheoretical support for the clinical treatment of pathogenic fungal infections.