Study On The Mechanism Of Histone H3K14 Acetylation Regulating The Growth And Sporulation Of Porphyridium Violaceum

P.lavendulum is a nematode biocontrol fungus which has been commercialized.In this study,the Ku80 knockout strain was used as the starting strain.Firstly,homologous recombination was used for point mutation of histone H3K14 site.Secondly,the histone acetyltransferase gcn5,which acetylated H3K14 site,was knocked out by homologous recombination.The growth and sporulation of Ku80 knockout strains and mutants were determined and compared.The mechanism of histone H3K14 acetylation regulating the growth and sporulation of P.lavendulum was studied by transcriptional histology and gene function analysis.Through the above studies,the molecular mechanism of histone modification in the regulation of sporogenesis of P.lavendulum can be preliminarily elucided.The main research results of this paper are as follows:1.Obtaining H3K14 mutant strainThe H3 fragment which was mutated to R and Q at the H3K14 site was first obtained,and then the plasmid H3-3'-pPK2-sur-GFP was digested,and the mutant fragment was ligated to the other end of sur gene via an In-fusion enzyme ligation.The constructed mutant vector was transformed into P.lavendulum by agrobacterium-mediated method,and transformants were screened by chlorimethanol.The transformants were verified by sequencing and Western blot.2.Obtaining gcn5 knockout strainThe plasmid pPK2-sur-GFP was first digested,and then the gcn5 upstream and downstream homologous fragments were ligated to both ends of sur gene via an In-fusion enzyme ligation.The constructed knockout vector was transformed into P.lavendulum by agrobacterium-mediated method.The transformants were screened by chlorimethanol and verified by PCR.3.Growth and sporulation of mutant and knockout strains were affectedCompared with the Ku80 strain,the spore production of both mutant and knockout strains was reduced.In addition to PDA medium,the growth rates of H3K14Q mutant and gcn5 knockout strains were lower than those of Ku80 strain.However,on PDA medium,the growth rate of H3K14R mutant was higher than that of Ku80 strain,and there was no significant change on other media.4.Transcriptome analysis of each mutant and knockout strainPost-translational modification of histones affects the structure of chromatin and thus affects gene expression.Whether it is an H3K14R mutant or a H3K14Q mutant,or a gcn5 knockout strain,the acetylation level of histone H3K14 is in an abnormal state compared with the Ku80 strain.This abnormal acetylation or deacetylation state will affect some growth and sporulation-related genes of P.lavendulum.This resulted in the decrease of growth and sporulation ability of H3K14Q mutant and gcn5 knockout strain,and the decrease of sporulation ability of H3K14R mutant strain.In order to find these genes related to growth and sporulation in different strains,the above phenotypes were analyzed,and the time point of sample collection was determined.The samples of Ku80 strain and transformants were compared by transcriptome analysis.The differential genes between strains with distinct phenotypic differences were analyzed to identify the metabolic related genes dependent on epigenetic modification.The main innovation of this paper:It is an important survival strategy for fungi to stop growing and produce spores in nutrient deficient environment.The molecular mechanism of starvation-induced fungal life cycle transformation from the perspective of epigenetic regulation has not been reported.Starting from histone H3K14 mutant,we tried to elucidate the molecular mechanism of sporulation induced by nutritional stress mediated by histone H3K14 by screening nutrients under nutritional stress in PDA,combining transcriptome analysis and mutant library screening.To lay a theoretical foundation for comprehensively understanding the survival strategies of fungi adapting to environmental stress to regulate life cycle changes.