Study On The Epigenetic Mechanism Of H3K9 Sporulation Induced By Nutritional Stress Of Porphyridium Violaceum

Asexual sporulation(conidiation)is the most common form of fungal reproduction.Starvation induced sporulation is also an important strategy of environmental adaptation.The nematophagous fungus Purpureocillium lavendulum(formally Paecilomyces lilacinus)is an important biocontrol agent of plant-parasitic nematodes.Previous studies showed that when the fungus was cultured on PDA medium,colony growth was restricted and a large number of spores were produced.When the nutrients of the fungal basic medium(MM medium)are added to the PDA medium,the radial growth of colony was increased.In order to study how the nutritional stress induced fungal sporulation through epigenetic regulation,we mutated the ninth lysine(H3K9)of the histone H3 in the P.lavendulum to alanine using the ?ku80 strain as the starting strain.A PlH3K9A mutant strain was obtained,and then the PlH3K9A mutant strain and the?ku80 strain were subjected to comparison,and it was found that the colony of the PlH3K9A mutant strain was increased on the PDA medium,and the sporulation amount was decreased.We proposed that the fungus may be stressed by starvation to some extent and produce conidia when grown on PDA media,and this process of sporulation would be regulated epigenetically via histone modification.To confirm this proposal,we want to firstly confirm which ingredient were lacked in PDA,and then study the molecular function of its metabolism pathway in sporulation.Then we continue mutate H3K9 to simulate several epigenetic marks,such as methylation or acetylation.The histone modification enzymes were also studied to confirm which epigenetic mark were used by starvation to induce sporulation.The transcriptome analysis will be also carried out to find more genes involved in regulating this sporulation process.The results of this project will help us more comprehensively understanding the molecular mechanism of lifestyle switching induced by starvation via epigenetic regulation and make a solid base for better bio-control of plant-parasitic nematodes.The main results of this study were described as follows:1.Histone H3K9 point mutation in P.lavendulumKnockin vector was constructed by using sur as a screening marker,transformed by Agrobacterium tumefaciens and knocked in by homologous recombination.As a result,it was found that the expression of sur gene did not affect the expression of histone H3 and H4.On this basis,the upstream fragment of histone H3 gene(containing the histone H3 gene)was ligated into the pEASY-Blunt Zero Cloning plasmid.Then the modified plasmid used as a template to mutate H3K9 into alanine,arginine,leucine and glutamine.Next,PlH3K9A,PlH3K9R,PlH3K9L and PlH3K9Q mutant strains were obtained.3.P.lavendulum dim5 gene knockoutKnockout vector was constructed by using sur gene as a screening marker,transformed by Agrobacterium tumefaciens and knocked out by homologous recombination.4.Phenotypic analysis of PlH3K9 mutant strain and related enzyme gene knockout strainsPhenotypic analysis was carried out on four mutant strains of PlH3K9A,PlH3K9R,PlH3K9L and PlH3K9Q,Pldim5,Plgcn5 knockout strain and ?ku80 strain.After observation and statistics,it was found that compared with the ?ku80 strain,the vegetative and sporulation of mutant strain and the knockout strain were different in MM and PDA media.While,the difference could be compensated by adding KNO3,or(NH4)2SO4 into PDA medium,respectively.5.Transcriptome sequencing analysis of PlH3K9 mutant strain and related enzyme gene knockout strainDifferent mutant strains and knockout strains were on different media,and no less than 0.8 g of hyphae were collected.The collected hyphae were frozen in liquid nitrogen for transcriptome sequencing analysis.The main innovation of this paper:The molecular mechanism of the transformation of life history of starvation-induced fungi from the perspective of epigenetic regulation has not been reported.This study starts from histone H3K9 mutant.By screening nutrient stress nutrients in PDA,point mutation simulation of histone modification and transcriptome comparison,the molecular mechanism of histone H3K9-mediated nutrient stress-induced sporulation were elucidated.This study lays a theoretical foundation for a comprehensive understanding of the survival strategy of fungi adapting to environmental stress and regulating life history transformation.