Study On Interaction Between LaeA And Gcn5 In M.ruber

Previous studies on Monascus.ruber in our laboratory found that the global regulator Lae A affected the level of histone acetylation.Compared with the wild-ty pe strain M7,The histone acetylation level of ?Mrlae A decreased significantly,w hile the histone acetylation level of overexpression strain trp C(p)::lae A increased si gnificantly.This suggests that Lae A may regulate various life activities by regulati ng histone acetylation.Based on this,this study further analyzes the similarities an d differences of strain M7,?Mrlae A,?Mrgcn5,trp C(p)::lae A,?Mrlae A::trp C(P)::Mrg cn5,?Mrgcn5::trp C(P)::Mrlae A in environmental stress and transcriptome.To lay afoundation for exploring the regulatory mechanism and interaction between Lae A and Gcn5 on metabolism and development,the specific research results are as foll ows:1.Construction of overexpression strain ?Mrlae A::trp C(P)::Mrgcn5 and ?M rgcn5::trp C(P)::Mrlae A.In this study,the overexpression vectors p CMrgcn5 and p CMrlae A were constructed,and then the overexpression vector was transformed int o strain ?Mrlae A and ?Mrgcn5.325 and 272 transformants were obtained respecti vely,and then strain No.65?Mrlae A::trp C(P)::Mrgcn5 and No.31?Mrgcn5::trp C(P)::Mrlae A were screened by PCR.The identification results showed that the Mrgcn5 overexpression cassette in strain No.65 was integrated into the genome of the start ing strain ?Mrlae A in the form of homologous recombination,while Mrlae A overe xpression cassette in strain No31 was integrated into the genome of the ?Mrgcn5strain in a non homologous recombination manner.2.Phenotypic analysis of ?Mrlae A::trp C(P)::Mrgcn5 and ?Mrgcn5::trp C(P)::Mrlae A strain.Original strain Mrgcn5 and Mrlae A were used as control and anal yzed the growth of strain ?Mrlae A::trp C(P)::Mrgcn5 and ?Mrgcn5::trp C(P)::Mrlae A under the medium conditions of high osmotic pressure,high oxidative pressure and cell wall inhibitor.The results showed that Mrgcn5 overexpression could slight ly affect the oxidative regulation and osmotic pressure regulation of ?Mrlae A,andMrgcn5 overexpression might acetylate proteins related to oxidative stress and os motic pressure tolerance.Overexpression of Mrlae A in ?Mrgcn5 strain had no sig nificant effect on environmental stress tolerance.3.Transcriptome analysis to explore the expression changes of Mrlae A andMrgcn5 regulatory genes.Transcriptome data were obtained by RNA-seq and the n differentially expressed genes were screened.Finally,GO and KEGG pathway w ere enriched and analyzed.We found that Mrlae A directly regulated sexual develop ment and secondary metabolism.Mrgcn5 regulates nuclear migration and primary metabolism,and then affects its secondary metabolism level.Compared with ?Mrl ae A,strain ?Mrlae A::trp C(P)::Mrgcn5 has up-regulated transcription in signal transd uction and transport activities,and it has a negative impact on cell cycle and me iosis.