Preliminary Study On The Interacting Proteins In The Lignocellulase Transcriptional Regulation Of Penicillium Oxalicum

At present,crazy consumption of oil,coal and other non-renewable resources have made it difficult to meet the rapid development of human economy.Exploring new green renewable resources has become an urgent problem to be solved.Using modem biotechnology to convert lignocellulosic biomass into liquid fuels and bulk chemicals can effectively alleviate the shortage of resources.Filamentous fungi can produce lignocellulases to degrade biomass,so it has been used for industrial-scale ligonocelluase production for many years.However,the high cost of expression of lignocellulases restricted its large-scale application.Therefore,it is of great scientific significance and application value to explore the mechanism of lignocellulase expression regulation in order to further improve lignocellulase expression level of filamentous fungi.Penicillium oxalicum is an important industrial filamentous fungus to produce lignocellulases.In the regulation system of cellulase expression of Penicillium oxalicus,regulatory proteins play an important role in the regulation of cellulase gene transcription level.The cellulase transcription factors and their interacting proteins are important components of cellulase expression regulation system.Therefore,it is important to explore the transcription factors and their interacting proteins of Penicillium oxalicus in the system of cellulase expression regulation.In this paper,we used yeast two-hybrid technique and tandem affinity purification technique to screen the interacting proteins of main transcriptional regulators of Htf,ClrB2,CreA,ClrB,AmyR and XlnR.The main transcription factors can combine to specific DNA sequences by recruiting related proteins,to change the chromatin structure of the promoter region,to regulate the target gene expression.Chromatin remodeling protein complexes act as the recruited proteins by transcription factors may play an important role.Therefore,the other work of this paper is to study the function of the main chromatin remodeling protein in regulating the cellulase expression.1.Screening the interacting protein of transcription factor Htf by yeast two-hybrid techniqueFor using yeast two-hybrid technique to screen the interacting protein of transcription factor Htf of Penicillium oxalicum,first of all,we constructed the Y2HGold-PGBKT7-Htf yeast strain to interact with the Y187-PGADT7-cDNA yeast strain to screen interacting protein with Htf.And then we studied the function of the interacting protein.Five proteins which may interact with transcription factor Htf were found and these proteins are PDE₀1231,PDE₀2486,PDE₀4682,PDE₀6164,and PDE₀8762.PDE₀1231 contains a C6_like binuclear cluster DNA binding domain similar to GAL4 in its N-terminal,and may play a role in the regulation of gene expression or replication.PDE₀2486 contains the LSM domain.LSM domain is mainly involved in the assembly of SnRNP and responsible for the splicing of mRNA.The functional prediction shown that the PDEe04682 protein is a member of the GPI-membrane anchored protein superfamily,and therefore may play a role in the MAPK signaling pathway.PDE₀6164 was annotated as a PRP1-N superfamily protein,may involved in mRNA splicing and RNA nicleation processes.PDE₀8762 was annotated as a MFS superfamily protein,involving secondary transport process.The interaction between these proteins is still required for intracellular experiments.2.Screening the interaction protein of main transcription factors of cellulase by tandem affinity purificationIn order to screen the interaction protein of the five main transcriptional regulators ClrB2,CreA,ClrB,AmyR and XlnR,we used Penicillium oxalicum 114-2 as the original strain to construct five stains with recombinant transcription regulators labled by FLAG-HA.The labeled transcription factor protein complexes were obtained by two-step affinity purification.And then we use the mass spectrometry technique to identified the interaction protein of five transcription factors.The results shown that,PDE₀1024 encoded RcoA protein and PDE₀3177 encoded Cyc8 protein were present in the interacting protein system of ClrB and AmyR.The RcoA protein encoded by PDE₀1024 has two conserved domains:the N-terminus is the Tup domain and the C-terminal is WD40 domain,and its function may involve in histone binding,histone deacetylase binding,and transcription repression.PDE₀3177 encoding Cyc8 protein contains a SNAP superfamily and TPR repeat domain,may be involved in RNA polymerase ? regulated transcription process,chromatin remodeling process,and a variety of biological processes by binding to Tup proteins to form co-inhibitors.PDE 09266 encoding Swi6 protein was present in the interacting protein system of ClrB.The Swi6 protein contains the ANK superfamily domain and the KilA-N domain.The ANK domain has an ankyrin function that can repeatedly mediate protein-protein interactions in different protein families.KilA-N proteins can bind to the upstream regulatory sequences of target genes to regulate cell growth and development.The unknown functional proteins encoded by PDE 08646 were present in the interacting protein system with CreA.PDE₀8646 protein contains a GAL4-like zinc finger binuclear DNA binding domain in the N-terminal,but its biological function is unknown.The interacting protein of ClrB2,CreA,ClrB,AmyR and XlnR were analyzed.Except ClrB2 transcription factor labeled strain,all other labled strains can obtain proteins interacting with the regulatory factor protein which may regulate cellulase expression.The proteins screened by TAP technique were divided into four groups:catalytic protein,structural protein,transport protein and other protein.The propotion of the catalytic protein selected by the labeled strain was lower than that of the negative control strain,but the propotion of glycoside hydrolase was increased,including ?-glucosidase and xylanase.The proportion of structural protein and transport protein were also reduced,while the proportion of other protein was increased.It is important to explore the mechanism of cellulase transcription factor in the regulation of cellulase gene transcription by tandem affinity purification.3.Study on the regulation of cellulase expression by chromatin remodeling proteinChromatin remodeling protein complex plays a role in transcription factor regulation of gene expression.The chromatin remodeling protein complex changes the chromatin structure by the energy produced by hydrolyzing ATP,thereby affecting the expression of gene.In order to explore the role of chromatin remodeling protein in the regulation of cellulase expression in Penicillium oxalicum,Penicillium oxalicum 114-2 was used as the original strain,and the homologous protein of the yeast remodeling protein was selected as the basis.The chromatin remodeling protein gene PDE₀4736,PDE₀7819 and PDE₀8304 were studied in this study.The phenotypic analysis and cellulase activity of knockout and overexpressing strains were carried out to analyse the role of chromatin remodeling protein in the regulation of cellulase expression.The results showed that the knockout of PDE₀4736,PDE₀7819 and PDE₀8304 had little effect on the growth,development and cellulase activities.The overexpression of PDE₀4736,PDE₀8304 increase the expression level of cellulase of strain.But overexpression of PDE₀7819 has no significant effect on the growth,development and the expression of cellulase of Penicillium oxalicum.It is speculated that chromatin remodeling proteins play a role by forming of eomplexes.The absence of a single chromatin remodeling protein does not significantly affect the action of chromatin remodeling complex,which may be caused by the functional compensation of other constituents.Some core subunit of the chromatin remodeling protein complex plays a key role in the chromatin remodeling complex,therefore,it is relatively difficult to study the function of the core subunit by gene knockout.