Study On The Transcriptional Regulation Of Aspergillus Niger On A Genomic Scale

Aspergillus niger,generally regarded as a save(GRAS)strain,is widely used in industrial factory for organic acids,industrial enzyme,and foreign protein expression.The Fungal Transcription Factor Database(FTFD)collects 775 transcription factors predicted by the genome sequencing in A.niger.Of these,only 5% of the transcription factors have been functionally characterized by traditional methods.The functional studies of most transcription factors are still blank,and little research has been done on the genome-wide transcription factor binding site and its regulatory mechanism.In filamentous fungi,TFs play key biological regulatory functions,such as growth and development,cellular processes,and stimulus response.Therefore,the analysis of its transcriptional regulation mechanism is of great value for the host transformation and genetic regulation mechanism of A.niger.Due to the above reasons,this thesis selected different Aspergillus strains as the research objects providing a new tool for the targeted modification of its genetic regulatory sites and combining high-throughput sequencing technology to analyze the binding sites of different types of transcription factors at the genome-wide level and their regulatory mechanisms.The specific research content and results are as follows:(1)Construction of a single base editing platform suitable for A.niger based on CRISPR/Cas9Based on the CRISPR/Cas9 system,the Cas9 protein was inactivated into nCas9 with DNA single-strand cleavage activity,which was fused with cytosine deaminase(r APOBEC1)and uracil glycosylation inhibitor(UGI)into base editing complex that can be normally expressed in A.niger and has base editing function.The new system can achieve amino acid inactivation(pyr G and fwn A)or synonymous mutations(fwn A)of a gene through base site-directed mutations.The editing efficiency ranged from 47.36% to 100%,and the editing window is the base at position 2-9.So we provided site-specific editing tools for genetic modification and regulation mechanism study of A.niger.(2)Construction of ATAC-seq platform for filamentous fungiThe possibility of developing the ATAC-seq technique in filamentous fungi by three methods including liquid nitrogen grind,INTACT and protoplast preparation were discussed,and the protoplast preparation was determined to be the optimal method.Based on the established ATAC-seq platform,the signal distribution characteristics of the open chromatin regions of filamentous fungi were initially determined,and the relationship between chromatin accessibility and gene transcription was determined in combination with RNA-seq.(3)Identify chromatin accessibility regions in filamentous fungiThe TFs deletion strains including creA,amyR,cpcA,pacC and prt T of A.niger and laeA knockout and overexpressing strains of A.oryzae were constructed by molecular biological means.The ATAC-seq library was constructed for different Aspergillus species,different culture conditions,and different TF mutant strains.The changes in chromatin accessibility under various conditions were systematically studied.(4)Mining TF-binding sites from the accessibility chromatin regionsA large number of trans-acting factors interact with DNA cis-regulatory elements exist in the chromatin accessibility region.First,analysis of the digestion frequency of the known motif transcription factor in the accessible region revealed that the imbalance of DNA double strand is an important standard for judging the TF binding site.Then,we studied the regulatory sites at the genomic-scale on known motif TFs and combined with RNA-seq to determine the regulatory functions of Cre A,Amy R,Prt T and Pac C.Then using HOMER software to perform de novo prediction of transcription factor binding sites for A.niger CBS513.88 and SH2,and found 15 and 17 new transcription factor binding motifs.The predicted TF instances were functionally verified in vivo by the artificially designed Aspergillus minimal promoter.(5)Study on the regulation mechanism of protease regulatory factor Prt TCharacterization of a series of prt T mutants showed that Prt T related with protease activity,of which Thr(T)in position 142 is not the key amino acid that influence Prt T activity.Besides,the u ORFs in 5'UTR inhibit Prt T translation.RNA-seq analysis revealed Prt T were involved in proteolysis,nitrogen metabolism regulation and so on.Among the DEGs,32 protease genes were identified and 8 targeted by Prt T directly.Through the EMSA and the knockout of starch hydrolase regulatory factor Amy R,the competitive regulation of Prt T and Amy R was determined.