Chromatin Immunoprecipitation Analysis The Function Of Transcription Regulation Protein Hac1 In Protein Secretion Regulation

Filamentous fungi has the remarkable ability of protein secretion and it has the important status in the production of industrial enzymes.Transcription regulatory proteins Hac1 from filamentous fungi is important to unfolding protein response pathway.Hac1 increased secretions of extracellular protein by increasing expressive quantity of genes such as molecular chaperone BIP1 of UPR,PDI1.So the function research of transcription regulation protein Hac1 continue to analyzing high protein secretion mechanism of filamentous fungi and improving the ability of protein secretion by genetic modification.Filamentous fungi 114-2 is the material of our study,we tracked out the target of transcription regulatory proteins Hac1 and studied its function in unfolding protein response.We measured the extracellular protein concentration and CMC enzyme activity,and we detected relative expression of UPR response gene bipA,pdiA by RT-qPCR,determining the optimum time of UPR response.Exploring the hyphae crosslinking time and ultrasonic broken condition,we establish the ultrasonic disruption method of chromatin immunoprecipitation in Penicillium decumbens.Using chromatin immunoprecipatation sequencing technology to find out target gene of Hac1.Screening to target gene3379,constructing gene 3379 knockout box by fusion PCR and nested PCR and protoplast transform to host strains filamentous fungi 114-2,to build gene 3379 knockout strain.Through out phenotype observation and the determination of protein concentration and enzyme activity,we identified the function of target gene 3379.The main results of the thesis are as follows:1.Filamentous fungi that was induced by DTT treatment produce unfolded protein response mechanism,and we set to join DTT time gradient.The determination results of extracellular protein concentration and CMC enzyme activity show that extracellular protein concentration and the enzyme activity reached the maximum value after joining DTT 15 h.we detected expression of UPR response gene bipA and pdiA,The results show that the relative expression of bipA and pdiA reached maximum value between 14 and 17 h after joining DTT.So we can determine the unfolded protein response mechanism was fully activated aboutjoining DTT 15 h.2.In this study,Penicillium decumbens was used to detect the expression of Hac1,a transcriptional regulation factors,which was induced by DTT treatment.Mycelium were treated using 1% formaldehyde crosslinking solution and ultrasonic was used to break the chromatin.We found that the best CHIP conditions for penicillium were listed as follows:Using 1% formaldehyde crosslinking solution to treat mycelium,and crosslinking time should be controlled within 20 min.The optimal fungus suspension concentration for sonication was 10 ml of CHIP buffer within 1 g hyphae.The optimal sonication condition is using 10% of the power working for a total of 30 times,each time for 5s,and there is an interval of 40 s between each time.According to this conditions,DNA fragments of size in100 to 1000 bp were collected,which insure the success of subsequent chromatin immunoprecipitation.3.The chromatin immunoprecipitation of DNA fragments after RAPD-PCR amplification for high-throughput sequencing.Sequencing of the data obtained are of good quality and compare rate is high,meets the requirement of subsequent analysis.Analysis results show that the target genes regulated by Hac1 mainly distributed on the 8 scaffold,combined with the characteristics of sequence length of 78 bp,combined with the base sequence for multiple pyrimidine and purine type,regulation of gene function mainly involved in glucose metabolism,amino acid metabolism,fatty acid metabolism,cell cycle,protein synthesis,secretion,gene expression regulation of biological processes,etc.4.Constructing gene 3379 knockout box using fusion PCR and nested PCR,protoplast transform to host strains filamentous fungi 114-2,we delet gene 3379 in Penicillium decumbens successful.Deletion of gene 3379 had no effect on morphological characteristics,such as colony diameter,colour of spores,volume of spores,and width of mycelia.Compared to the original strain 114-2,extracellular protein and enzyme activities were significantly decreased in 3379 deletion strain.We come to the conclusion that the function of gene 3379 accelerate protein secretion and it is indispensable to the protein secretion pathway.