Functional Study Of Oxidative Sterol Binding Protein OSBP5 In Aspergillus Oryzae

Aspergillus oryzae is the safety of traditional brewing soy sauce and wine strains,a kind of filamentous fungi,in Asia is widely used in production of beverages and traditional food,such as wine,soy sauce and bean paste has been for thousands of years of research history,although has completed the whole genome sequencing in 2005,but the understanding of it confined to the gene sequences,there are many important genes function subject to research and explore.In order to develop and use Aspergillus oryzae as a highly efficient heterologous expression strain,improve production efficiency and save cost,the research on the function of related genes is particularly important.Oxidative sterol binding is a class of conserved proteins in eukaryotes,which have functions in lipid and glucose metabolism,Akt signal transduction,steroid generation,cell adhesion,migration and proliferation.Therefore,the function of Aspergillus oryzae is of great significance.In this study,oxidative sterol binding protein of Aspergillus oryzae 3.042 was taken as the research object,and the following four aspects were mainly studied1.By constructing a cDNA library,the target gene OSBP5 was obtained by PCR,and the prokaryotic expression vector of sterol oxide binding protein OSBP5 of Aspergillus oryzae was constructed,which was transformed into Escherichia coli DE3 receptive state.The prokaryotic expression strain IPTG induced expression was obtained,and the protein was purified in vitro.By making standard protein curve,the specific concentration of purified protein was determined.Calculated according to the concentration of various ligands required protein concentration,using trace surge of hot technology,detect the protein in vitro and ergosterol,cholesterol,beta,sterol,stigmasterol and seven base cholesterol etc,the combination of different sterol KD values,to analyze the expression of protein in vitro activity and with the combination of different sterol in vitro.2.PCR from a CDNA library,gene expression vector chose in PEX2 B,interference carrier choice p EX1 successful build Aspergillus oryzae oxidized cholesterol binding protein OSBP5 expression and the interference of the carrier,and by using protoplast reforming process,turn to Aspergillus oryzae 3.042 uracil defect type strains,screening are successfully received express strains and interference strains all five strains,using q RT-PCR method,fluorescence quantitative,respectively be expressed quantity minimum and maximum strain of # 4 and # 11 phenotype changes.The expression pattern of OSBP5 gene at different time,different temperature and different ethanol concentration was analyzed.3.The overexpressed strain #11,interference strain #4 and control strain CK were sent to Guangzhou Gidio Company for sequencing.Firstly,the quality of the extracted RNA was analyzed,and the differential gene expression was analyzed,and it was found that the differential genes were concentrated in each metabolic pathway,indicating that OSBP5 was closely related to the metabolism of Aspergillus oryzae.4.After saponification of the obtained two strains,the ergosterol content in mycelia was detected by HPLC.It was found that the ergosterol content in mycelia of Aspergillus oryzae increased both after overexpression and after interference,revealing the possible gene function of OSBP5 in Aspergillus oryzae that may be related to sterol transport.5.The base sequence of the target gene was obtained by NCBI,and the protein sequence was obtained by BLAST alignment.Spring was used to predict the proteins that might interact with the protein,and five proteins were predicted.The role of genes in lipid transport of Aspergillus oryzae was explored by transforming yeast AH109 strain with yeast double hybrid technique.