Study On The Transformation System And RNA Interference Of Aspergillus Oryzae NiaD300

With the rapid development of molecular biology and genetic engineering in the field ofmicrobes,the genome of more and more filamentous fungi are analyzed and published.But compared with yeast,the research of filamentous fungi are obviously droped behind in thedepth,breadth and the research scale.Aspergillus oryzae as one of the importantindustrial strain in filamentous fungi has been used in food industry for a very long time.Thegenome sequencing of A.oryzae was accomplished in2005, so it is urgent to conduct thefundmental biological research of A.oryzae.One of the basic strategy to study the biologicalfunction of the target gene is to inhibit its activity and detect inhibition-derived effect.RNAinterference (RNAi) is an advanced technology based on such strategy to study the function offungal gene,which has many advantages than other techniques.RNAi technology hasshown great potential in high-throughput research of gene function.In this study,PEG-mediated protoplast transformation system of Aspergillusoryzae niaD300was established and optimized.The results showed that pyridine thiaminecould effectively screen transformants with the concentration of0.1μg/mL,where20mmol/LCsCl could reduce the growth background.We constructed RNAi vector bearing theintron-containing HairpinRNA (ihpRNA) in which amylase intron VIII was used,with thepyridine thiamine resistant gene as the selection marker.The regulatory factor gene ofunfolded protein (hacA) and glucose repressor factor (creA)was selected as the target gene toconstruct their RNAi vector.The RNAi vector of hacA gene and creA gene was successfully transformed intoAspergillus oryzae separately by PEG-mediated protoplast transformation.Transformants wereidentified by mycelium PCR and gene sequencing.The transcriptional change of target geneswere detected by real time quantitative PCR.Results indicated that the expression level of thetarget genes are reduced to a certain extent when the RNAi vector was introduced into thehost strain.