Deletion Of The Brsa-25 Gene In Aspergillus Niger Which Relates With Its Morphology

Citric acid is an organic acid,which is one of the indispensable ingredient of solid and liquid drink.In addition to the food industry,it has been used in the fields of modern agriculture,industry,textiles and so on.Citric acid is generally obtained by liguid fermentation.Aspergillus niger is used in the fermentation process.Studies showd that citric acid production is greatly affected by the different hypha forms of Aspergillus niger.When the hypha of Aspergillus niger is rendered as pollet,citric acid is largely accumulated,but enzymes is barely produced.In order to obtain high-yield strains of citric acid,we need to use molecular method for the transformation of Aspergillus niger.Studies had reported that brsa-25 gene can control the morphology of Aspergillus niger.This gene make it easier for Aspergillus niger to form a dispersion mycelium,which is to the disadvantage of the accumulation of citric acid.This study hopes to obtain a high-producing strain by deleting the gene of brsa-25,using the principles of homologous recombination.Results are as follows:1.By optimizing the process of overlapping PCR reaction,we successfully got a homologous recombination sequence,which contained the brsa-25 upstream homology arm,the hygromycin resistance gene expression cassette and brsa-25 gene downstream homology arm.2.We successfully connected the homologous recombination sequence with T-vector by using Infution enzyme,which is more efficient compared with the method of T-A connecton.3.We successfully transformed the homologous recombination sequence to Aspergillus niger by the method of liposomes transformation for the first time.4.We successfully transformed the homologous recombination sequences to the spores of Aspergillus niger by the method of electric conversion.We finally got 3 transformant strains,which had the right integration position.