Abstract:Abstract：An efficient method of gene knockout system was developed in Aspergillus niger 10142，an hyperproducer of citric acid fermentation.It constructed a binary vector containing lethal genes that a herpes simplex virus thymidine kinase（HSVtk）gene in T-DNA.By using Agrobacterium tumefaciens-mediated transformation（ATMT），the ectopic transformants did not grow on the medium addition of 10 μmol/L 5-fluoro-2'-deoxyuridine.This method increased the proportion of positive transformants to select gene knockout transformants.Gene knockout experiments on trehalose-6-phosphate synthase（tpsA）in Aspergillus niger were performed using double-tagged vector containing lethal gene and hygromycin.The results showed that the frequency which refered to positive number account for total transformants was 63%.The successful construction of Aspergillus niger gene knockout system has provided an efficient technology platform for gene knockout experiments and researchment on the gene mechanisms of Aspergillus niger.