Abstract:In this study，the nir gene locus of Bacillus subtilis 168 was replaced with selective marker genes flanked by loxP sequences in the same orientation through homologous recombination. Cre recombinase was then used to excise the entire sequences between the two loxP sites. This successfully led to the knockout of nasB，nasD，and nasE，the three subunit genes of nitrite reductase（NiR）in B. subtilis 168. The B. subtilis 168 without the nasD gene did not exhibit reduced nitrite content in the culture medium at different growth stages.It indicated that the NiR activity of this nasD gene-defective strain had been eliminated，and this strain no longer possessed the ability to degrade nitrite.