Bacillus subtilis 168中亚硝酸盐还原酶基因的敲除及表型鉴定

Knockout and Identification of Nitrite Reductase Gene in Bacillus subtilis 168

该研究首先通过同源重组方式将Bacillus subtilis 168菌株nir基因位点置换为两端具有同向排列的loxP序列选择性标记基因,再运用Cre重组酶将两个loxP间的全部序列切除,成功敲除了Bacillus subtilis 168菌株亚硝酸盐还原酶(nitrite reductase,NiR)的3个亚基基因nasB、nasD和nasE。其中,敲除nasD基因的B.subtilis 168菌株在不同生长时期均未分解培养基中亚硝酸盐,表明该nasD基因缺陷型菌株NiR活性已被完全消除,不再具备降解亚硝酸盐的能力。

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 lon⁃ger possessed the ability to degrade nitrite.