Knockout Of NarGHJI, The Gene Encoding The Nitrate Reductase In Denitrifier DNF409

The deterioration of aquarium water has become an important limiting factor of national aquiculture development in our country.Especilly to the aquiculture, the accumulation of nitrite is more harmful. Biodenitrification is one of the effective methods to remove nitrite from water in theory.The research begins with a bacillus cereus DNF409 separated by our laboratory as the original beginning bacterial strain. It has a strong denitrification capability which can reduce the consistency of nitrate in nitrate polluted water body from 40ug/mL to 5 ug/mL below within 72h. However, serious nitrite accumulation occurs during the process of purifying water. The purpose of this subject is to knockout nitratase gene of the bacterial strain by molecular biological methods to make it a single degraded nitrite bacterial strain. The specific research results are as follows:This research is based on a denitrifier named DNF409 which is isolated in our lab. DNF409 can drop the nitrate from 40μg/mL to 5μg/mL in 72 hours in the pollution water. But it can also accumulate the nitrite while puring the water.The aim of this research is to disrupt the gene of narGHIJ in DNF409 through the moleculer biological method. Then the DNF409 can change to another stain that can drop the nitrite only. The results were showed as below:(1) The vector of narGHIJ gene targeting pDGLSR-Ts was constructed from shuttle plasmid pDG646. It had a temperature-sensitive replicon,chloramphenicol resistance gene and two fragments which were 1 Kb,926 bp, respectively matching part the sequence of narGHIJ gene. And the erythromycin resistance gene, which could be expressed in denitrifier DNF409, was inserted in to the two fragments.(2) The vector of narGHIJ gene targeting pDGLSR-Ts was introduced into denitrifier DNF409 by electrotransformation. The Sm~ΓKan~s resistant marker isolate double overcross strain was obtained and narGHIJ gene disruption was confirmed by PCR.(3) In the denitrification experiment conducted in simulative polluted water that is rich in nitrate and nitrite with the introduction of double exchange knockout bacterial strain, it is discovered that 70% of nitrate's reducing capacity has been lost while much of nitrite's reducing capacity has been lowered.