Application Study Of CnorB Gene, Encoding The Catalyzing Subunit, In Population Analysis For Denitrifying Bacteria

Nitric oxide reductase is an obligatory intermediate during the respiratory denitrification. As a well-known cytotoxic compound, the accumulation of the NO has a potential of causing significant cell damage. So, to mitigate the toxity of NO appears to be very essential. The protein responsible for NO reduction, NO reductase(Nor), catalyzes the reaction. What is more, NO plays a significant role in biodenitrification system. So, the study on denitrifying bacteria based on nor gene as a functional marker is technically feasible.Gene-specific primer sets were developed previously to detect narG, narH, nirK, nirS and nosZ for nitrifying bacteria and denitrifying bacteria, but it is untill most recently, a PCR primer set specific to norB has ever been developed by Gesche Braker et al., which casts light on the phylogenetic study on denitrifying bacterias.Nor consists of two subunits: NorB and NorC, among which NorB, encoded by cnorB gene, is revealed to be the catalyzing subunit and embrace the active site. So this Nor is designate cNor. A prototype of a novel class of nitric oxide reductase was discovered by Cramm in 1997. This single-component enzyme, which suggests to be the homologue of the NorB subunit of cNor, accepts electrons from quinols. So it is referred as qNor, encoded by qnorB gene.In this present study, a cnorZB-specific PCR primer set was developed to test the native denitrifers from environmental samples. 17 cloned cnorB PCR products were screened by RFLP, and 5 clones were sequenced and aligned finally. Sequences were explored in EMBL and NCBI with the Blast and Fastx program. And a phylogenetic tree was drawn by Emboss and Clustal program as a result.