Cloning and expression of N2O reductase structural gene nosZ

摘要

Denitrification is the geochemical process to reduce nitrate, the main form of nitrogen contaminants in groundwater, into gaseous nitrogen. N2O reductase is the enzyme of the final step of biological denitrification. Expression of nosZ gene can lead to the formation of the N2O reductase holoenzyme. This study describes the cloning and expression of nosZ. Genomic DNA was extracted from Pseudomonas. stutzeri HS-03 as the template for PCR, and the PCR product was inserted into plasmid pMD18-T.The DNA fragment containing nosZ was subcloned into expression vector pET28. The recombinant plasmid containing the open reading frame of nosZ was expressed in Escherichia coli BL21. SDS-PAGE showed that the expression product was about 21 KD, meaning the target gene was only partly expressed. Analysis of this gene sequence indicated that the BLAST level of similarity to nosZ sequence in GenBank was 90%. The reason for incorrect expression was that the basic group had been mutated into a terminator.