Cloning And Functional Identification Of Key Enzyme Of Denitrification And Construction Of Strain With High Capability Of Denitrification

Sinorhizobium sp. NP1,which was isolated and purified by our lab,was used as the original strain to obtain the full-length ammonia monooxygenase gene and nitrite reductase gene by homology clone and Tail-PCR methods. The amo gene is 1089 bp,encodes 362 amino acids and the secondary structure is similar with that of Sinorhizobium meliloti 1021. The AMO has one signal peptide sequence, position of mature protein is between 37-38 of the VLA-SP and nine transmembrane regions.The nir gene is 1137 bp,encodes 378 amino acids and the tertiary structure is similar with that of Achromobacter cycloclastes. The NIR has one signal peptide sequences and one transmembrane region in the first 50 amino acids. And the mature protein position is between 33-34 of AHA-AG.The NP1 amo gene knockout plasmid named pJQ200SK-amo-Tc was constructed by the use of pJQ200SK plasmid, transformed into the original strain NP1. And then obtained the amo gene knockout strain named NP1::amo by the use of three parents hybridization method. The denitrification capacity of NP1::amo decreases about 35% compared to NP1 by Berthelot test. These results show that the amo gene is the key one in the nitrogen removal genes.Constitutive promoter dld was used to enhance the expression of amo genes, plasmid p5:dld-amo was constructed by the use of pBBR1MCS-5 plasmid. And then were transformed into the original strain NP1 using three parents hybridization to obtain the amo gene knockout strain NP1::dld-amo.The denitrification capacity of NP1::dld-amo increases about 10% compared to NP1 by Berthelot test. And the plasmid stability test showed that NP1::dld-amo can stably exist in NP1 after 12 generations of propagation.Cloning of gfp gene and connect with dld-amo to express fusion protein, plasmid named p5:dld-amo-gfp was constructed by the use of pBBR1MCS-5 plasmid. And then were transformed into E.coli to discover the right location of AMO. We can see faint green fluorescence under fluorescence microscope. This result indicates that AMO is membrane protein.