Cloning Of Key Genes Of Phosphate Metabolism And Research Of Construction Of Engineering Strain With High Capability Of Accumulating Polyphosphate

In natural conditions the phosphorus concentration in water is balanced. However, if the input of phosphorus to waters is higher than can be assimilated by a population of living organisms, the problem of phosphorus surplus occurs, which is the most important factor of eutrophication. The human production and daily life is irreversibly speeding up the process of such excess phosphorus content, which caused a series of ecological disasters. When the global water scarcity and water quality deterioration have been a serious threat to the survival of all humankind, efficient and low-cost biological phosphorus removal methods has become a global research hotspots.Sinorhizobium NP1 which was isolated and purified by our laboratory had been used as the original strain to do the research of clone of key genes of phosphorus metabolism and construction of engineering strains. There were systemic exploratory researches of biological phosphorus removal in this thesis.The results of physical and chemical characteristics and functions of research show that whether in high-phosphorus or low-phosphorus medium and whether in 30 or 10 temperature, Sinorhizobium NP1 is of good efficiency of phosphorus removal(up to 81.2%). Sinorhizobium NP1 is an excellent phosphate accumulating bacteria.By homologous cloning, NP1 ppk was obtained by PCR, it is identical to that of stain 1021 at 86% level, and similarity of its PPK is 93%. Tertiary structure prediction indicates that NP1 PPK is similar with Escherichia coli PPK monomer. Analysis by SignalP, NP1 PPK has a signal peptide sequence. Analysis by DAS, NP1 PPK has several transmembrane segments.By homologous cloning, NP1 ppx was obtained by PCR, it is identical to that of stain 1021 at 82% level, and similarity of its PPK is 87%. Tertiary structure prediction indicates that NP1 PPX is similar with Aquifex aeolicus PPX monomer.The NP1 ppx gene knockout plasmid pJQ200SK-ppx-Tc was constructed. By electroporation transformation, pJQ200SK-ppx-Tc was transformed into NP1, then screening recombinant mutant on sucrose selective medium plates. The results of PCR and RT-PCR proved that the recombinant mutant occurred double crossover of ppx and its ppx gene was replaced by ppx::Tc, which is named NP1::ppx.The capacity of phosphorus removal of NP1::ppx is significantly higher than that of NP1. Especially under conditions of low phosphorus and low temperature, NP1::ppx exhibit excellent capability of phosphorus removal, which increases by 8.2% compared to NP1; polyphosphate content in NP1::ppx increases by 25.4% compared to NP1.