Study On The Metabolism Of Cytochrome P450to Organic Pollutants By Transgenic Technology

Cytochrome P450(CYP) enzymes catalyze oxidative metabolism of a wide array of endogenous and exogenous compounds, including many environmental contaminants. They are widely distributed in various organisms including animals, plants and microorganisms. At present in vitro protein expression and transgenic technology are widely applied in the research of metabolism of CYPs to organic pollutants.Three parts contained in this thesis:(1) house fly cytochrome P4506A1(CYP6A1) was expressed in E.coli to study its metabolic ability to aldrin and the influence of human NADPH-P450reductase (CPR) on the catalytic activity of CYP6A1;(2) the binary expression vector of human cytochrome P4501A2(cypla2) was constructed and transformed into rice Nipponbare. The positive To transgenic Nipponbare;(3) Regenerated transgenic tobacco plants with rat cytochrome P4501A1were examined for tolerance and metabolism to benz[a]pyrene by HPLC and HPLC-MS. The main results are as follows:(1) House fly cytochrome P4506A1(CYP6A1) or both house fly cytochrome P4506Aland human NADPH-P450reductase (CPR) was expressed in Escherichia coli cells to study the influence of CPR enzyme on the catalytic activity of CYP6A1and the biodegradation of the recombinant cells to the aldrin. The results showed that CYP6A1had epoxidation effect on aldrin and CPR enzyme played a key role in the catalytic activity of CYP6A1. When5μM aldrin was added to the recombinant E. coli cell culture,163.36nmol/L dieldrin (the epoxidation production of aldrin) was detected in8h, which indicated the prokaryotic cells expressing house fly cytochrome P4506A1could be applied to the bioremediation of aldrin-contaminated soil.(2) Human cytochrome P4501A2gene have first cloned into the binary expression vector pCAMBIA1302, then transformed into rice Nipponbare by using Agrobacterium-mediated plant transformation. The positive transgenic plant have characterized by PCR analysis.(3) Transgenic rat cytochrome P4501A1tobacco, wild-type tobacco and binary vector without P4501A1tobacco were cultured on the water medium which contains50μM benz[a]pyrene. After5days the amount of benz[a]pyrene and its metabolic products in the root, stem, leave of tobaccos and the culture residue were analyzed by HPLC and HPLC-MS. The results indicated that the possibility of the transgenictobacco plants for tolerance and metabolism to benz[a]pyrene.