Prokaryotic Expression Of 2-Cys Prx Gene And Gpx Gene From Ditylenchus Destructor And The Establishment Of 2-Cys PRX Inhibitor In Vitro High-throughput Screening System

Crop losses caused by plant parasitic nematodes is about $ 125 billion per year in the world. At present, the main methods of preventing and controlling nematode are through the cultivation techniques, crop rotation, selection of resistant varieties and combined with some chemical which are still used today.Although these chemicals nematicides have positive effects on nematode control, but there are few varieties of nematode for nematicides in China now. the research and development of nematicides, both theoretical research and technology development, are still very weak in the field of pesticide. Because of environmental pressure, the development of new, low toxicity and high efficiency of nematode nematicides is the inevitable trend. The deep-going research on the mechanism of antioxidant system in plant nematode provides a theoretical basis and a new starting point for the development and utilization of drug target.1.In this paper, we used the Ditylenchus destructoras as the research object, a glutathione peroxidase gene was cloned and The prokaryotic expression vector pET-41b-DdGpx was constructed and then transformed into BL21(DE3). The expression conditions were optimized including optimal the concentration of IPTG and temperature. SDS-PAGE and Mass spectrum analysis indicated that the recombinant protein of Dd-GPX were successfully expressed. There were 7 peptides matched with D. destructor GPX protein and molecular weight of expressed fusion protein was 60 kD. IPTG concentrations cannot affect the expression of the Dd-GPX, but the temperature had a significant effect on the expression of the proteins. Dd-GPX recombinant proteins expressed in soluble form at 18?, the expression of the part soluble protein at 25 ?, the entire expression in inclusion bodiesat 37?;2. a 2-Cys PRX gene was cloned and the prokaryotic expression vector pET-41b-DdPrx was constructed and then transformed into BL21(DE3). The expression conditions were optimized including optimal the concentration of IPTG and temperature. SDS-PAGE and Mass spectrum analysis indicated that the recombinant protein of Dd-PRX was successfully expressed. There were 2 peptides matched with D. destructor PRX protein and molecular weight of expressed fusion protein was 55 kD. IPTG concentrations cannot affect the expression of the Dd-PRX, but the temperature had a significant effect on the expression of the proteins, the recombinant Dd-PRX protein expressed in soluble form at 18? and 25 ?, the soluble protein expression partly at 37 ?.3. Analysis of the recombinant Dd-PRX protein by enzyme kinetics,used ferric thiocyanate method.Optimization and set up 2-Cys PRX inhibitors in vitro high-throughput screening system. Enzyme kinetics analysis shows that, the recombinant Dd-PRX protein not only has a high affinity for H2O2, but also has a strong ability to remove H2O2.The Michaelis constant, catalytic constant and catalytic efficiency were 22.25 mol/L,1315.41s-1 and 5.91 × 107.4. Through the known to the PRX has inhibitory activity of ConoidinA on the screening system for in vitro and in vivo testing.The results showed that, the inhibition rate of Dd-PRX were 22%-41% at the ConoidinA concentrations were 11.25?mol/L ?180?mol/L,while the control 2,3-bis(bromomethyl) quinoxaline had inhibition rate of 9%-29% at the same concentrations, the differences between the two inhibitory effectt were obviously;In vivo testing showed that Conoidin A exhibit increased inhibitory effects trend with the concentration increasing. Which after six days treatment, the inhibition effect of 50?mol/L Conoidin A on Meloidogyne graminicola reached to 64.8%, after 12days, the rate dropped to 29.0%. While under the same conditions, the inhibitory rate of 2,3-bis(bromomethyl) quinoxaline were only 33.0% and 4.7%,significantly lower than the equivalent dose Conoidin A.In summary, this study had constructed pET-41b-Prx and pET-41b-Gpx expression plasmid and established the method of preparation and purification of 2-Cys PRX protein in Escherichia coli and established the 2-Cys PRX inhibitor in vitro high-throughput screening system based on 2-Cys PRX as the targets which can be used for screening inhibitors of plant nematode infection.The screening system laid the foundation for the further development of new nematicides.