多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrumsp.mp-4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS-5载体上,构建重组质粒pTPD,在辅助质粒pRK2013的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putidaKT2440中,获得的工程菌PseudomonasputidaKT2440-DOP可以降解多种有机磷农药及芳香烃化合物;KT2440-DOP的有机磷水解酶活较出发菌株MP-4提高了一倍左右,且遗传性状稳定。

Construction of a versatile degrading bacteria Pseudomonas putida KT2440-DOP and its degrading characteristics

Triazophos is a kind of organophosphorous pesticide which was widely used by farmers all over the world in 1990's.It is effective in controlling pesticide but harmful to human beings.Bioremediation is an effective and economic method to treat environment that has been polluted by hazardous organic compounds,so researchers paid much attention in this area.Most of which focused on isolating functional bacteria,studying its degrading mechanism,and cloning degradation-related genes.MP-4 was isolated from soil polluted by Triazophos for a long time and identified as Ochrobactrum sp..The triazophos hydrolase(tpd) gene was cloned by the method of shotgun cloning,and the sequences were determined and analyzed.In the former tests it was found that there was only 18 base pairs different in tpd gene from mpd gene,which was isolated from methyl parathion degrading strain Pseudomonas putida DLL-1.Enzyme TPD can hydrolyze triazophos and methyl parathion while MPD cannot hydrolyze triazophos.Pseudomonas putida KT2440 is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes.This bacterium is known for its diverse metabolism and potential for development of biopesticides and plant growth promoters because of its ability to colonize rhizosphere of crop plants.Tpd gene was isolated from the genomic DNA of Ochrobactrum sp.MP-4 by PCR amplification.Recombinant plasmids pTPD was constructed by ligating tpd gene into broad host vector pBBRMCS-5.With the help of plasmid pRK2013,pTPD was transferred into Pseudomonas putida KT2440 to construct KT2440-DOP.KT2440-DOP can degrade many organophosphate pesticides and aromatics compounds.The specific activity of organophosphate hydrolase of KT2440-DOP was approximately 2 times of MP-4.Later,parameters affecting bioremediation of Organophosphate pesticide in soil using KT2440-DOP will be studied.