Bioremediation Of Isoproturon-Contaminated Soil By Combinational Transgenic Soybean(Demethylase Gene PdmAB)-Microbe

Isoproturon（IPU）is widely used in agricultural production as a representative substituted urea herbicides.Due to its long-term extensive use and long half-life in the soil,the hazards to the environment are constantly exposed,so the removal of residual IPU in soil has attracted increasing attention.The N-demethylation product1-（4-isopropylphenyl）-3-methylurea（N-（4-isopropylphenyl）-N-methylurea,MDIPU）is the main degradation product of isoproturon,and the N-demethylation is also the rate-limiting step of IPU degradation.Sphingobium sp.YBL2 was an efficient isoproturon-degrading strain isolated in our previous study,and the gene pdmAB responsible for N-demethylation was cloned.Moreover,pdmAB could be used to construct herbicide-resistant transgenic crops and confirm its potential for transgenic phytoremediation of contaminated soil.This paper studied the effects of IPU and its N-demethylation product MDIPU on plant physiological and biochemical indexes and soil enzyme activity,and compared the ecological toxicity of IPU and MDIPU on plants and soil.At the same time,by comparing the tolerance of pdmAB transgenic soybean and its wild type to IPU,the characteristics of resistance of transgenic soybean to isoproturon toxicity were explored.Through pot experiment,the synergistic relationship between plants and rhizosphere inoculated MDIPU degrading strain Sphingobium sp.1017-1（pdmAB inactivation mutant of strain YBL2）was used to study the removal effect of pdmAB transgenic soybean combined with microbial degrading strain 1017-1 on isoproturon in soil.Finally,Mi Seq sequencing technology was used to further study the diversity and abundance of indigenous bacterial communities in soil under different remediation treatments,and the changes of bacterial community structure in soybean rhizosphere soil were analyzed.The above studies provide theoretical and application basis for the combined remediation of transgenic plants and microorganisms in soil contaminated by phenylurea herbicides.1.Ecotoxicity of IPU and its N-monodemethylated product MDIPU on wheat and soilThe effects of IPU and MDIPU on wheat seedling growth,photosynthesis,physiological indexes,antioxidant system and rhizosphere soil enzyme activities were studied by pot experiment.The concentrations of IPU and MDIPU were designed to be once,twice,five times and ten times the concentration of isoproturon applied in the field.The results showed that the growth of wheat under MDIPU treatment was more vigorous than that under IPU treatment,especially under high concentration pollution.The same concentration of IPU treatment on wheat photosynthesis inhibition was significantly higher than MDIPU treatment.Under low concentrations of IPU and MDIPU stress,the contents of H₂O₂and MDA in wheat seedlings increased,which had an activation effect on the antioxidant enzyme system of wheat seedlings.When the stress concentration was too high,the antioxidant enzyme activity of wheat leaves decreased,indicating that the external stress had exceeded the defense level of wheat itself.On the 24th day,high concentration of IPU inhibited phosphatase activity in wheat rhizosphere soil.Urease and sucrase in wheat rhizosphere soil were sensitive to IPU and MDIPU,and the activities of urease and sucrase in MDIPU treatment were higher than those in IPU treatment.2.Bioremediation of Isoproturon-Contaminated Soil by Combinational pdmAB Transgenic Soybean-degrading strain 1017-1 RemediationThe comparative study on the tolerance of pdmAB transgenic soybean and its wild type to isoproturon showed that there was no significant difference in the growth of transgenic soybean plants and non-transgenic lines without IPU stress.However,compared with wild-type soybean,transgenic soybean showed tolerance to 0.19-1.9 mg/kg IPU.It indicated that the single demethylase gene pdmAB had great application prospects in bioremediation.1.9 mg/kg IPU was added to the soil by indoor pot experiment.The results showed that under 1.9 mg/kg IPU stress,the wild type soybean appeared chlorosis and yellowing,and the growth of transgenic soybean was in good condition.The inoculation of Sphingobium sp.1017-1 in the rhizosphere of transgenic soybean plants could alleviate the toxic effect of MDIPU on soybean and promote the growth of transgenic soybean.The natural degradation rate of IPU within 21 days was 17.06%,and the removal rate of IPU by single bioaugmentation was 70.28%.However,the presence of IPU was not detected in the combined treatment of transgenic soybean and microbial enhancement.The viable counts of pdmAB transgenic soybean roots inoculated with degrading strains was significantly higher than that of strain YBL2 in single bioaugmentation treatment,indicating that transgenic soybean plants can provide more suitable rhizosphere niche for the survival of inoculated strains,establish a mutually beneficial cooperative relationship,and improve the survival rate of strains.The dynamic changes of bacterial community in soybean rhizosphere soil were further studied by high-throughput sequencing.The results show that 1.9 mg/kg IPU had no significant effect on soil bacterial community.The treatment of soybean planting at 21 days significantly increased the richness and diversity of rhizosphere soil bacterial community.Degrading strain YBL2 or planting crops can reduce the relative abundance of Firmicutes and Chlorofexi in soil,and increase the relative abundance of Proteobacteria.It is possible that some strains in the Proteobacteria phylum can use isoproturon as carbon source for growth,while rhizosphere secretions or exogenous strains inhibit the growth of some strains in the Firmicutes and Chlorofexi phylum.In the treatment group of soybean planting,the relative abundance of MM2 was significantly increased,suggesting that transgenic plants could help Sphingobium and MM2 bacteria colonize and promote the degradation of isoproturon.