Isolation And Degradation Function Of An Endophytic Bacteria Degrading Di-N-Butyl Phthalate

With the rapid development of industry,phthalates?PAEs?are widely usedas plasticizers,causing serious environmental pollution and endangering human health.Di-n-butyl phthalate?DBP?is an important member of PAEs.DBP enriched in soil is easily absorbed by plants and affects the growth of crops.In this study,the effects of DBP on plant stress were studied by studying the growth of plants and the difference in antioxidant enzyme activities after transplanting Brassica rapa var.chinensis on DBP contaminated soil.A plant endophyte capable of degrading DBP was isolated from the sorrel plants grown on DBP contaminated soil and named HB-T2.The strains were identified by physiological and biochemical tests and16SrDNA analysis,and their growth characteristics,degradation characteristics and influencing factors of DBP,and broad-spectrum studies of substrates of other family members of PAEs were studied.Preliminary exploration of the metabolic pathways of strains degrading DBP.After sequencing the whole genome of HB-T2,the esterase gene of DBP was degraded by this strain.The prokaryotic expression was used to study the degradation characteristics of other family members of DBP and PAEs.Colonization of HB-T2 into leafy vegetables Brassica rapa var.chinensis,study the colonization of the strain and the residue of DBP in Shanghai Green.The effect of the strain on the stress resistance of the plant was studied by detecting the change of antioxidant enzyme activity in Brassica rapa var.chinensis.The main findings are as follows:1.An endophytic bacterium capable of growing with DBP as the sole carbon source was isolated and purified from the sorrel plant.The similarity with Bacillus subtilis was 99%by physiological and biochemical tests,16SrDNA sequence and phylogenetic tree analysis.This strain was named HB-T2.The optimum conditions for degradation of DBP by HB-T2 in vitro were 35?,pH was 8.0,initial DBP concentration was 10 mg/L,and bacterial inoculation was 2%.Degradation efficiency of 6h degraded DBP reached 98.9%.The pathway for the degradation of DBP by this strain is DBP to monobutyl phthalate?MBP?to phthalic acid?PA?to protocatechuic acid?PCA?and eventually mineralized.A broad-spectrum study of the substrate found that HB-T2 has good degradation ability for most members of the PAEs family.Among them,tolyl phthalate?BBP?,dipentyl phthalate?DPP?,dimethoxyethyl phthalate?DMEP?,diisobutyl phthalate?DIBP?The degradation effect is very good.2.In this chapter,the genome-wide sketches of HB-T2 were sequenced,and the functions of each gene were annotated.At the same time as NCBI,the literature was selected to select multiple esterase genes for homology analysis,and finally five high homologous genes were screened.After the prokaryotic expression of these 5genes in vitro,DBP substrate was used to successfully screen the esterase gene with PAEs degradation ability and named EST-3.At the same time,it was found that the protein has good degradation characteristics for other members of the PAEs family,which indicates that the protein has high research value.3.DBP was artificially added to the soil,and endophytic HB-T2 was colonized into Shanghai green and transplanted into soil containing DBP for 21days.Determination of DBP in Brassica rapa var.chinensis by Liquid chromatography tandem mass spectrometry?LC-MS?with no colony as control group.Under DBP stress,DBP was found in roots and stems and leaves,and the root content was higher.The root length and biomass of the plants were significantly reduced by DBP stress,and there was no significant difference in chlorophyll content;the plant antioxidant system was also affected.Hydrogen peroxide?H₂O₂?,peroxidase?POD?and catalase?CAT?activities were significantly increased,and there was no significant difference in superoxide dismutase?SOD?and plant malondialdehyde?MDA?content.After inoculation treatment,it was found that inoculation of HB-T2 not only can degrade DBP content in plants,but also enhance the ability of plants to resist stress,and also improve the growth of plants.It shows that HB-T2 has established a good mutual symbiotic relationship with Brassica rapa var.chinensis.It is a multifunctional endophytic degrading bacteria that can cooperate with crops to avoid PAEs pollution.