| Microbial combined phytoremediation technology can give full play to the respective advantages and deficiencies of plant and microbial remediation technology through the symbiotic effect of soil microorganisms and vegetation,so as to greatly improve the phytoremediation efficiency of soil pollution.These technologies have been widely used in the treatment of heavy metal contaminated soil.Bacillus thuringiensis selected in this study is a bacterium isolated from the soil of Xiangtan Manganese Mine in Hunan Province.Previous studies found that the strain not only has certain heavy metal resistance,but also promoted plant growth.In this study,the Cadmium(Cd)resistance characteristics of Bacillus thuringiensis was analyzed by FTIR analysis,SEM analysis and RNA-Seq,and the Cd resistance functional gene of the strain was screened out,and the overexpressed sensitive mutant strain was constructed,and the overexpressed Bacillus thuringiensis-Broussonetia papyrifera combined system for the remediation of Cd contaminated soil was studied.In order to provide a theoretical basis for bioremediation of heavy metal contaminated soil.The main results of the study are as follows:(1)By analyzing the growth of Bacillus thuringiensis under Cd stress,it was found that the strain had good tolerance to heavy metal Cadmium,and its tolerance concentration reached 400 mg/L.FTIR and SEM analysis showed that the functional groups(hydroxyl,carbonyl,amide,aldehyde,etc.)on the cell surface of the strain were mainly involved in the Cd2+biosorption process.After biosorption of Cd2+,the strain adapted to Cd toxicity by changing cell morphology and producing extracellular secretions,forming the main mechanism of alleviating Cd toxicity.(2)Transcriptome data of Bacillus thuringiensis in response to Cadmium stress were obtained by Illumina Hiseq sequencing technology.A total of 25,169 differentially expressed genes were annotated into 6 databases respectively.The results of GO and KEGG enrichment analysis of differentially expressed genes showed that Bacillus thuringiensis could resist heavy metal damage through a variety of ways and most of differentially expressed genes were functional genes related to transportation system,metabolism and protein synthesis.Among them,the ABC transporter differentially expressed gene ugpC,which is significantly enriched in the ABC transport pathway,is significantly up-regulated and relatively stable,and responds positively to Cd stress.Based on the experimental results,the resistance gene ugpC was finally selected as the candidate functional gene of the overexpressed strain constructed in this study.(3)The overexpressed sensitive mutant strain of resistance gene ugpC was successfully constructed by electrical transformation method and the empty plasmid vector strain was used as control.The biosorption experiments of the strains on Cadmium showed that the three strains(wild-type strain,empty plasmid vector strain and overexpressed strain)had certain resistance to Cd,and the overexpressed strain had better removal effect on Cadmium.At 4 d,the increase of Cd2+ removal rate of overexpressed strain was the largest,which was 31.86%higher than that of the control group(wild-type strain).Therefore,wild-type strain,empty plasmid vector strain and overexpressed strain were finally determined as the candidate strains for the subsequent pot experiment.(4)The pot experiment of overexpressed Bacillus thuringiensis-Broussonetia papyrifera to repair Cd contaminated soil showed that the three strains(wild-type strain,empty plasmid vector strain and overexpressed strain)had certain effects on the root structure,physiological response,enrichment and transport characteristics and soil nutrients of Broussonetia papyrifera.When the stress concentration was 500 mg/kg,the TOC content of the treatment group inoculated with overexpressed strain increased by 40.96%compared with the control group(wild-type strain),which was conducive to promoting the nutrient cycle of Broussonetia papyrifera.Cd stress has a certain inhibitory effect on the synthesis of Broussonetia papyrifera chlorophyll.Inoculation with overexpressed strain can promote the synthesis of Broussonetia papyrifera chlorophyll.When the Cd stress concentration was 50 mg/kg,the contents of chlorophyll a,chlorophyll b and total chlorophyll of the overexpressed strain were increased by 52.61%,45.16%and 50.69%respectively compared with the control group(wild-type strain).In terms of physiological response,compared with the control group(wild-type strain),the antioxidant enzyme activity after inoculating the overexpressed strain was improved.By improving the antioxidant enzyme activity,it actively participated in the process of oxidative stress,cleared the excess reactive oxygen free radicals in Broussonetia papyrifera,and reduced the toxic effect of Cd stress on Broussonetia papyrifera.In terms of heavy metal enrichment and transport,inoculation with overexpressed strain reduced the enrichment of heavy metal Cd in Broussonetia papyrifera,and improved the ability of Broussonetia papyrifera to transfer Cd from the root to the aboveground part.With the increase of Cd stress concentration,the transport coefficient of the treatment group inoculated with overexpressed strain increased by 75.65%and 47.76%respectively compared with the control group(wild-type strains).The overexpressed strain can reduce the toxicity of Cd by improving the ability of Cd transfer from the root of Broussonetia papyrifera to the aboveground part.The results further illustrate the potential of Bacillus thuringiensis-Broussonetia papyrifera combined remediation system for the remediation of heavy metal contaminated soil. |
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