Screening,Cloning And Expression Of BDE-47 Aerobic Degradation Gene

Polybrominated diphenyl ethers(PBDEs)are widely used flame retardants that cause great harm to human health and the ecological environment.In this study,BDE-47 was used as the target pollutant,and a bacterial strain,Acinetobacter pittii GB-2,which was screened by the research group to degrade BDE-47 efficiently,was used as the experimental strain to study its growth and degradation.Characterization,analysis of the products generated during the degradation of BDE-47 and deduction of effective pathways,analysis of significantly up-regulated genes,screening of degraded genes and verification of their functions by cloning and expression.The main research results are as follows:(1)Study the growth and degradation characteristics of degrading bacteria GB-2:when the p H is 6.0?8.0,the temperature is 25?35?,the BDE-47 concentration is50?200 ?g/L,and the inoculum amount is 1?5%,Bacterial activity is strong and growing well.The optimum growth conditions of GB-2 were: 30?,p H 6.0,initial concentration of BDE-47 was 100 ?g/L,and the inoculum amount was 5%;GB-2degrading bacteria were at 30?,p H was 6.0,and the inoculum amount was 3%,500?g/L BDE-47 degradation system has a significant degradation effect.(2)In the process of GB-2 degrading BDE-47,three hydroxylated products and two bromophenols were detected.According to the detection results,GB-2 degraded BDE-47 basically in three ways: hydroxylation,debromination and ether.The key is broken.This process is dominated by hydroxylation,and it is deduced that the1,2-dioxygenase involved in the hydroxylation is the key enzyme in the reaction.(3)By analyzing the differentially expressed genes in the process of GB-2degradation of BDE-47,we speculated a pathway of GB-2 degradation and transformation of BDE-47.Under the action of transmembrane proteins,BDE-47 enters the bacterial cell through the cell membrane;undergoes hydroxylation,reductive dehydration,and a series of oxidoreductases such as 1,2-dioxygenase,phenol hydroxylase,and monooxygenase.The catabolic process of bromine and ring-opening mineralization;the up-regulated expression of genes such as ribosomal proteins,two-component systems and ATP transporters helps the bacteria to respond to pollutant stress,thereby protecting cells and avoiding death.(4)Combining the functions of GB-2 degradation pathway and differentially expressed genes,it was speculated that 1,2-dioxygenase Ant A was the functional gene of the initial step of degradation.In this study,the PMD-18 T vector was used to construct the cloning vector of the degradation gene,which was verified by colony PCR.It was introduced into Escherichia coli BL21(DE3)by heat shock transformation.The degradation rate of BDE-47 can reach 52.6%,which lays a theoretical foundation for the subsequent construction of genetically engineered bacteria.