南方医科大学学报2017，Vol.37Issue(10)：1290-1295,6.DOI:10.3969/j.issn.1673-4254.2017.10.02
ChrA基因在大肠杆菌中的表达及其抗铬特性
Characterization of chromate resistance in genetically engineered Escherichia coli expressing chromate ion transporter ChrA
摘要
Abstract
Objective To construct a genetically engineered Escherichia coli expressing chromate (Cr) ion transporter ChrA and test its Cr resistance capacity. Methods ChrA gene was cloned by PCR from the DNA template of Serratia sp. S2 and linked with the prokaryotic vector pET-28a(+).The recombinant vector was transformed into E.coli BL21(DE3)cells for expression of ChrA protein. Cr (VI) risistance and Cr (VI) uptake and efflux of the engineered bacteria were tested, and the effects of Cr loading time, oxyanions (ulfate, molybdate, vanadate, tungstate), and respiratory inhibitors (valinomycin, CN-, oligomycin, and NADH) on Cr (VI) efflux were examined to analyze the pathway of Cr (VI) transport by ChrA protein. Results The engineered E.coil strain was successfully constructed.Experiments using cell suspensions showed a lowered Cr2O72-uptake but an increased efflux capacity of ChrA-engineered bacteria compared with the control strain(P<0.05).The engineered E.coil cells in exponential growth incubated for 30 min in the presence of 50 mg/L Cr2O72-showed a total displacement of Cr(VI)of 20% after resuspension in PBS at 10 min,but chromate efflux decreased subsequently as the incubation time extended.Oxyanions sulfate and molybdate significantly inhibited chromate efflux in the engineered bacteria (P<0.05), whereas tungstate and vanadate did not obviously affect chromate efflux; chromate efflux was significantly inhibited by K+ionophore valinomycin and CN-,enhanced by NADH(P<0.05),but not affected by oligomycin,suggesting the role of chromate transporter ChrA as a chemiosmotic pump that extrudes chromate using the proton-motive force.Conclusion ChrA can efficiently transport chromate ions from the cytoplasm to enhance chromate resistance of the genetically engineered E.coli.关键词
ChrA/工程菌/铬(Ⅵ)抗性/外排Key words
ChrA/engineered bacteria/chromate resistance/efflux引用本文复制引用
周思敏,董兰岚,何元,肖虹..ChrA基因在大肠杆菌中的表达及其抗铬特性[J].南方医科大学学报,2017,37(10):1290-1295,6.基金项目
国家自然科学基金(21403021) （21403021）
重庆市教委科学技术研究项目(KJ1500216) Supported by National Natural Science Foundation of China(21403021). （KJ1500216）
