Espression,Purification And Metal-binding Properties Of Zea Mays Metallothionein 1 In E.coli

Metallothioneine（MT）as a class of metal binding proteins with low molecular weight（about 6-7kDa in molecular weight）is found in plants,animals and microorganisms which contains no histidine and aromatic amino acids except for cysteine richness and can be metal induced to synthesize with little difference in physicochemical properties.MT plays an important role in detoxifying heavy metals,regulating intracellular balance,and maintaining the steady state of metal ion concentration.Compared with vertebrate MT,plant MT has been extensively studied due to its sequence diversity and complexity of species,and the results are remarkable.In this paper,the recombinant plasmid pGEX-6p-1-ZmMT1 was transported into the gene-defective E.coli BL21（DE3）,the prokaryotic expression system of Zea mays Metallothionein1（ZmMT1）was constructed by means of genetic engineering and high-purity expression of ZmMT1 is achieved outside the plant.On this basis,the mechanism of ZmMT1accumulation in plants with different metal ions and rare earth ions,tolerance mechanism and the interaction mechanism of ZmMT1 with different metal ions and rare earth ions outside the plant were studied by UV spectroscopy.Investigating the binding ability of different metal ions with ZmMT1 and the distinction of the binding strength,and the effect of thiol compounds on the binding properties of ZmMT1 with metal ions by DTNB oxidation rate.The full text is divided into six chapters:（1）Mainly reviews the physicochemical properties of plant MT,the classification of plant MT,the detection method of plant MT,and the function of plant MT.When discussing the function of plant MT,the role of plant MT in detoxification of heavy metals,maintaining internal environment stability,participating in gene regulation,removing active oxygen,and participating in environmental protection was focused discussion.（2）The prokaryotic expression system of ZmMT1 was constructed by means of genetic engineering and the high purity expression of ZmMT1 was achieved outside the plant.Simultaneously,the effect of Zn²⁺ion concentration,IPTG induction concentration,IPTG induction temperature,IPTG induction time and other physical and chemical factors on the expression and purification of ZmMT1 was investigated.The recombinant plasmid p GEX-6p-1-ZmMT1was finally successfully transferred into E.coli BL21（DE3）.In LB medium with a volume of 500mL,the concentration of Zn²⁺was 50μM,the final concentration of IPTG was 1mM,the temperature was 37℃,and the induction time was 3h,the large-scale,high-purity ZmMT1 was finally obtained by digesting GST-ZmMT1 on a Prescission protease column.The problem of obtaining large quantities of metallothionein outside the plant was effectively solved,which lays a solid foundation for further research on the properties of metallothionein in maize.（3）Based on the binding properties of plant MT and different metals,a comparative method was used to study the accumulation and tolerance mechanism of ZmMT1 in plants and different metal ions.Experiments show that ZmMT1 can tolerate metal ion Zn²⁺,Cd²⁺,Pb²⁺,Cu²⁺well in plants,at the same time,these metal ions can induce the production of ZmMT1.Simultaneously,the binding mechanism of ZmMT1 to different metal ions is also studied founding that ZmMT1 can bind specifically with Zn²⁺,Cd²⁺,Pb²⁺,Cu²⁺,etc.in plants.The intensity of binding of ZmMT1 to heavy metal Cd²⁺,Pb²⁺and physiological element Zn²⁺was intensively investigated by DTNB oxidation rate and the order of binding ability of ZmMT1 to different metal ions was:Cd²⁺>Pb²⁺>Zn²⁺.It provides a basis for detecting the degree of pollution in the ecological environment and effectively and timely controlling heavy metal ion pollution.（4）UV spectroscopy was used to investigate the spectrum changes caused by different metal ions titrating apo-ZmMT1 in the presence of thiol compounds.The research shows that the presence of thiol compounds will enhance the metal ion binding ability of ZmMT1 to some extent.At the same time,it is found that increasing the S^2-concentration can enhance the ability of ZmMT1 to bind to metals to a certain extent.The order of“apo-ZmMT1+10eq S^2-+xCd²⁺”can succeed in the formation of S^2-doping into thiol clusters and enhances the binding ability of metallothioneins.Therefore,the presence of thiol compounds can indeed increase the metal ion binding capacity of ZmMT1,which is not only related to the order of addition but also the concentration of sulfur ions.（5）Mainly studies the interaction mechanism of ZmMT1 with rare earth ions La³⁺and Lu³⁺in plants and out of plants.The experiments show that ZmMT1 can tolerate rare earth ions La³⁺,Lu³⁺and rare earth ions in plants can also induce ZmMT1 production,however,it does not effectively combine with rare earth ions in plants providing references for studying the correlation between metallothionein and rare earth biological effects.（6）Summarizes the research work of this thesis,highlights the innovation of this research,and lists the content of the future research work.