Study On The Induction Of Metallothionein In Crassostrea Plicatul

Metallothionein?MT?,ranging from bacterium to mammals,is a c lass of low molecular weight,high sulfhydryl contents and metal-binding protein,which plays important roles in detoxifying heavy metals,scavenging free radicals,fighting against ionizing radiation,apoptosis resistance and strengthening the bad state o f adaptive capacity and so on.MT owns broad market prospect in medicine,environmental monitoring,health care,cosmetics and other related fields.Yet the complex production process,low yield and expensive price of MT purified from rabbit liver in local market have become the bottleneck restricting the development of MT industry seriously.In this study,Crassostrea plicatul were used as materials to investigate MT expression under the single and combined metal exposure pathway based on the 96 h acute to xicity experiment,and we would get the optimum induction conditions.The optimum extraction and separation method was established to obtain the purity oyster MT.The main results are as follows:1.Individual toxicity of zinc sulfate?ZnSO₄·7H₂O?,cadmium chloride?CdCl₂·2.5H₂O?,cupric acetate?Cu?CH₃COO?₂·H₂O?,potassium dichromate?K₂Cr₂O₇?,sodium hydrogen selenite?Na₂SeO₄?to Crassostrea plicatul with a semi-static test method was addressed under 18±1?,salinity 26²8,pH 8.0±0.2.The result showed that the median lethal concentrations(LC₅₀)of Zn²⁺,Cd²⁺,Cu²⁺,Cr⁶⁺,Se⁴⁺for 96 h to Crassostrea plicatul were 2.59?4.10?8.83?81.84?378.06 mg/L.2.Based on the acute toxicity results,Crassostrea plicatul MT was determined at 1,2,3,4,5 day after Zn²⁺,Cd²⁺,Cu²⁺,Se⁴⁺,Cr⁶⁺induction which was individually selected 1/16,1/8,1/4,1/2 of 96 h-LC₅₀ as concentration gradients.The result showed that Cd²⁺and Se⁴⁺exposure can increase MT synthesis,with obviously time-response and dose-response.Under exposure to 0.5mg/L Cd²⁺,MT content was increased to 0.165 mg/g wet weight,106.3%inductivity and reached the highest level,and to 0.103 mg/g wet weight,39.2%inductivity under exposure to 1mg/L Se⁴⁺.3.Based on single induction results and security,significantly induced dose levels,uniform test method was adopted to design the concentration gradients.The MT expression induced under combined metal exposure pathway within 5 days was then investigated and the best conditions?element species,concentration,temperature?for Crassostrea plicatul MT induction was also selected.The result showed that MT could be expressed in short time under combined metal exposure pathway.However,the absorption and metabolism channel between each metal would be affected due to the interaction between the metals.Therefore,MT concentration varies so fluctuant with time and dose that is not conducive to the induced-progress implementation and management.Zn²⁺-Cd²⁺and Se⁴⁺-Cd²⁺exposure can increase MT synthesis under certain limit.The levels of MT reached to the highest point 0.203 mg/g wet weight at 0.2 mg/L Se combined with 0.5 mg/L Cd²⁺under 20?.4.Tris-HCl as buffer extract,the content of MT of per gram oyster tissue as response value,four most influenced factors on the response value?pH,temperature,buffer concentration,material ratio?was selected through the single factor test.Then optimization extraction conditions were determined on the basis of three levels in the four factors in the response surface test.The optimum extraction conditions:pH 9.0,T=30?,concentration of extract 0.25 mol/L,material ratio 1:4.5.Static adsorption and dynamic adsorption method were combined to study the nine different types of ion exchange chromatography resins upon MT purification capacity.The purification of the process conditions was determined at last.