Study On The Gene Cloning Of Ferredoxins Fd? And Fd? From Acidithiobacillus Ferrooxidans And The Heavy Metal Resistance

Acidithiobacillus ferrooxidans(A.f)is a kind of microorganism widely applicated in industry.Due to its excellent resistances to several heavy metals,A.f can be used for the extraction of precious metals and the control of environmental pollution.Widely existed in living organisms and participating in the important metabolic process of A.f such as iron sulfur cluster assembly,it is of great significance to study the structure and function of ferredoxins to understand the metabolic process,electron transport and heavy metal resistances of A.f.The contents and results of the research were as follows:(1)A total of four strains of A.f and a strain of leptospirillum ferrooxidans(L.f)were isolated from the samples collected from three typical ore district in Lanzhou city,Baiyin city and Longnan city of Gansu Province,and BYSW1 was selected to carry in following study because of short growth cycle and high enrichment of bacteria.The growth conditions were optimized by single factor experiments,including temperature,pH value,inoculum proportion and energy substrate types.Results showed that: the optimum growth conditions of BYSW1 were as follows: at pH 2.0,30 ? and inoculum proportion of 10%.The glucose and peptone can significantly inhibit the growth of A.f.(2)The sequences of ferredoxins genes(fd I,fd II)were collected from NCBI database,then amplified in BYSW1 and ligated to pET-32(a).The recombinant vector was transformed into Escherichia coli(E.Coli)DH5?.The sequence identity between the amplified gene of fd I,fd II and that from the sequence database were respectively 83.92% and 85.59%,and encoding protein sequence database were 89.47% and 85.59%,respectively.(3)Recombinant plasmids were transformed into express hosts E.coli BL21(DE3).Single factor experiments regarding culture temperature,culture duration and IPTG concentration were carried out to optimize the expression conditions of recombinant proteins.The recombinant proteins chromatography were validated by Western Blotwere,which were purified by nickel ion affinity column.Fd? and Fd? were separately induced by 0.5 mM IPTG at 25 ? for 6 h,0.3 mM IPTG at 25 ? for 6 h to achieve the best expression.Fd I and Fd II were eluted with 100 m M and 50 mM imidazole from affinity column,respectively.(4)Five kinds of heavy metals(Cd2+,Cu2+,Mn2+,Ni2+,Zn2+)were selected in the heavy metal resistance study.The changes of cell morphology and genome of A.f,the expression of recombinant proteins,the growth of host cells were respectively studied before and after adaptation in the presence of heavy metals(Cu2+ and Cd2+).The result showed that: BYSW1 was of excellent resistance to heavy metals,and good growth was observed at 0.04 mol/L Cd2+,0.04 mol/L Cu2+,0.15 mol/L Mn2+,0.08 mol/L Ni2+and 0.08 mol/L Zn2+,respectively.However,the growth conditions of BYSW1 was significantly inhibited by high Cd2+ and Cu2+(>0.04 mol/L).Under different concentrations of Cu2+ and Cd2+ stress,the BYSW1 cellswere noticed to be smaller and the surface was more rough,while the genome of A.f remains integrity.The expressions of ferredoxins were significantly different before and after cultivated in the presence of different concentrations of heavy metals,meanwhile,the harmful effect of Cd2+on A.f was higher than that of Cu2+.Different concentrations and different kinds of heavy metals have different effects on the growth of host cells,and 0.25 mM of Cd2+ and Cu2+ could significantly inhibit the growth of host cells.