تاثير بيان دگر ساخت ايزوفرم هاي كلاس I وII متالوتيونين برنج ﴿Oryza sativa﴾ بر روي تحمل باكتري Esherichia coli به فلزات سنگين

چكيده انگليسي :

The Effect of Heterologous Expression of Rice Oryza sativa Class I and Class II Metallothionein Isoforms on Metal Tolerance of Esherichia coli Rezvan Mohammadi Nezhad Rezvan m 1365@yahoo com Date of submission January 16 2012 Department of Agricultural Biotechonolgy Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi 1 A Mirlohi Ph D Proffesor Supervisor mirlohi@cc iut ac ir 2 A Shahpiri Ph D Assist Professor Supervisor a shahpiri@cc iut ac irAbstractThe contamination of heavy metals either by natural sources or industrial activities has brought significant deleteriousconsequences to the environment and organisms health To balance the concentration of toxic metals in the cells allorganisms possess different potential cellular mechanisms such as synthesis of metal binding peptides and proteins Metallothioneins MTs are a superfamily of small molecular weight 10 KDa and Cys rich proteins that can bindheavy metals via the thiol groups of their Cys residues A total of 13 genes encoding putative MT proteins have beenidentified in rice Oryza sativa which are classified into two major classes on the basis of Cys distribution in theiramino acid sequences Despite the plenty of studies on the expression of genes encoding MT isoforms the knowledgeis still lags behind on the individual function of each isoform at protein level The aim of this study was to clarify thespecific function of class I and II MT isoforms from rice in response to heavy metals as well as oxidative stress Thecoding sequence of OsMTI 1b and OsMTII 1a genes which belong to class I and II of rice MTs respectively weresynthesized in pUC57 coloning vector and after cleavage by appropriate restriction enzymes the inserts were ligatedinto pET41a plasmid The resulting constructs were transferred into expression host Escherichia coli Rosetta DE3 The considerable amount of recombinant form of GST GST OsMTI 1b and GST OsMTII 1a proteins were producedfrom cells harboring pET41a control pET41a OsMTI 1b and pET41a OsMTII 1a plasmids respectively afterinduction with Isopropyl D thiogalactopyranoside IPTG The tolerance of recombinant strains to ZnSO4 CdCl2 CuSO4 and NiCl2 salts was compared to control by plotting their growth curve in addition to determination of theamount of accumulated Zn 2 Cd 2 Cu 2 and Ni 2 ions in bacterial cells and culture medium using inductively coupledplasma atomic emission spectroscopy ICP AES The metal binding ability of purified recombinant proteins metalated both in vivo and in vitro was evaluated using Elman reagent The spectroscopic characteristics and metalcontent of in vitro metalated recombinant proteins were compared with control Whereas the tolerance of bacteriaexpressing GST OsMTI 1b protein increased to Cd Ni and Zn through sequestering more metal ions in bacterialcells the tolerance of cells expressing GST OsMTII 1a protein increased slightly only to Ni and Zn ions None of theMT isoforms confer tolerance to Cu From reaction with Elman reagent the metal binding affinity for GST OsMTI 1b protein was indicated as follows Cd Ni Zn Cu In addition our results showed that GST OsMTI 1b traps Ni Cd and Zn with a stochiometric ratio of 6 25 4 8 and 4 96 respectively To examine the ability of two isoforms forscavenging reactive oxygen species ROS the growth curve of recombinant bacteria were monitored in the mediasupplemented with hydrogen peroxide H2O2 or ethanol Expression of both GST OsMTI 1b and GST OsMTII 1aproteins increased the growth of recombinant bacteria in the presence of H2O2 while they had no significant effecttoward ethanol stress In summary the results presented here provided evidences indicating functional discriminationbetween class I and II isoforms which may reflect their different roles in rice cells Keyword Rice Metallothionein Heavy Metals Protein Expression Cloning Oxidative Stress

محمدي نژاد، رضوان

تاثير بيان دگر ساخت ايزوفرم هاي كلاس I وII متالوتيونين برنج ﴿Oryza sativa﴾ بر روي تحمل باكتري Esherichia coli به فلزات سنگين

همسانه سازي , بيان پروتئين , تنش اكسيداتيو