مشخصه يابي آنزيم هاي درگير در سيستم تيوردوكسين وابسته به NADPH از گياه برنج (Oryza sativa L.)

چكيده انگليسي :

Characterization of Enzymes Involved in Rice Oryza sativa L NADPH Thioredoxin System Ehsan Shaykholeslam Esfahani e shaykholeslam@ag iut ac ir Date of Submission 2013 13 03 Department of Agricultural Biotechnology Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi A Shahpiri Ph D Assist Professor Supervisor a shahpiri@cc iut ac ir A Mirlohi Ph D Professor Advisor mirlohi@eng ui ac ir Abstract An NADP thioredoxin system NTS consisting of NADPH NADP thioredoxin reductase NTR and thioredoxin h Trx h plays a posttranslational regulatory role by reducing disulfide bonds in target proteins involved in different cellular processes In contrast with prokaryotic and mammalian cells plants have a complex NTR Trx system comprising several Trx h and NTR isoforms Therefore one important question to be addressed is whether there is specificity in the interactions between different NTR and Trx h isoforms Through search in rice oryza sativa L genome database we identified nine potential genes encoding Trx h and four potential genes encoding NTR The recombinant forms of two isoforms of OsNTR OsNTR1 and OsNTR2 and three isoforms of Trx h OsTrx1 OsTrx20 and OsTrx23 were heterologously produced in E coli as His tag proteins in considerable amounts and then purifed using affinity chromatography This enabled us to analyze in vitro interaction between different isoforms of NTR and Trx h from the same organism To this end the kinetic parameters of OsNTRs towards three isoforms of OsTrx were determined using DTNB assay The results of this assay showed that in contrast to OsTrx1 and OsTrx23 the isoform OsTrx20 was not reduced by OsNTR Both OsNTR1 and OsNTR2 had five times more affinity towards OsTrx1 than OsTrx23 The amount of kcat however was almost similar towards both OsTrx isoforms These results indicate that there is specificity in interaction of NTR and Trx isofroms in NTS Furthermore to test whether OsTrx isoforms can be reduced by glutathione GSH the reduction of insulin was measured in the presence of GSH and each Trx isoform OsTrx20 was efficiently reduced by GSH indicating that glutathione system may be responsible for reduction of OsTrx20 In addition in the present study the tolerance of cells expressing recombinant proteins in response to oxidative stresses such as H2O2 and ethanol were compared with control by plotting their growth curve In comparison to OsNTR2 the expression of OsNTR1 yielded more growth in the presence of H2O2 and ethanol Moreover the strains overexpressing OsTrx23 and OsTrx1 showed higher tolerance compared to OsTrx20 in the presence of H2O2 respectively whereas the tolerance to ethanol did not differ significantly among these strains We also for first time prepared the strains which were able to co express OsNTR1 with each OsTrx isoform after induction with IPTG To this end the PCR products including promoter ribosome binding site and the gene encoding each OsTrx isoform were ligated to plasmid pET 28a containing gene OsNTR1 Therefore three constructs containing genes encoding OsNTR1 OsTrx1 OsNTR1 OsTrx20 and OsNTR1 OsTrx23 were produced and transferred to E coli The preparation of these starins enabled us to analyze the interaction of OsNTR1 with each OsTrx isoform in vivo Among these strains tolerance of strain coexpressing OsNTR1 OsTrx1 in response to H2O2 was higher than the strains expressing OsNTR1 or OsTrx1 solely This result indicates that OsNTR1 has higher interaction towards OsTrx1 in vivo which corresponds to the results of in vitro assay Key Words Rice NTR Thioredoxin System Kinetic Parameters Glutathione Oxidative Stress Co expression PDF created with pdfFactory trial version www pdffactory com

شيخ الاسلام اصفهاني، احسان

مشخصه يابي آنزيم هاي درگير در سيستم تيوردوكسين وابسته به NADPH از گياه برنج (Oryza sativa L.)

سيستم NTR/Trx , پارامترهاي كلينيكي , گلوتاتيون , تنش اكسيداتيو , بيان هم زمان