موتاسيون زايي هدايت شده در توالي تيوردوكسين نوعTrx h)h ﴾ از گياه برنج و اثر آن بر فعاليت كينتيكي

چكيده انگليسي :

Site directed mutagenesis of rice thioredoxin h sequence and its impact on the kinetic activity Mitra Roodgar Nashta m roodgarnashta@ag iut ac ir Date of submission 2012 16 10 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 M Mohammad beigi Ph D Assist Professor Advisor majid beigi@eng ui ac ir Abstract Thioredoxins Trxs are ubiquitous low molecular mass proteins that are characterized by the resence of p an exposed active site with the amino acid sequence of WC G P PC Trxs participate n numerous redox i processes via the reversible disulfide dithiol reduction reaction involving ctive site cysteines Plants have a multiple isoforms of Trx as opposed to animals bacteria fungi he multiple Trx isoforms in plants are T classified on the basis of both their primary structures and ubcellular localization Trxs f m x and y are s found in the chloroplasts Trx o is localized in itochondria and Trx h isoforms are distributed in multiple m cell compartments Multiple h type rx are present in higher plants In the genome of rice Oryza sativa T nine genes encoding Trx h ave been identified A multiple alignment between these isoforms h demonstrated some emarkable differences between OsTrx20 with other eight isoforms The active site r sequence of hese eight isoforms corresponds to classical Trx active site motif WCG PPC whereas t Gly Pro as replaced with Thr in the active site of OsTrx20 In addition in the close of active site a w onserved Asp is present in Trx h isoforms which has been replaced with a positive charge amino cid c a Lys in OsTrx20 In the present study to understand the critical role of conserved Gly and sp on the A activity of Trx h Thr and Lys was replaced with Gly and Asp in OsTrx20 espectively and Gly41 was 55 48 r replaced with Thr in OsTrx23 using site directed mutagenesis ethod For this aim on the basis of this m method the reactions of PCR was performed using ET 15b carrying the gene encoding the wild type of p OsTrx20 as starting material The PCR product as transferred to DH5 after treatment with DpnI After w confirmation of mutagenesis using equencing or digestion with a marker restriction enzyme the plasmid s containing mutant genes ere transferred to Rosetta DE3 The mutant recombinant proteins his6 w OsTrxh20 T55G sTrx20 K48D OsTrx20 T55G K48D and OsTrx23 G41T were found in the soluble fraction of O the E coli ransformant culture after induction with isopropyl b D thiogalactopyranoside IPTG The t ecombinant proteins in the supernatant were purified by affinity chromatography using His rap r T columns The recombinant Trxh mutant isoforms as well as wild types were analyzed in ifferent pH for d their ability to reduce Insulin a general substrate for Trxs In addition the bility of reduction of the a mutants by OsNTR1 and glutathione was tested and was compared ith wild types The results show that w the activity OsTrx20 wt is not stable in different pH owever the mutants OsTrxh20 T55G OsTrx20 K48D H OsTrx20 T55G K48D showed pH stability ccordingly the replacement of Gly41 with Thr in OsTrx23 result A in loss of OsTrx23 pH stability In ddition the results show that the interaction of OsNTR1 with a OsTrx20 was slightly improved by eplacement of Thr55 with Gly and Lys48 with Asp Accordingly the r interaction of OsNTR1 with OsTrx23 ecreased considerably by replacement of Gly41 with Thr n order d I to study the role of N terminal Cysteins in OsTrx20 each cysteins were replaced ith serine using site w directed mutagenesis The results show that whereas OsTrx20 Wt and sTrx23 Wt is present in mixture of O monomer and dimer orms the mutant OsTrx20 C3S C4S and sTrx23 C11S is dominantly in monomer f O form The activity of mutant was lmost similar to ild type These results suggest that Cys 3 Cys4 in a w 11 OsTrx20 and Cys at the N terminal of sTrx23 has a key ole in dim

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موتاسيون زايي هدايت شده در توالي تيوردوكسين نوعTrx h)h ﴾ از گياه برنج و اثر آن بر فعاليت كينتيكي

جايگاه فعال , پايداري , دايمرشدن