جداسازي ، همسانه سازي و بيان دو ژن كد كننده تيوردوكسين ردوكتاز وابسته به NADPH از گياه برنج ﴿Oryza sativa﴾

چكيده انگليسي :

Abstract An NADP thioredoxin system consisting of NADPH NADP thioredoxin reductase NTR and its thioredoxin thioredoxin h Trxh plays important role in the cell by reducing disulfide bonds in target proteins involved in different cellular process NTR utilizes NADPH to catalyse the conversion of oxidized Trx into reduced Trx Reduced Trx provides reducing i Trx peroxidase wich breaks down H2O2 to water ii ribonucleotide reductase which reduces ribonucleotides to deoxyribonucleotides for DNA synthesis and transcription factors which leads to their increased binding to DNA and altered gene transcription In addition Trx increases cell growth and inhibits apoptosis Plants contain multiple forms of Trx that are classified based on their primery and sub cellular localization The reduction of cytosolic and mitochondrial types of Trx is dependent on NADPH and catalyzed by NADPH dependent reductase In contrast to 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 Trxh isoforms Through search in rice oryza sativa genome database we identified nine potential genes encoding Trxh and four potential genes encoding NTR We aim to produce purified recombinant forms of rice NTR which will enable us to analyze invitro interaction between different isoforms of NTR and Trxh from the same organism and other organisms Here we describe isolation cloning expression purification and biochemical characterization of two full length cDNAs encoding NTR OsNTR1 and OsNTR2 from rice The polypeptide deduced from these cDNAs shows high similarity with plant cytoplasmic or mitochondrial type NTR A B NTR containing an FAD and NADP binding domain and an active site For OsNTR1 coding sequences then extraction of total RNA from shoot and RT PCR amplified fragments was cloned in pJET1 2 blunt cloning vector Since OsNTR2 was not expression in 7 old rice tissues OsNTR2 coding sequences was synthesis in pUC57 cloning vector with Genescript Company The restriction sites EcoR I and Hind III were introduced the ends of the OsNTR1 and OsNTR2 coding sequences after cleavage by the appropriate restriction enzymes the inserts were ligated into pET28a expression vector Sequences were verified by sequencing then constructs transformed into the expression host Rosetta DE3 and subsequently induced with IPTG to produce the recombinant proteins Analysis of expressed proteins by SDS PAGE indicated that OsNTR1 and OsNTR2 were present as soluble and insoluble fraction but were found mostly in the insoluble fraction We were increasing solubility of recombinant proteins with optimization of growth condition by reducing of temperature and concentration of IPTG In the next steps recombinant proteins carrying an amino terminal His6 tag were produced in Escherichia coli His6 OsNTR1 and His6 OsNTR2 were purified from by nickel affinity chromatography Proteins were yellow color with absorption similar to another flavoproteins Optimization of reaction for investigation Kinetic activity of OsNTR1 and OsNTR2 were performed using DTNB as the final disulfide at 25 The production of purified forms of OsNTR1 and OsNTR2 will provide the investigation of their interaction with different OsTrxh isoforms Keyword Trx NTR cloning expression purification Kinetic activity

اسلام پناه سيدي، هديه السادات

جداسازي ، همسانه سازي و بيان دو ژن كد كننده تيوردوكسين ردوكتاز وابسته به NADPH از گياه برنج ﴿Oryza sativa﴾

بيان خالص سازي , فعاليت كينيتيكي