Expression And Purification Of γ-glutamylcysteine Synthetase (GSHI) In Plants And Its Activities In Response To The Temperature Variation

γ-glutamylcysteine synthetase(GSHⅠ),also known as glutamyl-L-cysteine ligase(GLCL) is the first enzyme in the process of glutathione(GSH) synthesis.GSHⅠis considered to be the rate-limiting enzyme in the process because of its feedback suppression by GSH.But it is controversial whether it is still the rate-limiting enzyme under some certain stress(GSH synthease(GSHⅡ) becomes the rate-limiting enzyme instead under the cadmium stress).Our research mainly concentrates on the resistance of GSHⅠto temperature stress at the level of transcription.The enzymes related to GSH synthesis were firstly cloned from Arabidopsis Thaliana in 1996 by Ullmann.Afterwards they were cloned in succession from Pisum sativum,Medicago truncatula,Glycine max,Zea mays,Triticum aestivum and Zinnia elegans etc.It was firstly proved by Bloch that GSH synthesis is a sequential ATP-consuming reaction process from L-Glu ,L-Cys and Gly with catalysis ofγ-ECS(GSHⅠ,EC 6.3.2.2) and GLCL(GSHⅡ,EC 6. 3.2.3) in cell.So it is necessary to have the high activities of GSH synthetase system and ATP-supply to synthesize GSH efficiently.The extra and intracellular concentration of GSH increases with the acceleration of GSH synthesis catalyzed by high activities of GSHⅠ.The activities of GSHⅠcan be regulated at the levels of gene transcription,after-transcription and translation,but mainly at transcription.Our research is trying to express the GSHⅠand GSHⅡgenes from Arabidosis Thaliana with pETM plasmids with or without fusion protein.Both cDNA genes were found from the cDNA library of Arabidopsis Thaliana.GSHⅠwith fusion protein TrxA was over-expressed in Rosetta gami in our experiment.It is detected on SDS-PAGE and purified with Ni-NTA agarose successfully.The relative quantities of GSHⅠtranscription from tobacco(Nicotiana megalosiphon) under limit temperature were detected by realtime PCR.The results showed that the GSHⅠtranscription from tobacco was greatly induced at 4℃the same temperature as the freezing tolerance temperature of Arabidopsis Thaliana.The temperature of thermotolerance in Arabidopsis Thaliana was also used to induce GSHⅠtranscription in tobacco,but there was no over-expression,inversely the amount of GSHⅠtranscription gradually decreased with time. The exact thermotolerance temperature of tobacco needs further exploration.The design of a plasmid to express two ORFs at the same time was explored and E.coli Integration Host Factor was over-expressed.The successful expression provides a feasible vector-design to express two target proteins at the same time which can also be used to express relative enzymes in two-enzyme catalysis system.It has perspectives in plant genetic engineering and also provides a good method to over-express exogenous relative-genes in engineering bacteria.The data showed that GSHⅠhas some functions to resist to the extreme temperature,so it can be used to transform plants as exogenous gene to enhance the tolerance to environment stress.