植物谷胱甘肽合成酶（GSHI）的表达纯化及其对温度变化的应答
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摘要
γ-谷氨酰半胱氨酸合成酶GSHⅠ(γ-glutamylcysteine synthetaseγ-ECS),又称谷氨酰-L-半胱氨酸连接酶,是谷胱甘肽(GSH)合成过程第一步的催化酶,由于合成产物GSH可通过反馈抑制调控GSHⅠ的活性,GSHⅠ被认为是GSH生物合成途径中的限速酶(在某些逆境条件下,如在Cd胁迫过程中GSHⅡ(GSH合成酶)可能成为GSH合成途径的限制酶)。本论文将主要对GSHⅠ在温度上的活性变化及其在转录水平上的响应进行初步探索。
Ullmann等在1996年首次从拟南芥(Arabidopsis thaliana)中中克隆了植物GSH合成酶基因。此后,植物谷胱甘肽合成酶基因陆续在豌豆(Pisum sativum)、蒺藜状苜蓿(Medicago truncatula)、大豆(Glycine max)、玉米(Zea mays)、小麦(Triticumaestivum)和百日菊(Zinnia elegans)等植物中被克隆。Bloch最早证实细胞内的GSH是由GSHⅠ(EC 6.3.2.2)与GSHⅡ(EC 6.3.2.3)在ATP存在下催化L-谷氨酸(Glu)、L-半胱氨酸(Cys)和甘氨酸(Gly)的一个序贯反应合成的。因此,要实现GSH的高效合成,GSH合成酶系的高活性和ATP的有效供给是两个必须满足的条件。细胞内GSHⅠ活性提高使GSH合成加速,从而提高细胞内外GSH浓度。GSHⅠ活性的调节可在基因转录水平、转录后水平、翻译后水平等环节,但主要在基因转录水平。
本论文首次尝试在pETM载体系统中进行拟南芥(Arabidopsis thaliana)的GSHⅠ和GSHⅡ的融合和非融合表达。从拟南芥的cDNA文库中分别克隆GSHⅠ和GSHⅡ,采用原核表达技术,将其转入pETM系列原核表达载体中,与载体中的硫氧还蛋白基因(TrxA)相融合,便于融合蛋白表达后的纯化。SDS-PAGE分析表明,只有融合基因GSHⅠ-TrxA通过E coli在适当温度下经IPTG诱导后得到了过量表达的融合蛋白。
本论文通过应用实时荧光定量PCR对烟草(Nicotiana megalosiphon)在极限温度胁迫下进行相对定量。结果表明,烟草同拟南芥一样在4℃低温条件下,GSHⅠ被大量诱导表达,但是在40℃时表达受到抑制,表达量降低,其机理有待进一步的研究工作。
本实验成功对拟南芥中的GSHⅠ进行了融合表达和纯化,为了对GSHⅠ和GSHⅡ在细胞中的动态研究奠定基础,对在一个质粒中同时表达两个酶做了尝试性的工作,并成功的表达了一个双亚基的酶E.coli Integration Host Factor(IHF);对在一个载体中同时表达合成链中两个酶积累了实验依据,对载体合成酶系构建提供了良好的基础,也对用构建工程菌表达植物细胞内相互作用或关联的蛋白或酶提供了很好的思路和方法,在植物基因工程研究方面具有重要的意义。同时,本论文首次对能够诱导烟草谷胱甘肽合成酶表达的温度进行了初步探索,证明了GSHⅠ可能具有协同抵御极限温度胁迫的功能,为其在今后提高作物对环境抗逆作用的转基因应用提供了可靠依据。
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.
Ullmann等在1996年首次从拟南芥(Arabidopsis thaliana)中中克隆了植物GSH合成酶基因。此后,植物谷胱甘肽合成酶基因陆续在豌豆(Pisum sativum)、蒺藜状苜蓿(Medicago truncatula)、大豆(Glycine max)、玉米(Zea mays)、小麦(Triticumaestivum)和百日菊(Zinnia elegans)等植物中被克隆。Bloch最早证实细胞内的GSH是由GSHⅠ(EC 6.3.2.2)与GSHⅡ(EC 6.3.2.3)在ATP存在下催化L-谷氨酸(Glu)、L-半胱氨酸(Cys)和甘氨酸(Gly)的一个序贯反应合成的。因此,要实现GSH的高效合成,GSH合成酶系的高活性和ATP的有效供给是两个必须满足的条件。细胞内GSHⅠ活性提高使GSH合成加速,从而提高细胞内外GSH浓度。GSHⅠ活性的调节可在基因转录水平、转录后水平、翻译后水平等环节,但主要在基因转录水平。
本论文首次尝试在pETM载体系统中进行拟南芥(Arabidopsis thaliana)的GSHⅠ和GSHⅡ的融合和非融合表达。从拟南芥的cDNA文库中分别克隆GSHⅠ和GSHⅡ,采用原核表达技术,将其转入pETM系列原核表达载体中,与载体中的硫氧还蛋白基因(TrxA)相融合,便于融合蛋白表达后的纯化。SDS-PAGE分析表明,只有融合基因GSHⅠ-TrxA通过E coli在适当温度下经IPTG诱导后得到了过量表达的融合蛋白。
本论文通过应用实时荧光定量PCR对烟草(Nicotiana megalosiphon)在极限温度胁迫下进行相对定量。结果表明,烟草同拟南芥一样在4℃低温条件下,GSHⅠ被大量诱导表达,但是在40℃时表达受到抑制,表达量降低,其机理有待进一步的研究工作。
本实验成功对拟南芥中的GSHⅠ进行了融合表达和纯化,为了对GSHⅠ和GSHⅡ在细胞中的动态研究奠定基础,对在一个质粒中同时表达两个酶做了尝试性的工作,并成功的表达了一个双亚基的酶E.coli Integration Host Factor(IHF);对在一个载体中同时表达合成链中两个酶积累了实验依据,对载体合成酶系构建提供了良好的基础,也对用构建工程菌表达植物细胞内相互作用或关联的蛋白或酶提供了很好的思路和方法,在植物基因工程研究方面具有重要的意义。同时,本论文首次对能够诱导烟草谷胱甘肽合成酶表达的温度进行了初步探索,证明了GSHⅠ可能具有协同抵御极限温度胁迫的功能,为其在今后提高作物对环境抗逆作用的转基因应用提供了可靠依据。
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.
引文
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