谷胱甘肽基因的克隆表达及其抗紫外辐射功能的研究
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摘要
谷胱甘肽(glutathiore,GSH)是一种由甘氨酸、半胱氨酸和γ—谷氨酰氨组成的小分子硫醇类抗氧化剂。本研究详细探讨了谷胱甘肽基因在溶原菌中的表达及对λ原噬菌体紫外诱导的影响。以野生型λ溶原菌为出发菌株,通过增加γ-谷氨酰半胱氨酸合成酶(GSH-Ⅰ)、谷胱甘肽合成酶(GSH-Ⅱ)基因拷贝数的方法构建了过量合成谷胱甘肽的突变菌株,通过缺失gshⅠ基因的方法构建了谷胱甘肽合成缺陷菌株。检测结果显示,在相同的紫外辐射条件下(60J/m~2,254 nm),λ原噬菌体紫外诱导的频率与其宿主菌细胞内的谷胱甘肽含量呈反相关,谷胱甘肽含量最低的合成缺陷株诱导率最高,约为出发株的4.8倍,而谷胱甘肽含量最高的突变株诱导率最低,仅为出发株的28%。相应地,溶原菌抗紫外辐射的能力与细胞内的谷胱甘肽含量呈正相关,谷胱甘肽含量最高的突变株对紫外辐射的抗性也最强,其存活率是出发株的8.5倍,而缺陷株对紫外辐射最敏感,其存活率仅为出发株的16%。本研究显示外源谷胱甘肽同样能够抑λ原噬菌体的紫外诱导,抑制效果与浓度相关。当外源谷胱甘肽达到3 mol/L时,抑制效果高达93%以上。应用电子自旋共振法及自旋捕捉技术,在λ原噬菌体的紫外诱导过程中检测出明显的自由基信号,而且信号的强度则受到内源或外源谷胱甘肽浓度的影响。以上结果说明,自由基在入原噬菌体紫外诱导中起着重要的作用,内源和外源的谷胱甘肽均可以有效地清除诱导过程中产生的自由基,从而抑制λ原噬菌体的紫外诱导。
Glutathione (GSH) is a kind of low-molecular-weight thiol made up by L-glutamic acid, L-cysteine and glycin. The effect of GSH on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli in present study. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was over 93 % with 3.0 mol/L GSH. The effect was also measured in different lambda lysogens: a wild-type strain (wt), an isogenic glutathione-deficient strain isolated by disrupting the gamma-glutamylcysteine synthetase (gshI) gene, and three isogenic strains producing increased amounts of GSH due to the overexpression of gshI and/or gshII gene. The result showed that when subjected to UV irradiation (254 nm, 60 J/m~2), GSH-deficient strain was approximately five fold more sensitive to be lysed than the wild-type strain, whereas the strain with highest intracellular GSH levels was only 28 % susceptible to be lysed. Correspondingly, the UV-resistance of different lambda lysogens is correlated to their intracellular GSH levels. The survival rate of the strain with highest intracellular GSH levels was more than eight fold when compared to the wild-type strain, while the survival rate of the GSH-deficient strain was determined to be only 16 %. With electron spin resonance (ESR) and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated in the process of UV irradiation.
Glutathione (GSH) is a kind of low-molecular-weight thiol made up by L-glutamic acid, L-cysteine and glycin. The effect of GSH on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli in present study. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was over 93 % with 3.0 mol/L GSH. The effect was also measured in different lambda lysogens: a wild-type strain (wt), an isogenic glutathione-deficient strain isolated by disrupting the gamma-glutamylcysteine synthetase (gshI) gene, and three isogenic strains producing increased amounts of GSH due to the overexpression of gshI and/or gshII gene. The result showed that when subjected to UV irradiation (254 nm, 60 J/m~2), GSH-deficient strain was approximately five fold more sensitive to be lysed than the wild-type strain, whereas the strain with highest intracellular GSH levels was only 28 % susceptible to be lysed. Correspondingly, the UV-resistance of different lambda lysogens is correlated to their intracellular GSH levels. The survival rate of the strain with highest intracellular GSH levels was more than eight fold when compared to the wild-type strain, while the survival rate of the GSH-deficient strain was determined to be only 16 %. With electron spin resonance (ESR) and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated in the process of UV irradiation.
引文
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朱素华,方呈祥。朱石桥.抗坏血酸对λ原噬菌体紫外诱导过程的影响.分析科学学报,2001,17(5):383-386
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沈立新,魏东芝,赵哲峰.谷胱甘肽合成酶在大肠杆菌中的高效表达及性质。华东理工大学学报,2000,26(4):342-345
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张健.曹恩华,秦静芳等.抗氧化剂对DNA损伤的保护作用机制的研究,生物物理学报,1997,13(1):123
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