铜离子对嗜酸氧化亚铁硫杆菌ATCC 23270谷胱甘肽相关酶基因表达水平的影响
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摘要
嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans, A.ferrooxidans)是重要的生物浸出功能菌,它通过氧化亚铁、元素硫和还原性含硫化合物获得生长所需能量,其亚铁和硫氧化活性决定着它的生物浸出能力。谷胱甘肽(GSH)及其相关酶系在A.ferrooxidans硫氧化过程中发挥着重要作用,但易受到重金属离子的影响,目前相关的影响机制仍不清楚。因此,开展重金属离子对A.ferrooxidans ATCC 23270谷胱甘肽相关酶基因表达水平的影响具有重要的理论实践指导意义。
本文针对铜离子对A.ferrooxidans ATCC 23270谷胱甘肽相关酶基因表达水平的影响开展如下两部分研究工作：
一,研究铜离子浓度对细菌生长,GSH含量以及相关酶活性变化的影响。结果表明：(1)在设定的铜离子浓度范围内并没有抑制A.ferrooxidans的硫氧化活性；(2)随着铜离子浓度的增高,GSH含量以及相关酶活性都会降低。但是这个降低并不与铜离子浓度呈单纯的线形关系,总体上表现为先下降后上升的趋势；(3)当铜离子浓度低于0.12mol／L时,GSH的含量随之缓慢减少,A.ferrooxidans处于耐受状态,此时清除活性氧基团(ROS)主要是由GSH完成的。但随着铜离子浓度的增高,GSH的清除能力已经不能满足细菌体的需要,此时超氧化物歧化酶(SOD)与过氧化氢酶(CAT)的活性开始上升,直至细菌死亡
二,用实时荧光定量PCR方法研究铜离子对谷胱甘肽相关酶基因表达的影响。结果表明：(1)铜离子胁迫时所有10个谷胱甘肽相关酶的基因均表达上调,在铜离子0.08 mol／L时表达量达到最大值,特别是编码谷胱甘肽硫转移酶(GST)和谷胱甘肽还原酶(GR)的基因表达上调最为明显。尤其在铜离子为。(2)在铜离子胁迫的60 min内,细菌中的谷胱甘肽相关酶基因先随时间增加呈下调表达,尔后基因表达量迅速上调,说明A.ferrooxidans的相关快速自我调节机制可以使其对铜离子胁迫具有适应性。
这些结果可能有助于了解A.ferrooxidans应答重金属离子刺激时的细胞保护机制,从而用于指导高抗重金属菌株的分离和选育。
As an important biological leaching bacteria, Acidithiobacillus ferrooxidans grown by oxidation of ferrous, elemental sulfur and reduced sulfur compounds, the oxidation activity of the ferrous and sulfur determines the ability of its bio-leaching.Glutathione (GSH) and related enzymes plays an important role in the A.ferrooxidans sulfur oxidation, but vulnerable to the effects of heavy metal ions, the current impact of the relevant mechanisms remain unclear. Therefore, it is a great theoretical significance to evaluate the effect of heavy metal ions on the gene expression of the GSH-related enzymes in A.ferrooxidans ATCC 23270.
In order to know the effect of copper exposure on expression of GSH-related genes in A.ferrooxidans ATCC 23270,two parts of works were completed in this study.
1. Evaluation of the effect of Cu2+ on growth, content of GSH and activity of GSH-related genes encoding enzymes.The results show that:(1) At the setting series of concentrations Cu2+ did not inhibit the growth of the A.ferrooxidans; (2)With the increasing of Cu2+ concentration, the content of GSH and the activity of GSH-related enzymes decrease.(3)With addition of less than 0.12 mol/L of Cu2+,the content of GSH decrease slowly,but maintaining basically the resistance to Cu2+ stress through possible removal of reactive oxygen species (ROS) by GSH. With addition of higher Cu2+, however, the superoxide dismutase(SOD) and catalase (CAT) activity begin to rise,when the scavenging of ROS by GSH can no longer meet the need of cells, and the cells tent to be dying.
2.Evaluation of the effect of Cu2+ exposure on expression of GSH-related genes by Real-time PCR.The result shows that:(1)The expression of all the 10 GSH-related genes, especially the glutathione-S transferases (GST) encoding gene gst and the glutathione reductase (GR) encoding gr, were up-regulated, and with maximal expression at 0.08 mol/L of Cu2+ exposure.(2) During the exposure of Cu2+in 60 min, the expression of the GSH-related genes down-regulated at the beginning and finally up-regulated, indicating A.ferrooxidans could be adaptive to the Cu2+ stress in a relatively quick self-adjust way.
These results could contribute the evaluation of cellular protection mechanisms of A.ferrooxidans from the stress of some heavy metals, and thus useful to guide the isolation and breeding of bacterial strains with high-resistance to heavy metals.
本文针对铜离子对A.ferrooxidans ATCC 23270谷胱甘肽相关酶基因表达水平的影响开展如下两部分研究工作：
一,研究铜离子浓度对细菌生长,GSH含量以及相关酶活性变化的影响。结果表明：(1)在设定的铜离子浓度范围内并没有抑制A.ferrooxidans的硫氧化活性；(2)随着铜离子浓度的增高,GSH含量以及相关酶活性都会降低。但是这个降低并不与铜离子浓度呈单纯的线形关系,总体上表现为先下降后上升的趋势；(3)当铜离子浓度低于0.12mol／L时,GSH的含量随之缓慢减少,A.ferrooxidans处于耐受状态,此时清除活性氧基团(ROS)主要是由GSH完成的。但随着铜离子浓度的增高,GSH的清除能力已经不能满足细菌体的需要,此时超氧化物歧化酶(SOD)与过氧化氢酶(CAT)的活性开始上升,直至细菌死亡
二,用实时荧光定量PCR方法研究铜离子对谷胱甘肽相关酶基因表达的影响。结果表明：(1)铜离子胁迫时所有10个谷胱甘肽相关酶的基因均表达上调,在铜离子0.08 mol／L时表达量达到最大值,特别是编码谷胱甘肽硫转移酶(GST)和谷胱甘肽还原酶(GR)的基因表达上调最为明显。尤其在铜离子为。(2)在铜离子胁迫的60 min内,细菌中的谷胱甘肽相关酶基因先随时间增加呈下调表达,尔后基因表达量迅速上调,说明A.ferrooxidans的相关快速自我调节机制可以使其对铜离子胁迫具有适应性。
这些结果可能有助于了解A.ferrooxidans应答重金属离子刺激时的细胞保护机制,从而用于指导高抗重金属菌株的分离和选育。
As an important biological leaching bacteria, Acidithiobacillus ferrooxidans grown by oxidation of ferrous, elemental sulfur and reduced sulfur compounds, the oxidation activity of the ferrous and sulfur determines the ability of its bio-leaching.Glutathione (GSH) and related enzymes plays an important role in the A.ferrooxidans sulfur oxidation, but vulnerable to the effects of heavy metal ions, the current impact of the relevant mechanisms remain unclear. Therefore, it is a great theoretical significance to evaluate the effect of heavy metal ions on the gene expression of the GSH-related enzymes in A.ferrooxidans ATCC 23270.
In order to know the effect of copper exposure on expression of GSH-related genes in A.ferrooxidans ATCC 23270,two parts of works were completed in this study.
1. Evaluation of the effect of Cu2+ on growth, content of GSH and activity of GSH-related genes encoding enzymes.The results show that:(1) At the setting series of concentrations Cu2+ did not inhibit the growth of the A.ferrooxidans; (2)With the increasing of Cu2+ concentration, the content of GSH and the activity of GSH-related enzymes decrease.(3)With addition of less than 0.12 mol/L of Cu2+,the content of GSH decrease slowly,but maintaining basically the resistance to Cu2+ stress through possible removal of reactive oxygen species (ROS) by GSH. With addition of higher Cu2+, however, the superoxide dismutase(SOD) and catalase (CAT) activity begin to rise,when the scavenging of ROS by GSH can no longer meet the need of cells, and the cells tent to be dying.
2.Evaluation of the effect of Cu2+ exposure on expression of GSH-related genes by Real-time PCR.The result shows that:(1)The expression of all the 10 GSH-related genes, especially the glutathione-S transferases (GST) encoding gene gst and the glutathione reductase (GR) encoding gr, were up-regulated, and with maximal expression at 0.08 mol/L of Cu2+ exposure.(2) During the exposure of Cu2+in 60 min, the expression of the GSH-related genes down-regulated at the beginning and finally up-regulated, indicating A.ferrooxidans could be adaptive to the Cu2+ stress in a relatively quick self-adjust way.
These results could contribute the evaluation of cellular protection mechanisms of A.ferrooxidans from the stress of some heavy metals, and thus useful to guide the isolation and breeding of bacterial strains with high-resistance to heavy metals.
引文
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