Expression and characterization analysis of type 2 metallothionein from grey mangrove species (Avicennia marina) in response to metal stress
Expression and characterization analysis of type 2 metallothionein from grey mangrove species (Avicennia marina) in response to metal stress
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DOI：
10.1016/j.aquatox.2010.04.004
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发表时间：
2010-08-01
影响因子：
4.5
通讯作者：
Wang, You-Shao
中科院分区：
文献类型：
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作者：
Huang, Guo-Yong;Wang, You-Shao
金属硫蛋白 (MT) 是一类低分子量富含半胱氨酸的蛋白质，被认为在金属代谢或解毒中发挥可能的作用。为了评估金属硫蛋白在白骨壤金属稳态或耐受性中的作用，开发了实时定量PCR方案来直接评估当白骨壤幼苗暴露于不同浓度的锌（Zn）、铜（Cu）或铅（Pb）3和7天时AmMT2 mRNA的表达。实时定量PCR结果表明，Zn、Cu和Pb对AmMT2 mRNA表达的调节强烈依赖于浓度和暴露时间。至少在某些实验条件下，还发现对 Zn、Cu 和 Pb 的响应，AmMT2 基因的转录物显着增加。当 AmMT2 作为谷胱甘肽-S-转移酶 (GST) 的羧基末端延伸在大肠杆菌 BL21 中过表达时，与对照菌株相比，转基因细菌对 Zn、Cu、Pb 和 Cd 暴露的耐受性增强。此外，GST-AmMT2是从在400μM Zn、Cu、Pb或Cd存在下生长的大肠杆菌细胞中纯化的。与单独的 GST 相比，纯化的 GST-AmMT2 融合蛋白可以结合更高水平的所有四种金属。总而言之，这些数据支持这样的假设：AmMT2 可能参与 A. marina 的金属稳态或耐受过程。 (C) 2010 Elsevier B.V. 保留所有权利。
Metallothioneins (MTs) are a family of low-molecular-weight cysteine-rich proteins and are thought to play possible roles in metal metabolism or detoxification. To evaluate the roles of metallothioneins in metal homeostasis or tolerance in Avicennia marina, a real-time quantitative PCR protocol was developed to directly evaluate the expression of AmMT2 mRNA, when A. marina seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 3 and 7d. Real-time quantitative PCR results indicated that the regulation of AmMT2 mRNA expression by Zn, Cu and Pb was strongly dependent on concentration and time of exposure. A significant increase in the transcripts of AmMT2 gene was also found in response to Zn, Cu and Pb, at least under some experimental conditions. When AmMT2 was overexpressed in Escherichia coli BL21 as a carboxy-terminal extension of glutathione-S-transferase (GST), the transgenic bacteria showed an increased tolerance to Zn, Cu, Pb and Cd exposure as compared to control strains. Moreover, GST-AmMT2 was purified from E. coli cells grown in the presence of 400 mu M Zn, Cu, Pb or Cd. The purified GST-AmMT2 fusion protein could bind higher levels of all four metals than GST alone. Taken together, these data support the hypothesis that AmMT2 may be involved in processes of metal homeostasis or tolerance in A. marina. (C) 2010 Elsevier B.V. All rights reserved.