Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects.
Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects.
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DOI：
10.1007/s10534-014-9704-0
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Gutiérrez JC
中科院分区：
文献类型：
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作者：
Amaro F;Turkewitz AP;Martín-González A;Gutiérrez JC
金属（类）化合物作为环境污染物的重要性使其成为生态毒理学的重点，其目的是尽量减少与动物或人类的接触。因此，有必要开发灵敏且廉价的方法来有效检测和监测环境中的这些污染物。传统的分析技术具有成本高且复杂的缺点。另外，原核或真核全细胞生物传感器（WCB）是环境监测中使用的最新分子工具之一，它使用细胞作为整合报告基因，将报告基因融合到重金属响应启动子上。在本文中，我们报告了在纤毛虫嗜热四膜虫中表达由报告 gfp 基因组成的结构的结果，该基因在 MTT1 金属硫蛋白启动子的转录控制下与完整的 MTT1 或 MTT5 蛋白编码区融合，该启动子在该纤毛虫的重金属应激中发挥着关键作用。当暴露于 Cd2+ 时，此类细胞会过度表达 GFP 报告转基因和连接的金属硫蛋白基因。我们报告说，对于 GFPMTT5 菌株，与野生型细胞（24h LC50 ~ 1.73 µM Cd2+）相比，这种金属硫蛋白过度表达导致对镉毒性的显着抵抗（24h LC50 ~ 15 µM Cd2+）。这些结果提供了第一个实验证据，证明纤毛虫金属硫蛋白与其他生物体一样，具有保护细胞免受有毒金属离子侵害的功能。由于这些菌株作为 WCB 可能具有新的优势，因此我们将它们的特性与之前报道的其他四膜虫 WCB 的特性进行了比较。
The significance of metal(oid)s as environmental pollutants has made them a priority in ecotoxicology, with the aim of minimizing exposure to animals or humans. Therefore, it is necessary to develop sensitive and inexpensive methods that can efficiently detect and monitor these pollutants in the environment. Conventional analytical techniques suffer from the disadvantages of high cost and complexity. Alternatively, prokaryotic or eukaryotic whole-cell biosensors (WCB) are one of the newest molecular tools employed in environmental monitoring that use the cell as an integrated reporter incorporating a reporter gene fused to a heavy metal responsive promoter. In the present paper, we report results from expressing, in the ciliate Tetrahymena thermophila, constructs consisting of the reporter gfp gene fused to the complete MTT1 or MTT5 protein coding regions under the transcriptional control of the MTT1 metallothionein promoter, which plays a critical role in heavy metal stress in this ciliate. When exposed to Cd2+, such cells overexpress both the GFP reporter transgene and the linked metallothionein gene. We report that, for the GFPMTT5 strain, this metallothionein overexpression results in marked resistance to cadmium toxicity (24h LC50 ~ 15 µM of Cd2+), compared to wild type cells (24h LC50 ~ 1.73 µM of Cd2+). These results provide the first experimental evidence that ciliate metallothioneins, like in other organisms, function to protect the cell against toxic metal ions. Because these strains may have novel advantages as WCBs, we have compared their properties to those of other previously reported Tetrahymena WCBs.