Development of Combinatorial Technology To Simultaneously Accomplish Detection and Purification of the Heavy Metal Contamination.

低水平、大范围的大气重金属污染是未来环境问题之一，将严重危害人类健康。在本研究中，开发了同时检测和净化重金属污染的组合技术。首先，将重金属转运活性和报告基因表达系统引入细菌细胞，建立了污染重金属敏感检测系统。其次，利用汞离子转运体基因和多磷酸合成基因ppk基因在烟草细胞中培育出具有重金属去除活性的植物。第三，研究了将汞离子转运体基因引入酵母细胞的具体方法，以了解该基因在酵母细胞中的表达不均匀性。本研究的实验结果表明，所研制的汞生物传感器具有很高的灵敏度（对无机汞离子的检测限为1 nM，对有机汞化合物的检测限为0.5 nM），并且该绿色荧光蛋白融合生物传感器装置不需要任何特殊的汞检测设备，可以适用于实际环境场所汞污染的测量。通过对转基因植物（烟草）的研究，了解到ppk基因与汞转运基因merT的克隆对面包抗汞和积累汞植物是有效的。通过酵母细胞表达系统的研究发现，所表达的重金属转运蛋白MerC与合胞素家族蛋白Vam3p的融合蛋白特别位于酵母细胞的液泡膜上，并赋予酵母细胞抗重金属和积累能力。

As one of environmental problems in future, low level and wide aerial contamination of heavy metals will be serious for human health. In this study, combinatorial technology for simultaneous detection and purification of the heavy metal contamination was developed. First, sensitive detection system of contaminated heavy metals was developed by introducing heavy metal transportation activity and a reporter gene expression systems into bacterial cells. Second, plants that have heavy metal removal activity were developed using mercury ion transporter gene and polyphosphate synthesis gene, ppk gene, into tobacco cells. Third, specific methods of introduction of a mercury ion transporter gene into yeast cells were investigated to know maldistribution of the gene expression in the yeast cells.The experimental results obtained from the research showed that the developed mercury biosensor has very high sensitivity (with detection limit of 1 nM for inorganic mercury ion and 0.5 nM for organic mercurial compound) and that the gfp fusion biosensor device does not required any special equipment for the mercury detection and it can be applicable for measurement of the mercury contamination in actual environmental sites. From the study using transgenic plant (tobacco), it was understood that ppk gene cloning with a mercury transporter gene, merT, is effective to bread mercury resistant and accumulation plants. Form the study using yeast cell expression system, it was shown that the expressed fusion protein of heavy metal transporter protein, MerC, and a syntaxine family protein, Vam3p is specially located at the vacuole membrane of the yeast cell, and it confers heavy metal resistance and accumulation capability to the yeast cell.