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重金属膜輸送遺伝子とキナーゼ遺伝子の共クローニングによる重金属とリン除去の研究

共克隆重金属膜转运基因与激酶基因去除重金属除磷的研究

基本信息

批准号：
07808063
负责人：
ENDO Ginro
金额：
$ 1.15万
依托单位：
Tohoku Gakuin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07808063/
关键词：
Heavy metal removal Mercury transport gene Polyphosphate kinase gene Co-cloning, and Genetic Engineering heavy metal mercurials genetic engineering plyphosphate gene cloning stable expression

项目摘要

The purpose of the research project was to develop a technology for simultaneous removal of phosphorus and heavy metals with biotechnological methods. The researcher focused on the phenomenon that bacteria accumulate divalent cations such as Mg^<2+> and Ca^<2+> when they are accumulate polyphosphate in the cells. Therefore, bacterial potential capability of bio-accumulation of divalent heavy metal cations such as Hg^<2+> and Cd^<2+> was investigated.Hg^<2+> and Cd^<2+> resistance of bacterial plasmids were characterized at the first stage of this research project. Phosphate transport genes, a pst operon, were cloned in the most stable cells of an E.coli strain, in the second stage of this project. Thereafter, a mercury resistance operon of Pseudomonas strain were challenged to co-clone the mercury influx transport genes with a polyphosphate kinase gene in a same E.coli strain. The research results obtained from this project were as follows.(1) Transcriptional regulatory protein of the product of cadC gene was characterized as a repressor protein for Cd resistance.(2) One of E.coli strain can possess pst operon and polyphosphate kinase gene stably and can grow actively in diluted LB broth. This bacterial strain uptook phosphate from the medium constitutively.(3) Hyper-sensitive E.coli strain for mercury chloride and methyl mercury was constructed by sitederected deletion of mercury reducing gene (merA).(4) The possibility of simultaneous accumulation of polyphosphate and mercury in the co-cloned E.coli cells was recognized.

该研究项目的目的是开发一种利用生物技术方法同时去除磷和重金属的技术。研究者关注的是细菌在细胞内积累多聚磷酸盐时积累二价阳离子如Mg^<2+>和Ca^<2+>的现象。因此，本研究的第一阶段主要研究了细菌对二价重金属离子Hg^2+和Cd^2+的生物富集能力，并对细菌质粒的Hg^2+和Cd^2+抗性进行了表征。在本项目的第二阶段，在大肠杆菌菌株的最稳定细胞中克隆了磷酸盐转运基因，即一个pst操纵子。此后，对假单胞菌菌株的汞抗性操纵子进行挑战，以在同一大肠杆菌菌株中共克隆汞流入转运基因与多磷酸激酶基因。该项目取得的研究成果如下。(1)cadC基因产物的转录调控蛋白是镉抗性的阻遏蛋白。(2)其中一株大肠杆菌能稳定地携带pst操纵子和多磷酸激酶基因，并能在稀释的LB肉汤中生长。该菌株从培养基中组成性地摄取磷酸盐。(3)通过定点缺失汞还原基因（merA），构建了对氯化汞和甲基汞高度敏感的大肠杆菌菌株。(4)多磷酸盐和汞在共克隆的大肠杆菌细胞中同时积累的可能性得到了确认。