SGER: The Rapid Evolution of MerA Encoded Metal Ion Reduction: A Novel Mutagenesis Protocol

9415295 Meagher重金属污染在世界许多地方已达到有毒水平，已成为一个紧迫的问题。如果能够操纵植物的单基因性状，这将是有用的，这些性状可以处理重金属，并通过抗性、隔离来修复重金属污染，甚至可能有助于从环境中清除这些污染物。细菌merA基因的产物，汞离子还原酶（merA）可以通过将离子汞还原成毒性较小的金属形式Hgo来解毒。一个高度诱变的merA基因在转基因高等植物中具有显著的抗Hg++能力。据推测，MerA可以发生突变，以指导各种金属离子的有效还原，用于收获或修复。本研究的具体目的是#1：探索目前MerA的底物范围；#2：开发基因选择或强大的筛选减少金，Au++，银（Ag+），铅（Pb++）和镉（Cd++）在细菌中；#3：产生具有新型底物特异性的merA突变体（例如，增强对Au++的还原，以及对Pb++和Cd++的新型还原）；#4：开发一种强大的新型RE-PCR（重组聚合酶链反应）诱变方案，其中成千上万的突变克隆是唯一的幸存者。该方法利用了土壤细菌钙酸不动杆菌（Acinetobacter calcoaceticus）具有不可思议的自然转化性和高频率的同源重组。一旦开发出来，这种强大的方法将有利于许多领域的生物研究和开发，但特别是，它应该加速生物修复的进展。＊＊＊

9415295 Meagher Heavy metal contamination has reached toxic levels in many parts of the world and has become an urgent problem. It would be useful to be able to manipulate single gene traits in plants, which could process heavy metals and remediate heavy metal pollution by resistance, sequestration and perhaps even assist in the removal of these contaminants from the environment. The product of the bacterial merA gene, mercuric ion reductase (MerA) can detoxify ionic mercury by reducing it to its less toxic metallic form, Hgo. A highly mutagenized merA gene confers significant levels of resistance to Hg++ in transgenic higher plants. It is hypothesized that MerA can be mutated to direct the efficient reduction of a wide variety of metal ions for harvest or remediation. The specific aims of this research are #1: to explore the present substrate range of MerA; #2: to develop genetic selections or powerful screens for the reduction of gold Au+++, silver (Ag+), lead (Pb++), and cadmium (Cd++) in bacteria; #3: to generate merA mutants with novel substrate specificities (e.g. enhanced reduction of Au+++ and novel reduction of Pb++ and Cd++; and #4: to develop a powerful new RE-PCR (recombination polymerase chain reaction) mutagenesis protocol in which tens of thousands of mutant clones are the only survivors. The method takes advantage of the incredible natural transformability and high frequency of homologous recombination with in certain strains of the soil bacterium, Acinetobacter calcoaceticus. %%% Once developed, this powerful approach will benefit many areas of biological research and development, but in particular, it should accelerate progress in bioremediation. ***