Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.
Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.
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DOI：
10.1073/pnas.87.24.9903
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发表时间：
1990-12
影响因子：
11.1
通讯作者：
W. V. Camp;C. Bowler;R. Villarroel;W. Tsang;M. MONTAGUt;Dirk INZt
中科院分区：
文献类型：
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作者：
W. V. Camp;C. Bowler;R. Villarroel;W. Tsang;M. MONTAGUt;Dirk INZt
大肠杆菌的超氧化物歧化酶（SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1）缺陷突变体可以通过与异源SOD编码序列的功能互补来恢复其在最小培养基上不能有氧生长的缺陷。基于这一特性，已经开发了一种表型选择系统，用于分离含有真核SOD cdna的克隆。利用电穿孔法将烟叶和拟南芥的cDNA表达文库转化为缺乏sod的大肠杆菌菌株，并在最小培养基上培养出含有功能性sod的克隆。对这些克隆的分析揭示了编码铁形式超氧化物歧化酶（FeSOD）的cdna的身份-这是第一个从真核生物中克隆的这种类型的sod。这种酶在这两种不同植物物种中的存在挑战了以前认为FeSOD仅在少数植物科中发现的观点。此外，这些结果显示了通过细菌突变体的互补来鸟枪克隆真核基因的潜力，特别是当它与高效的转化方法（如电穿孔）相结合时。
The inability of superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1)-deficient mutants of Escherichia coli to grow aerobically on minimal medium can be restored by functional complementation with a heterologous SOD-encoding sequence. Based upon this property, a phenotypic selection system has been developed for the isolation of clones containing eukaryotic SOD cDNAs. cDNA expression libraries from both Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed into a SOD-deficient E. coli strain by electroporation, and clones containing functional SODs were selected by growth on minimal medium. Analysis of these clones revealed the identity of cDNAs encoding the iron form of superoxide dismutase (FeSOD)--the first SODs of this type to be cloned from eukaryotes. The presence of this enzyme in these two divergent plant species challenges previous ideas that FeSOD is found in only a few plant families. In addition, these results show the potential for shotgun cloning of eukaryotic genes by complementation of bacterial mutants, particularly when it is combined with a highly efficient transformation method, such as electroporation.