Cloning and expression of a 5-enolpyruvylshikimate-3-phosphate synthase gene from Halomonas variabilis

文献类型： 外文期刊

第一作者： Liu, Zhu

作者： Liu, Zhu;Pan, Zhiwei;Xu, Yuquan;Dong, Zhiyang;Yang, Zhirong;Lin, Min

作者机构：

关键词： aroA gene;cloning and expression;EPSPS;glyphosate tolerance;Halomonas variables;HERBICIDE GLYPHOSATE;CONFERS RESISTANCE;PSEUDOMONAS SP;CELLS;MUTATION;STRAIN

期刊名称：DNA SEQUENCE （ 影响因子：0.408； 五年影响因子：0.594 ）

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收录情况： SCI

摘要： A novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene of 1.35 kb was cloned from a cosmid library of Halomonas variabilis HTG7, inserted into vector pET-28a (+) and transformed in Escherichia coli BL21 (DE3). EPSPS was over-expressed in soluble form after induction with IPTG at 30 degrees C and it showed a single band in SDS-PAGE, which corresponds to a molecular weight of 51 kD. Deduced amino acid sequence analysis showed that there is little homology with the aroA genes which encode glyphosate-tolerant EPSPS in known sources, such as E. coli K12 and Agrobacterium sp. CP4. The over-expressed EPSPS was purified on nickel-nitrilotri acetic acid resin and detected by Western blotting analysis. Enzyme activity measurements demonstrated that there were 4.27 units/mg in cell extract, compared with 0.049 units/mg of the control. There is an 87-fold increase in specific activity for EPSPS.

分类号： Q5

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