5-烯醇式丙酮酰-莽草酸-3-磷酸合成酶(EPSPS)基因AM79 aroA的活性位点分析

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摘要 AM79 aroA (WO/2009/059485)基因是从草甘膦高度污染的土壤中克隆的、具有我国自主知识产权的新型草甘膦抗性基因，具有重要应用价值。为进一步解析AM79 EPSPS蛋白的作用机制，本研究对此蛋白的活性位点进行了研究。进化树分析显示AM79 EPSPS属于Ⅰ型EPSPS。序列比对结果表明AM79 EPSPS中第107位的丙氨酸(Ala)，第114位的苯丙氨酸(Phe)，第355位的Ala和第356位的组氨酸(His)是特异的。采用重叠延伸PCR介导的核苷酸定点突变技术，对这些保守的氨基酸位点进行定点突变。将AM79 aroA及其突变基因转入大肠杆菌(Escherichia coli)aroA缺陷型菌株ER2799中，并进行草甘膦抗性鉴定。结果显示，Phe114、Ala355和His356等氨基酸的突变，使AM79 EPSPS蛋白的草甘膦抗性降低，表明这些氨基酸位点对于维持AM79 EPSPS高抗草甘膦的能力是必需的。同源建模结果表明，这些保守氨基酸位点的突变，使AM79 EPSPS蛋白的结构发生变化，这可能是导致突变蛋白草甘膦抗性能力降低的原因。两个Ⅰ型EPSPS(荧光假单杆菌(Pseudomonas fluorescens)G2 EPSPS和拟南芥(Arabidopsis thaliana) EPSPS)对应氨基酸突变后，其草甘膦抗性没有明显变化，表明AM79 EPSPS中几个特异的氨基酸位点的功能在Ⅰ型EPSPS中并不是保守的。本研究结果为解析AM79 EPSPS高抗草甘膦的作用机理及对AM79 EPSPS进行定向进化提供了理论依据。

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	曹高燚
	陈荣荣
	杜锦
	陆伟
	刘允军

关键词 ：草甘膦, 突变, 活性位点, 抗性

Abstract：AM79 aroA (WO/2009/059485), an novel 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) gene, is isolated from glyphosate-polluted soil using metagenomics method and showed high glyphosate resistance. However, the function mechanism of the high glyphosate resistance of AM79 aroA remains unknown. Site-directed mutagenesis to uncover the function of some specific amino acids in AM79 EPSPS was performed. Phylogenetic tree analysis showed that AM79 EPSPS was a typeⅠEPSPS. Sequence alignment indicated that the 107th Alanine (Ala), the 114th Phenylalanine (Ala), the 355th Alanine and the 356th Histidine (His) were symbols to distinguish the AM79 EPSPS from other EPSPS. To explain the function of these specific amino acids in AM79 EPSPS, point mutations were carried out via overlap extension PCR. AM79 aroA and the mutant gene were transformed into aroA defective strain ER2799, then the glyphosate resistance of the transformed strains were identified. The results showed that mutants of Phe114, Ala355 and His356, respectively, led to the decrease of glyphosate resistance, indicating that these amino acids were essential for the enzyme activity and glyphosate resistance of AM79 EPSPS. Homology modeling analysis showed that the mutation of these amino acids changed the structures of the mutated AM79 EPSPS and this might be the reason for the decreased glyphosate resistance. We hypothesized that the highlighted amino acids in AM79 EPSPS might have similar function in other typeⅠEPSPS and reverse mutations on these amino acids of Pseudomonas fluorescens G2 EPSPS and Arabidopsis thaliana EPSPS (AtEPSPS) were performed. However, the mutations of these key amino acids didn't enhance the glyphosate resistance of G2 EPSPS and AtEPSPS. This study can be useful for enhancing the glyphosate resistance of AM79 EPSPS by directed evolution process.

Key words： Glyphosate Mutation Active site Resistance

收稿日期: 2014-11-06 出版日期: 2015-03-17

基金资助:国家转基因生物新品种培育重大专项

通讯作者: 刘允军 E-mail: liuyunjun@caas.cn

引用本文:

曹高燚陈荣荣杜锦陆伟刘允军. 5-烯醇式丙酮酰-莽草酸-3-磷酸合成酶(EPSPS)基因AM79 aroA的活性位点分析[J]. , 2015, 23(5): 606-616.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I5/606

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