Soil and Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.
Pest Manag Sci. 2018 May;74(5):1184-1196. doi: 10.1002/ps.4624. Epub 2017 Aug 12.
Glyphosate is a herbicide that acts by inhibition of the enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), involved in the catalysis of an essential step in the biosynthesis of aromatic amino acids. The objective of this study was the isolation of glyphosate-resistant bacterial strains and subsequent characterization of the gene(s) encoding glyphosate resistance in these isolates. Using an enrichment culture technique, a glyphosate-resistant bacterium, Ochrobactrum intermedium Sq20 was isolated from glyphosate-contaminated indigenous soil and characterized.
An open reading frame (ORF) comprising of 1353 bp potentially encoding aroA was amplified from O. intermedium Sq20. It showed 97% homology with aroA genes from other Ochrobactrum spp. Physicochemical characterization revealed that aroA encodes a polypeptide of 450 amino acids with a calculated molecular mass of 48.9782 kDa and an isoelectric point of 5.21. Secondary structure prediction of AroA demonstrated a high percentage of random coils and α helices. Methodical optimization and validation of the protein structure helped to build a reliable protein model indicating the presence of 91.8% amino acid residues in most favoured regions. In addition, strain Sq20 was found to be capable of complete degradation of glyphosate at 500 mg L initial concentration as the sole carbon and energy source within 4 days.
A glyphosate-resistant bacterial strain O. intermedium Sq20 was discovered. Sequence analysis and structure modelling demonstrated that AroA closely resembles class II EPSPS and possesses high glyphosate resistance. This provides a good foundation for functional analysis of experimentally derived crystal structures. The cloning and characterization of AroA will further help in understanding its role at the molecular level and its potential use in the production of glyphosate-resistant transgenic crops. © 2017 Society of Chemical Industry.
草甘膦是一种除草剂，通过抑制参与芳香族氨基酸生物合成的关键步骤的酶 5-烯醇丙酮酰莽草酸-3-磷酸合酶（EPSPS）而起作用。本研究的目的是分离草甘膦抗性细菌菌株，并随后对这些分离株中编码草甘膦抗性的基因进行表征。使用富集培养技术，从草甘膦污染的土著土壤中分离出一株草甘膦抗性细菌 Ochrobactrum intermedium Sq20 并进行了表征。
从 O. intermedium Sq20 中扩增出一个包含 1353bp 的开放阅读框（ORF），该 ORF 可能编码 aroA。它与其他 Ochrobactrum spp. 的 aroA 基因具有 97%的同源性。理化特性分析表明，aroA 编码一个 450 个氨基酸的多肽，其计算分子量为 48.9782kDa，等电点为 5.21。AroA 的二级结构预测表明，无规则卷曲和α螺旋的比例很高。对蛋白质结构进行系统的优化和验证有助于构建一个可靠的蛋白质模型，该模型表明大多数有利区域中存在 91.8%的氨基酸残基。此外，Sq20 菌株在 500mg/L 初始浓度下可作为唯一碳源和能源源在 4 天内完全降解草甘膦。
发现了一株草甘膦抗性细菌 Ochrobactrum intermedium Sq20。序列分析和结构建模表明，AroA 与 II 类 EPSPS 非常相似，具有很高的草甘膦抗性。这为实验获得的晶体结构的功能分析提供了良好的基础。AroA 的克隆和表征将进一步有助于在分子水平上了解其作用及其在生产草甘膦抗性转基因作物中的潜在用途。 © 2017 化学工业协会。
