Nicolia A, Ferradini N, Molla G, Biagetti E, Pollegioni L, Veronesi F, Rosellini D
Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
J Biotechnol. 2014 Aug 20;184:201-8. doi: 10.1016/j.jbiotec.2014.05.020. Epub 2014 Jun 4.
The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GO(TP+)) and one without (GO(TP-)) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GO(TP+) lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation.
植物对除草剂草甘膦产生抗性的主要策略是过表达一种对除草剂不敏感的细菌5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）。基于降解除草剂能力的草甘膦抗性策略对于降低草甘膦对作物的植物毒性可能是有用的。在此，我们展示了表达来自枯草芽孢杆菌的经植物优化的甘氨酸氧化酶（GO）变体的转基因苜蓿（紫花苜蓿）中草甘膦抗性的特征，该变体通过蛋白质工程方法在体外进化以有效降解草甘膦。使用了两种构建体，一种带有（GO(TP+)），另一种不带（GO(TP-)）豌豆rbcS质体转运肽。分子和生化分析证实了转基因的稳定整合以及质体导入的GO蛋白的正确定位。对转基因苜蓿植株进行了体外和体内草甘膦抗性测试。两个GO(TP+)株系在两种条件下均表现出对该除草剂的中度抗性。对这种GO变体表达的优化可能会使其获得对草甘膦除草剂足够的田间抗性，从而提供一种基于除草剂降解的抗性策略。
