Baerson Scott R, Rodriguez Damian J, Tran Minhtien, Feng Yongmei, Biest Nancy A, Dill Gerald M
Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198, USA.
Plant Physiol. 2002 Jul;129(3):1265-75. doi: 10.1104/pp.001560.
The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.
尽管草甘膦除草剂已广泛使用20多年，但杂草物种中自发出现对其的抗性事件极为罕见。最近，在马来西亚发现了一种对草甘膦具有抗性的牛筋草（蟋蟀草）生物型，其半数致死剂量（LD50）值比从同一地区采集的敏感生物型高约2至4倍。比较从抗性（R）和敏感（S）生物型制备的提取物中草甘膦对5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）活性的抑制作用，发现（R）生物型的半数抑制浓度（IC50）（草甘膦）高约5倍。对两种生物型预测的EPSPS成熟蛋白编码区进行序列比较，发现四个单核苷酸差异，其中两个导致氨基酸变化。其中一个变化是（R）生物型中第106位的脯氨酸被丝氨酸取代，这与先前在鼠伤寒沙门氏菌的草甘膦不敏感EPSPS酶中鉴定出的取代相对应。重组酶的动力学数据表明，在（R）EPSPS中鉴定出的第二个取代对其降低的草甘膦敏感性没有显著贡献。表达（R）生物型成熟EPSPS酶的大肠杆菌aroA-（EPSPS缺陷）菌株相对于表达（S）生物型成熟EPSPS的菌株，对草甘膦的耐受性提高了约3倍。这些结果首次证明了EPSPS酶的改变是任何植物物种中进化出的草甘膦抗性的潜在组成部分。
