Ott K H, Kwagh J G, Stockton G W, Sidorov V, Kakefuda G
American Cyanamid Company, Agricultural Products Research Division, Princeton, NJ 08543-0400, USA.
J Mol Biol. 1996 Oct 25;263(2):359-68. doi: 10.1006/jmbi.1996.0580.
Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.
通过导入编码经合理设计的突变乙酰羟酸合酶（AHAS）的基因，已对具有针对单一类别除草剂的特异性抗性的植物进行了基因工程改造。从AHAS-抑制剂复合物的三维模型中鉴定出合适的取代突变。基于与丙酮酸氧化酶的同源性生成结构模型，并利用此类除草剂的构效数据将咪唑啉酮抑制剂定位在提议的结合位点。对在大肠杆菌中表达的突变蛋白进行生化分析，实现了对突变基因的迭代优化。重组蛋白在烟草植物中的表达赋予了体内抗性。这种将分子建模与分子生物学相结合的新方法比传统的随机诱变和选择方法具有许多优势，对于环境安全和可持续农业系统的未来发展至关重要。
