茉莉酸甲基转移酶代谢除草剂2,4-D的分子机制和理性改造

杂草危害造成作物减产，严重影响我国粮食安全。培育抗除草剂2,4-D（2,4-二氯苯氧乙酸）转基因作物是一种有效的杂草防治对策。我国除草剂抗性基因单一，缺乏2,4-D抗性基因专利，解决该问题的关键是开发具有自主知识产权的新型抗性基因。本团队前期研究中首次发现杨树茉莉酸甲基转移酶JMT催化2,4-D生成无活性的2,4-D甲酯，具有开发成为2,4-D抗性基因的巨大潜力。本项目将深入研究JMT代谢2,4-D的分子机制，通过解析JMT与2,4-D复合物的高分辨率结构明确活性中心，建立准确的酶-底物结合模型；基于结构信息，结合计算机QM/MM分子动力学模拟与体外酶学性质表征，清晰阐明催化反应机制；采用促进过渡态构象形成的策略合理设计突变体，提高催化反应效率，并筛选有效突变进行叠加组合获得高效的2,4-D抗性基因。研究结果将为开发具有自主知识产权的新型2,4-D抗性转基因作物提供理论依据。

Weed hazards have caused crop yield reduction every year which seriously affected our country’s food security. Cultivating herbicide 2,4-D-resistant crops is considered as an effective weed control strategy. The research on herbicide resistant genes in our country is still in its infancy. Indeed, the lack of 2,4-D resistant gene patents restricts the development of herbicide resistant crops. For solving this critical problem, the key point is to develop new resistance genes with independent intellectual property rights. In previous study, our team discovered for the first time that Populus trichocarpa jasmonate methyltransferase (JMT) could catalyze 2,4-D into inactive 2,4-D methyl ester, suggesting a great potential of developing into 2,4-D resistant gene. Here, this project will focus on the in-depth study of the molecular mechanism of JMT metabolizing 2,4-D through following strategy: crystal structure of JMT-2,4-D complex will be determined to elucidate the active center in the first step. Then an accurate enzyme-substrate binding model could be established. Based on the structure information, combination of computer QM/MM kinetic simulation with in vitro enzymatic properties characterization clearly clarify the molecular mechanism of the catalytic reaction. The strategy of promoting the formation of transition state conformation is adopted to rationally design mutants, which improve the catalytic reaction rate. Screen and combine effective mutations to obtain efficient 2,4-D resistant genes. Eventually, this project will lay the foundation for the development of new 2,4-D resistant genes and provide theoretical basis for development of herbicide resistant crops with independent intellectual property rights in our country.