四氢叶酸依赖型麦草畏脱甲基酶特性、结构和功能及其应用于抗除草剂转基因工程的可行性研究

麦草畏具有广谱高效、低毒和杂草抗性产生慢等优点，是理想的抗除草剂转基因靶标除草剂。孟山都利用微生物来源的麦草畏脱甲基酶基因DMO构建了抗麦草畏作物，即将商业化，这将大大推动麦草畏的应用。但目前对麦草畏微生物降解机制、环境行为和生态安全的研究不够深入。此外，我国缺乏自主产权的除草剂抗性/降解基因资源，制约了我国农业生物技术发展。申请人已分离到麦草畏降解菌Sphingomonas sp. Ndbn-20，发现该菌通过一个新的四氢叶酸依赖型脱甲基酶催化麦草畏脱甲基，且有两个麦草畏脱甲基基因簇dmt50和dmt66。本项目研究这两个脱甲基基因簇组成和功能，脱甲基酶Dmt50和Dmt66特性、结构和催化机制，针对植物密码子偏好的脱甲基酶基因优化及应用于植物转基因可行性。研究结果将揭示一个新的依赖四氢叶酸的麦草畏脱甲基分子机制，为麦草畏环境行为和生态安全研究提供理论依据，为抗除草剂转基因提供基因资源。

The herbicide dicamba is a widely used benzoic acid herbicide. Due to its low toxicity and residue, broad-spectrum and high efficiency, and rare occurrence of weed resistance, dicamba has been regarded as an ideal target of herbicide-resistant genetic engineering. Recently, the biotech giant Monsanto Company has successfully developed genetically modified (GM) dicamba-resistant crops by transferring a microbial-derived dicamba monooxygenase gene (DMO), and planned to put them into commercial application in 2015. Once this GM plants are planted on a large scale, the usage of dicamba would be increased dramatically. However, the microbial degradation process and mechanism, environmental behavior, toxicological and ecological security of dicamba has not been deeply elucidated. Furthermore, our country is lack of herbicide resistant or degradation gene resource with independent intellectual property right, which restricted the development of agricultural biotechnology in China. In our previous research, a dicamba-degrading strain Sphingomonas sp. Ndbn-20 was isolated, it initiated the degradation of dicamba by demethylation via a novel tetrahydrofolic acid (THF)-dependent demethylase to generate herbicidal inactive product 3,6-dichlorosalicylic acid, two dicamba demethlase gene clusters dmt50 and dmt66 were found in the genome of the strain. The target of this program is to study the function and regulation of the two dicamba demethylase gene clusters, the enzymatic characteristics, structure and function of the two dicamba demethylases, the functional verification of the genes involved in THF metabolism in the two demethylase gene clusters, optimization of the dicamba demethylase gene sequences and evaluation of their application potential in genetic engineering of herbicide-resistant crops. The results of this program will elucidate the mechanism of THF-dependent demethylation of dicamba in Sphingomonas sp. Ndbn-20, promote the study on the environmental behavior and ecological safety of dicamba, and provide gene resources for the genetic engineering of herbicide-resistant crops.