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

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.

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。

麦草畏是一种甲氧基取代苯甲酸类除草剂，具有广谱、高效、低毒、廉价和杂草抗性产生慢等优点。生物技术巨头孟山都利用微生物来源的麦草畏脱甲基酶基因DMO构建了抗麦草畏转基因作物，已成功规模化应用。课题组前期分离到麦草畏降解菌Sphingomonas sp. Ndbn-20，发现该菌通过一个新的机制即四氢叶酸依赖型脱甲基酶催化麦草畏脱甲基。本项目拟研究麦草畏脱甲基基因簇转录调控，脱甲基酶特性、功能和结构，以及脱甲基酶基因应用于植物转基因工程可行性和策略。结果表明基因簇dmt50在菌株中不表达，而基因簇dmt66为组成性表达。基因簇dmt66包含4个基因：脱甲基酶基因（dmt）和3个与四氢叶酸代谢相关的基因：mthfr、mthc和dhc。研究了Dmt50和Dmt66的酶学特性，发现Dmt50和Dmt66的催化活力受到产物5-methyl-THF的严重抑制。解析了Dmt50以及Dmt50-5-methyl-THF复合体三维结构，使用分子对接研究麦草畏结合位点，并进一步推测了Dmt50脱甲基催化机理。Dmt50包含三个结构域，形成与叶酸结合相关的中央空腔。对比Dmt50结构单体与与Dmt50-5-methyl-THF复合体结构发现，在催化过程中Dmt50会发生构象变化。在脱甲基过程中，THF的结合使Dmt50构象发生变化，将麦草畏锁在活性中心。Tyr253和His69与麦草畏的氧原子形成氢键，使其质子化，THF中N5上的孤对电子对麦草畏中的甲基进行亲核攻击，完成甲基转移反应，形成DCSA和5-methyl-THF。利用定向进化技术获得Dmt50的一个突变体，对麦草畏脱甲基催化活力提高了25.4%。将针对植物表达优化后dmt50和dmt66基因导入模式植物拟南芥和作物玉米中，并分别使植物表达的Dmt50定位在细胞质和叶绿体中。结果表明： dmt50和dmt66基因均可以赋予拟南芥和玉米麦草畏除草剂抗性；而且Dmt50和Dmt66定位于叶绿体中表达可以增强植物对麦草畏除草剂的抗性。本项目从生理、生化和遗传角度揭示Sphingomonas sp. Ndbn-20的麦草畏脱甲基机制，促进对四氢叶酸依赖型脱甲基酶的深入了解,为抗除草剂转基因工程研究提供理论依据和基因资源。

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