催化除草剂麦草畏脱甲基的细胞色素P450基因的克隆和功能鉴定

Cytochrome P450s can catalyze the detoxification of various herbicides via dealkylation or hydroxylation, thus has potential applications in the engineering of herbicide-resistant transgenic crops and bioremediation of herbicide contaminated environment. However, there is no report on the identification of cytochrome P450 catalyzing the dealkylation of dicamba. Dicamba can effectively kill glyphosate-resistant weeds and is considered a new target herbicide for the engineering of herbicide-resistant transgenic crops. The biotech company Monsanto has successfully developed the genetically modified (GM) crops that are resistant to dicamba by introducing a bacterial-derived dicamba demethylase gene DMO, and these GM crops have been commercially planted on a large scale in recent years. Therefore, it is of great important to study the microbial metabolism of dicamba and to clone new dicamba-degrading genes. In our preliminary study, we found that a new cytochrome P450 was responsible for the demethylation of dicamba in strain Dca-5, however, its coding gene remains unknown. This project was designed to clone the gene encoding the cytochrome P450 through protein purification and peptide fingerprint analysis, random and site-directed mutations and transcriptome analysis, study the enzymatic characteristics, kinetics and the key active sites of this new cytochrome P450. This project is expected to obtain the new cytochrome P450 gene resource, and provides new insight to the structural-functional diversity of cytochrome P450, and is of significance to promote the innovation of agricultural and environmental biotechnology in China.

细胞色素P450能够催化多种除草剂脱烷基或羟基化降解脱毒，因而在除草剂污染生物修复及抗除草剂转基因工程中有很好的应用前景，但目前还没有催化麦草畏脱甲基的细胞色素P450报道。麦草畏能杀死草甘膦抗性杂草，被认为是新的抗除草剂转基因靶标除草剂。孟山都公司利用细菌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，近年开始大规模应用。因此，研究麦草畏微生物降解过程，发掘新的麦草畏降解酶基因具有非常重要的价值。近期发现麦草畏降解菌Dca-5通过一个新的细胞色素P450催化麦草畏脱甲基，但其编码基因还未知。本项目拟采用蛋白纯化结合肽指纹图谱分析、随机和定点突变及转录组分析等方法从该菌克隆这一细胞色素P450基因，研究该酶酶学特性、动力学和关键活性位点。本项目预期获得新的细胞色素P450基因资源，为深入认识细胞色素P450结构-功能多样性提供依据，对推动我国农业和环境生物技术创新具有积极意义。

麦草畏是一种应用广泛杀草谱广的除草剂，因其能杀死草甘膦抗性杂草，被认为是新的良好的抗除草剂转基因靶标除草剂，因此，研究麦草畏微生物降解脱毒过程，发掘新的麦草畏降解脱毒酶基因具有非常重要的价值。本项目从土壤中分离到一株麦草畏降解菌株Sphingobium sp. Dca-5，前期发现麦草畏降解菌Dca-5是通过一个新的细胞色素P450催化麦草畏脱甲基。通过对Dca-5基因组测序及比对分析及定点突变的方法克隆到细胞色素P450基因cydD4，通过降解酶功能验证、降解基因敲除、转录研究以及代谢产物鉴定等实验证明cydD4具有麦草畏脱甲基酶功能，此外生物信息学分析发现在菌株Dca-5基因组中存在一整套编码麦草畏脱甲基产物3,6-二氯水杨酸降解相关酶的基因簇gtdA-dsmH2和dsmR1DABCEFGR2，通过荧光定量PCR表明这些基因簇参与麦草畏下游产物3,6-二氯水杨酸的降解。本项目从细胞和遗传学角度揭示了菌株Dca-5中麦草畏微生物降解代谢途径和机制，为深入研究土壤中麦草畏残留的分解转化过程及生物修复提供理论依据。