谷胱甘肽介导油菜耐受磺酰脲类除草剂的非靶标抗性机制研究

Farmland weed infestation has severely constrained the development of mechanized production of rapeseed (Brassica napus L.) and simplified crop cultivation in China. Chemical herbicide control is an effective weed management strategy. Sulfonylurea herbicides targeting acetohydroxyacid synthase are the most widely used selective herbicides worldwide and have many benefits, including broad-spectrum and high activity, and safety. Creating sulfonylurea herbicide-resistant germplasm resources and breeding resistance varieties could limit the chemical control of rapeseed by sulfonylurea herbicides. However, studies of sulfonylurea herbicide tolerance have primarily focused on target resistance areas; non-target resistance mechanisms remain to be explored. Our group used three different tribenuron-methyl (TM)-tolerant rapeseeds and, using RNA sequencing and iTRAQ analyses as well as physiological and biochemical indexes, determined that non-target site herbicide resistance to sulfonylureas is related to the ascorbic acid-glutathione cycle. Further studies assessing cell-reducing equivalents and relationships between key enzymes and metabolites belonging to related metabolic pathways will be performed to identify candidate gene(s) linked to non-target site herbicide resistance to sulfonylureas. Additionally, the candidate gene(s) will be cloned and their function identified by Arabidopsis thaliana transformation to reveal the molecular mechanisms of non-target resistance of TM tolerance regulated by glutathione in rapeseeds. The results of this study will provide a theoretical basis as well as technical support for the improvement of existing resources regarding the resistance of rapeseed germplasm to sulfonylurea herbicides.

农田杂草严重制约我国油菜机械化、轻简化栽培的发展，化学防除是遏制杂草危害最经济有效的手段。磺酰脲类除草剂是以乙酰羟基酸合酶为靶标开发的选择性除草剂，因广谱、高效、安全等优点而被广泛应用。创制抗性种质、培育抗性品种是利用此类除草剂进行油菜田化除的必然途径。然而，目前对油菜磺酰脲类除草剂抗性的研究主要集中在靶标抗性，非靶标抗性机制还不明确。项目组前期以3个对磺酰脲类除草剂苯磺隆抗性不同的油菜为材料，通过转录组、蛋白组并结合代谢过程中的生理生化指标进行分析，初步确定油菜的非靶标抗性与抗坏血酸-谷胱甘肽循环有关。本项目拟进一步研究油菜叶片细胞还原力、相关代谢通路关键酶基因和代谢产物的关系，以期获得其非靶标抗性的关键候选基因，并进行基因克隆和转基因功能验证，解析谷胱甘肽调控油菜耐受苯磺隆的非靶标抗性机理。本项目结果可为改善和提升现有抗磺酰脲类除草剂油菜种质的抗性水平提供一定的理论依据和技术支撑。

杂草防控难题一直困扰油菜生产，现有研究多集中于对油菜抗除草剂靶标抗性机制的挖掘，鲜有对油菜非靶标抗性的研究。谷胱甘肽（GSH）作为植物细胞中重要的解毒物质，是除草剂非靶标抗性机制组成中非常重要的一种。本项目通过表型考察、抗性鉴定、毒理学实验、转录组测序以及生化分子等研究手段明确了GSH能够缓解苯磺隆对油菜幼苗的胁迫作用；然后利用已经测序的中双11油菜参考基因组信息，对GST基因家族进行全基因组鉴定，确定基因筛选范围，对GST基因进行聚类分析并比较其结构特征。随后设计不同基因荧光定量PCR引物、结合除草剂苯磺隆处理，从89个GST基因家族成员中筛选到3个GST基因显著诱导表达，包括BnGST39、BnGST78和BnGST86。进一步以非靶标抗性品系NR146和敏感品系N131为材料，比较苯磺隆处理后，BnGST78和BnGST86基因的表达模式变化，结合生物信息学分析，设计共表达网络基因的荧光定量PCR引物，筛选到BnGST78的11个上调共表达基因、6个下调共表达基因，并初步注释分析了BnGST78基因的功能。本项目结果可为改善和提升现有抗磺酰脲类除草剂油菜种质的整体抗性水平提供一定的理论依据和技术支撑。