水稻P450s和GSTs在氯乙酰胺类除草剂代谢中的联合作用机理研究

The herbicide phytotoxicity to rice is an emergent scientific problem to be resolved for agricultural production in our country. It is known that GSTs in rice play an important role in metabolism of chloroacetamide herbicide, but the mechanism of synergistic herbicide metabolism between P450s and GSTs is quite unclear. On basis of our preliminary studies, the enzyme acitivity assay and SDS-PAGE will be applied firstly to discover the induction mechanism of P450s and GSTs induced in rice by chloroacetamide herbicides and fenclorim. Then LC-MS will be applied to analyze the degradation of chloroacetamide herbicides catalyzed by P450s and GSTs respectively or jointly in vitro. The RTLC, HPLC and LC-MS will be applied to identify the metabolites of radiolabeled chloroacetamide herbicides in rice,combining with the results in vitro,to deduce the possible metabolic pathway of chloroacetamide herbicides. At last, inhibitors of GSTs and P450s will be used in rice to confirm the the effects of GSTs and P450s in metabolizing chloroacetamide herbicides in rice. The purpose of this project is to elucidate the metabolism mechanism of chloroacetamide herbicides in rice，which will provide theoretical instructions for improving the safety of this type of herbicides to rice.

水稻的除草剂药害是目前我国农业生产中亟待解决的科学问题，水稻体内解毒酶GSTs在氯乙酰胺类除草剂的代谢中发挥着重要功能，但目前人们对P450s和GSTs的联合作用机理知之甚少。本项目拟在前期研究的基础上，采用酶活性检测和蛋白质SDS-PAGE法，首先明确氯乙酰胺类除草剂及解草啶对水稻体内P450s和GSTs的诱导机制；然后通过离体试验结合LC-MS等检测技术分析这两类酶单独或联合催化氯乙酰胺类除草剂降解的效果；并用放射性薄层色谱法（RTLC）、HPLC、LC-MS鉴定水稻体内放射性标记的氯乙酰胺类除草剂的代谢产物，结合离体降解效果推导该类除草剂在水稻体内可能的代谢途径；最后利用P450s和GSTs抑制剂进一步确证两类酶在氯乙酰胺类除草剂代谢中的作用。旨在阐明氯乙酰胺类除草剂在水稻体内的代谢机理，为提高该类除草剂对水稻的安全性提供理论依据。

作物体内的解毒酶在代谢除草剂的过程中发挥着重要作用，明确作物体内关键解毒酶P450s和GSTs在氯乙酰胺类除草剂代谢中的联合作用对除草剂的安全使用具有重要意义。本项首先采用酶活性测定的方法研究了氯乙酰胺类除草剂及安全剂解草啶对水稻幼苗体内P450s和GSTs的诱导效果，研究发现解草啶可以使水稻幼苗体内的GSTs活性显著提高，而氯乙酰胺类除草剂不能诱导水稻体内GSTs活性增强；氯乙酰胺类除草剂和解草啶能显著诱导P450s活性提高为对照的2倍以上。通过HPLC和GC-MS等分析技术分析了离体条件下P450s和GSTs对氯乙酰胺类除草剂代谢速率，结果发现解草啶处理的水稻P450s和GSTs都较对照具有对氯乙酰胺类除草剂更强的催化代谢能力；体内的动态降解试验也印证了安全剂对氯乙酰胺类除草剂的促进代谢作用。为了确证P450s和GSTs在氯乙酰胺类除草剂代谢中的联合作用机理，我们将丙草胺与两类酶的抑制剂组合处理水稻幼苗，发现在添加1-ABT或（和）利尿酸后，水稻幼苗对丙草胺均变得更为敏感，进一步说明P450s和GSTs均参与了氯乙酰胺类除草剂的代谢。此外，本项目还研究了丙草胺、解草啶以及二者的组合处理对8种代表性的P450和GST基因表达的影响，发现丙草胺对几种基因的表达均表现为显著的抑制作用，其中OsGSTU4和OsGSTF3基因在解草啶加入后，其表达量有明显的恢复。在本基金的资助下，我们已发表SCI论文1篇，2篇论文已被接受，获湖南省技术发明二等奖1项，申请专利2项，培养研究生3名，圆满地完成预定的各项目标。