蔬菜中马拉硫磷代谢的对映体选择性及酶催化机制

Malathion is commonly used on vegetables to control pests. In crops, malathion is metabolized into malaoxon, which is more toxic than the parent. Both malathion and malaoxon consist of two enantiomers. However, the mechanism of enantioselective metabolism of malathion remains unclear, which indicates a risk to the safe use of chiral pesticide on vegetables. The present study is proposed to conduct enantioselective metabolism of malathion in vegetables under trial plot conditions. The vegetable with the highest conversion rate of malathion-to-malaoxon would be selected as the model vegetable, which would be further performed with the cell suspension cultures under sterile conditions to investigate malathion metabolic transformation. The relationship between the enantioselective metabolism of malathion and the activity of cytochrome P450 enzymes would be estimated via cytochrome P450 enzymes activity analysis and cytochrome P450 specific inhibitor. Additionally, the relationship between the enantioselective metabolism of malathion and gene expression of cytochrome P450 enzymes would be assessed via transcriptome high-throughput sequencing. Finally, we would disclose the cytochrome P450 enzymatic mechanism of enantioselective catalytic metabolism of malathion. On the basis of the results, we aim to reveal how chiral pesticide is enantioselectively transformed to highly toxic metabolite in vegetables as well as the safe way to use malathion on vegetables.

马拉硫磷是当前我国蔬菜常用杀虫剂，在作物中通常发生增毒代谢生成马拉氧磷，马拉硫磷和马拉氧磷均具有一对手性对映体，然而此种伴随着手性立体增毒代谢的催化机制仍不清楚，不利于手性农药在蔬菜上的安全使用。本研究选择细胞色素P450酶为研究材料，基于模拟田间实验，筛选出马拉硫磷至马拉氧磷转化率最高的蔬菜，开展无菌条件下的细胞代谢实验，通过细胞色素P450酶活性测定、酶抑制剂和有参转录组高通量测序等技术，评价马拉硫磷手性增毒代谢同P450酶活性及P450酶基因表达的关系，揭示细胞色素P450酶对马拉硫磷代谢为马拉氧磷的对映体选择性催化机制。研究结果有助于解析蔬菜中手性农药增毒代谢的机制，为马拉硫磷的安全合理使用提供科学指导。

马拉硫磷是当前我国蔬菜常用杀虫剂，在作物中通常发生增毒代谢生成马拉氧磷，马拉硫磷和马拉氧磷均具有一对手性对映体，然而此种伴随着手性立体增毒代谢的催化机制仍不清楚，不利于手性农药在蔬菜上的安全使用。本研究选择细胞色素P450酶为研究材料，基于模拟田间实验，筛选出马拉硫磷至马拉氧磷转化率最高的蔬菜，通过细胞色素P450酶活性测定、酶抑制剂和有参转录组高通量测序等技术，评价马拉硫磷手性增毒代谢同P450酶活性及P450酶基因表达的关系，揭示细胞色素P450酶对马拉硫磷代谢为马拉氧磷的对映体选择性催化机制。在小区试验中，S-马拉硫磷、R-马拉硫磷、Rac-马拉硫磷在甘蓝、黄瓜、黄瓜叶、胡萝卜、胡萝卜叶中的降解半衰期在0.4-5.7天之间，而R-马拉氧磷、S-马拉氧磷、Rac-马拉氧磷的半衰期在1.2-7.6天之间，高毒代谢产物马拉氧磷较其母体马拉硫磷在目标蔬菜中降解更慢。通过EF值分析，在目标蔬菜中马拉硫磷和马拉氧磷大多以R对映体为残留主体，呈现明显的立体选择性降解转化过程。甘蓝幼苗代谢试验表明细胞色素P450酶抑制剂增效醚可抑制甘蓝幼苗中马拉硫磷的立体选择性降解行为，并且解除增效醚胁迫后，可观察到幼苗中马拉硫磷的立体选择性降解行为。此外，将甘蓝细胞液高温去活或者加入增效醚，可使甘蓝细胞液中马拉硫磷在培养前后并不发生降解转化，当不干扰甘蓝细胞液活性时，甘蓝细胞液中马拉硫磷发生选择性降解行为。由此可见，细胞色素P450酶在甘蓝马拉硫磷立体选择性代谢过程中具有重要作用。研究结果有助于解析蔬菜中手性农药增毒代谢的机制，为马拉硫磷的安全合理使用提供科学指导。