水稻中微囊藻毒素(MC-LR)转运蛋白基因OsOPTM的分离及功能解析

We have reported that Microcystin-LR (MC-LR) could be detected in rice grains collected from Taihu Lake region, which drawed great attention on the danger of MC-LR contamination in agricultural products. Due to the limited report on the detailed mechanism of MC-LR uptake by plants, the following experiments will be carried out: (1) Searching genes coding for proteins with potential ability to transport MC-LR by screening rice genome. (2) Cloning these genes and expressing them in E. coli. Isolating the candidate genes, named as OsOPTM, coding for proteins that are capable of transporting MC-LR by analyzing MC-LR uptake and accumulation in OsOPTM-transformed E. coli. (3) Analyzing the correlation between the spatiotemporal expression of OsOPTM and the accumulation of MC-LR in rice. (4) Constructing transgenic rice plants with down-expressed of over-expressed OsOPTM. Identifying the physiological function of OsOPTM by analyzing MC-LR accumulation in transgenic rice plants. These studies will contribute to the understating of molecular mechanism of MC-LR uptake by rice plants. And it will provide scientific basis for the evaluation of MC-LR health risk in agricultural products.

我们首次检测到微囊藻毒素 (MC-LR) 在太湖周边稻区的水稻籽粒中积累，是急需引起重视的食品安全隐患。鉴于植物吸收MC-LR的机理研究鲜有报道，且植物对大分子量多肽的吸收采取主动运输的特点，本项目拟以水稻为材料，完成以下工作：(1) 在水稻基因组中搜寻能够转运MC-LR的潜在蛋白基因；(2) 克隆上述基因并进行大肠杆菌原核表达，通过分析表达后大肠杆菌对MC-LR的吸收量，进一步筛选出能够转运MC-LR的候选蛋白基因 (命名为OsOPTM)；(3) 分析MC-LR在水稻各个器官的积累与OsOPTM时空表达的相关性；(4) 分别构建抑制表达和过表达OsOPTM的转基因水稻，通过分析转基因水稻对MC-LR的吸收量，确认OsOPTM转运MC-LR的生理功能。从而揭示水稻吸收MC-LR的分子机制，为MC-LR在农产品中的安全风险评估提供科学依据。

本文主要研究了MC-LR在水稻体内的累积特征与毒性之间的相关性；克隆了水稻体内MC-LR转运蛋白基因，并在生化和遗传学水平证明了其生理功能。主要结果如下：首先，从水稻中克隆到了哺乳动物中MC-LR转运蛋白基因OATP1的同源基因OsOPTM(Os04g44430)的完整序列。OsOPTM在水稻各个组织中均有表达，茎和叶中表达量相对较高，根中表达量相对较低。序列分析结果显示OsOPTM属于典型的MFS跨膜蛋白家族，负责转运多种大分子底物。原核表达OsOPTM后的大肠杆菌菌株对MC-LR的吸收量减少，耐受性增加。OsOPTM水稻突变体也表现为相同表型，说明其作用可能为外排MC-LR，负责MC-LR的跨组织转运。其次，累积到水稻体内的MC-LR能够通过影响内源生长素与NO信号之间的互作抑制根的发育。最后，证明水稻体内MC-LR的累积量与水稻生长成显著负相关，而与水稻的生化损伤正显著负相关。本研究结果解析了植物中首个MC-LR转运蛋白基因，并通过研究MC-LR累积效应与毒理学效应的相关性，初步揭示了水稻吸收、转运和累积 MC-LR 的分子机制，为MC-LR在农作物中的安全风险评估提供了重要的理论依据。