GST在佛手柑内酯解除水稻幼苗乙草胺药害中的功能分析

Acetochlor can easily cause phytotoxicity to rice seedlings when it controls the annual gramineous weed in paddy. Our research team has separated and purified a monomeric compound (i.e., Bergapten) which possesses a satisfactory detoxification activity to acetochlor phytotoxicity, from Notopterygium incisum. In our former research, we observed that the glutathione S-transferase (GST) activity has a significant improvement in rice seedlings during the detoxification process of acetochlor induced by Bergapten, which could plays a leading role for detoxification of acetochlor. However, the exact molecular mechanism of detoxification has not yet been elucidated. We further conduct the RNA-Seq analysis for different-treated rice seedlings and then, integrate the different-expressed genes, KEGG pathway and the response regularity of GSTs to make a conjoint analysis. Finally, we screen out five different GST genes as the candidate most likely to be involved in detoxification of acetochlor phytotoxicity. This project will clone these five candidate GST genes, and verify their spatiotemporal expressions in detoxification process with qRT-PCR method. Moreover, we will conduct transgenosis researches including promoter and enhancer cloning, over-expression and RNAi, to determine the roles of these candidate GST detoxification genes in rice plant. The successful application of this program will reveal the molecular genetics mechanism of the detoxification of acetochlor phytotoxicity. Thus we can lay a theoretical foundation for the development and application of novel safeners.

乙草胺在高效防控水田一年生禾本科杂草的同时会对水稻幼苗产生药害，本项目前期从羌活材料中分离纯化到具有保护活性的单体化合物—佛手柑内酯（Bergapten），并证实其对水稻的乙草胺药害有显著解毒效果，同时发现水稻幼苗在解除乙草胺药害过程中体内谷胱甘肽转移酶（GST）活力显著提升，可能对乙草胺解毒起主导作用，但确切分子机理尚未阐明。对不同处理组水稻幼苗进行RNA-Seq测序，结合差异表达基因、KEGG通路及GST解毒酶响应规律进行联合分析，找到5条候选GST解毒基因。利用qRT-PCR技术研究其时空表达规律；对筛选得到的特异性GST解毒基因进行全长克隆，开展启动子和增强子克隆、过表达、RNAi干扰等转基因研究，对解毒基因在水稻体内进行功能验证，阐明GST在佛手柑内酯解除水稻幼苗乙草胺药害中的作用，研究结果可为新型安全剂的开发及应用奠定理论基础。

酰胺类水田除草剂易对水稻幼苗产生药害，已发现多种植物源活性物质能够有效缓解水稻酰胺类除草剂药害，但其解毒机理研究相对较少。.在中期改进研究体系后，本课题明确了赤霉素对水稻幼苗酰胺类除草剂药害的缓解效果与最佳使用剂量，测定了主要解毒酶活性响应规律，探明了遭受药害及其缓解过程中的基因表达及代谢物差异，并通过蛋白质组挖掘了该过程中的差异蛋白，联合分析发现Os04g0286500基因编码谷胱甘肽转移酶（GST）受赤霉素诱导。克隆了特异性GST解毒基因（Os04g0286500）全长，构建了过表达和RNAi干扰载体，并进行了体外表达试验。我们的结果表明施用外源植物激素解除水稻幼苗酰胺类除草剂药害是可行的，本研究有助于深入阐明植物源活性物质介导的酰胺类除草剂药害解除机制，有较好的应用前景。