藏药川西獐牙菜环烯醚萜合成酶的克隆和功能验证

The Tibetan Swertia mussotii French is used commonly as a tibetan folk herbal called "zangyinchen", is one of the eight tibetan precious treasures in the Qinghai-Tibet Plateau. The wildlife resources of S. mussotii is lacking to meet the demand of the market. The main active ingredients swertiamarin and gentiopicroside are iridoid compounds, they can treat many diseases, such as anti-hepatitis, liver damage repair, inhibit the the central nervous system, anti-ulcer, insect-resistant and anti-cancer. This study will clone the key enzyme encoding gene and verify the function in the biosynthesis pathway of swertiamarin and gentiopicroside, we intend to separate the iridoid synthase encoding gene in S. mussotii; and then intend to verify the gene function and analyze the enzyme activity assay through the gene expression in prokaryotic E. coli and eukaryotic yeast; we will analyze the gene expression in different organs, and then do the gene overexpression and knock-down expression with genetic transformation in S. mussotii respectively, through analyzing the gene expression in different organs and iridoid compounds accumulation to further validate the function of the gene. This study will lay the foundation for clarify the biosynthetic pathway of iridoid compounds in S. mussotii, and have the important significance for improving the active ingredient content of S. mussotii with genetic engineering of secondary metabolites.

藏药川西獐牙菜（Swertia mussotii French）为藏族民间常用的一种草药，称为"藏茵陈"，是青藏高原中藏药八珍之一。川西獐牙菜野生资源匮乏，市场供不应求。主要有效成分獐牙菜苦苷及龙胆苦苷为环烯醚萜化合物，它们具有抗肝炎、肝损伤修复、抑制中枢神经系统、抗溃疡、抗虫、抗癌等作用。本研究将对獐牙菜苦苷及龙胆苦苷生物合成关键酶基因进行克隆和功能研究，拟分离川西獐牙菜环烯醚萜合成酶的编码基因；通过其在原核大肠杆菌和真核酵母中的表达，对基因功能进行验证及酶活力测定。通过研究该基因在不同器官中的表达，进而通过遗传转化将该基因在川西獐牙菜中进行过量表达和敲减表达，分析不同器官该基因的表达与环烯醚萜化合物积累量的相关性，在植物体内进一步验证该基因的功能，为阐明川西獐牙菜中环烯醚萜化合物生物合成途径奠定基础，对开展利用次生代谢的基因工程来提高川西獐牙菜有效成分含量的研究也具有重要意义。

藏药川西獐牙菜（Swertia mussotii French）为藏族民间常用的一种草药，称为“藏茵陈”，是青藏高原中藏药八珍之一。川西獐牙菜野生资源匮乏，市场供不应求。主要有效成分獐牙菜苦苷及龙胆苦苷为环烯醚萜化合物，它们具有抗肝炎、肝损伤修复、抑制中枢神经系统、抗溃疡、抗虫、抗癌等作用。本研究对獐牙菜苦苷及龙胆苦苷生物合成关键酶基因进行克隆和功能研究，分离了川西獐牙菜环烯醚萜合成酶的编码基因SmIS1和SmIS2；通过其在原核大肠杆菌中表达，对基因功能进行了验证及酶活力测定。研究了SmIS1和SmIS2在不同器官中的表达，构建了该基因的植物过表达载体，进而初步通过遗传转化将该基因在川西獐牙菜和烟草中进行过量表达，为阐明川西獐牙菜中环烯醚萜化合物生物合成途径奠定基础，对开展利用次生代谢的基因工程来提高川西獐牙菜有效成分含量的研究也具有重要意义。