基于鲜味受体T1R1/T1R3茶褐牛肝菌呈鲜的分子机制研究

Umami is one of the five basic tastes. The umami taste receptor （T1R1/T1R3）is a G protein-coupled receptor, which mediates the umami taste, similar to the sweet taste receptor. Since being identified more than 100 years ago, natural umami compounds are expressed by only a few known amino acids,such as glutamic acid, aspartic acid, and RNA. Known diversity of the umami compounds is far lower than for the natural sweeteners. Monosodium glutamate is still the most widely used umami compound in the world. Due to the fact that the umami taste receptor and the sweet taste receptor are expressed through a similar mechanism, and also to the diversity of sweetening compounds, we believe we can discover many more naturally umami compounds. Yunnan is famous for its rich wild mushrooms and is called China's "wild mushroom kingdom". However, the umami ingredients and molecular mechanism of most wild mushrooms is not clearly understood. The preliminary results show that wild edible mushrooms are likely to contain previously unknown umami molecules. As this reason this project aims to select Boletus brunneissimus as the investigated media,using modern separation technology and hight-throughtput screening technology to separate umami molecules.Then we will use humam-rat chimeras and mutation technology to further verify these molecules are responsible for the umami taste.This study will help us understand more about the structure and function relationship of the human umami taste receptor and the way it interacts with its various agonists,and to explore the molecular mechanism of umami taste of Boletus brunneissimus, as well as umami receptor.

鲜味是五大基本味觉之一,鲜味受体（T1R1/T1R3）作为G蛋白偶联受体,与甜味受体相似的机制传导味觉。鲜味自被发现百余年以来，天然的鲜味剂仅限于少数氨基酸如谷氨酸，天冬氨酸等，以及核糖核苷酸而已，多样性远远低于天然甜味剂。味精（谷氨酸钠）目前依然是单一且应用最为广泛的鲜味剂。鉴于鲜味受体与甜味受体的相似性以及甜味剂的多样性，我们相信还有很多天然鲜味分子有待发掘。云南是我国的"野生菌王国"，然而大多数的野生食用菌的鲜味活性成分和分子机制还不清楚。初步研究结果显示，野生食用菌中极有可能含有未知的新型鲜味活性分子。本项目拟选取茶褐牛肝菌为切入点，利用现代分离分析技术，结合鲜味受体筛选技术，分离鉴定其中的鲜味有效成分，再进一步通过人鼠受体嵌合模型，定点突变等技术，从分子水平上论证活性成分调节鲜味受体的作用位点和作用方式，阐明茶褐牛肝菌呈鲜味的分子机制，并为进一步探讨鲜味受体的作用机制提供依据。

项目以云南味道鲜美的茶褐牛肝菌为切入点，利用现代分离分析技术，结合鲜味受体（T1R1/T1R3）筛选技术，分离鉴定其中的鲜味有效成分，从分子水平上论证活性成分调节鲜味受体的作用方式，阐明茶褐牛肝菌呈鲜味的分子机制。. 主要内容为1）五种食用野生牛肝菌, 华美、红葱、美味、黄赖头和茶褐牛肝菌，分成菌盖和菌柄分别进行游离氨基酸、游离核苷及可溶性多糖测定，并进行鲜味的评定，感官滋味稀释分析初步评定鲜味活性。2）茶褐牛肝菌子实体的醇提物，经系统的分离纯化结构鉴定了18 个化合物；扩展研究了华美牛肝菌子实体的小分子化合物，系统分离纯化和鉴定了32个化合物。分离的化合物经感官稀释滋味测试实验进行初步活性测试。3）鲜味受体(T1R1-T1R3) 稳定细胞株的建立。4）T1R优化和活性筛选，应用新的技术，重新构建一个全新的人类鲜味(T1R1-T1R3)稳定细胞株。. 通过对茶褐牛肝菌中小分子成分进行化学成分的分离鉴定，结合感官实验和鲜味受体T1R细胞系的荧光成像筛选，从分子水平证明产生鲜味的物质基础是鲜味氨酸，鲜味核糖核苷酸和C-4 二羧酸，并且5′-核糖核苷酸如肌苷一磷酸（IMP）和鸟苷一磷（GMP）能显著成倍增强L-谷氨酸，L-天冬氨酸和C-4 二羧酸的鲜味。本项目的研究结果为研究小分子化合物和大分子甜蛋白受体之间协同作用的分子机理，进一步开发新型鲜味剂或者调节剂提供了重要的前期数据和理论支持。. 除了以上工作外，我们还利用超声有机溶剂萃取法萃取其中挥发性风味成分，并结合GC-MS分析不同干燥和酶解对茶褐牛肝菌中挥发性风味成分的影响，冷冻干燥与新鲜样品具有较高的相似性，其次是酶解，而其他真空干燥、热风干燥、微波干燥方式对挥发性成分有显著影响。. 截止至目前，已发表与项目相关的论文7篇，其中SCI3篇，中文核心4篇，另有1篇SCI论文已收录待发表；申请国家发明专利5项，其中3项获得授权；培养硕士研究生17名，其中毕业8名；另有大量实验数据和论文还在整理撰写。