野生与栽培罗汉果资源代谢组学比较与糖苷积累模式研究

Luohanguo (Siraitia grosvenorii), an herb has been shown to have broad development potentialities in food, medicine and health protection areas, is an important economical species endemic to Southern China, especially in Guangxi province. Phytochemistry in different parts of Luohanguo is very rich, but its development is challenged by increasing scarcity of wild resources and monotonous cultivated varieties, comprehensive comparisons of chemical compositions among roots, leaves and fruits of Luohanguo germplasms are thus required. Furthermore, the concentration of mogrosides, the major bioactive constituents of Luohanguo, are very low in fruits, intensively exploring their biosynthesis mechanisms is one of the most effective ways to its high production cost. Although studies on mogrosides have been gradually increased recently, the biosynthesis as well as intermediate products of mogrosides in fruits remain unclear. Therefore, taking advantages of the special geographic location of Guangxi, this proposal plans to fully utilize several wild and cultivated germplasms collected formerly, and designs several common garden experiments. Biochemical analysis and histochemical localization will be performed to establish growth curves of wild and cultivated Luohanguo germplasms. GC-MS combing with UPLC-QTOF metabolomic techniques will be employed to fully investigate the metabolomics profiles in roots, leaves, and fruits among Luohanguo germplasms. Based on these experiments, we plan to further perform integrative analyses of metabolomics and transcriptomics data to reveal the metabolic accumulation mechanisms of mogrosides in fruits. Our proposal will help us to fully understand phytochemical diversity in Luohanguo germplasms and metabolic networks of mogrosides, in turn providing theoretical guidance for sustainable exploitation of wild resources and planting industrialization of Luohanguo.

罗汉果是我国南方一种重要的特产经济作物，在食品、医药、保健领域具有广阔开发潜力，广西是主产区。其化学成分丰富，然却面临野生资源日益匮乏、主栽品种单一等问题，亟需对种质间根、叶、果实化学组成进行全面比较研究。此外，果实主要活性甜味物质（糖苷）含量低，深入研究其生物合成是解决其成本高的有效途径之一。虽然近年来关于糖苷的报道逐渐增多，其代谢规律与中间产物仍不完全清楚。基于此，本项目利用广西特殊的地理优势，拟利用已收集的野生与栽培种质资源，采用同质园实验结合生化分析、组织化学定位，构建野生与栽培种生长曲线；再利用GC-MS、UPLC-QTOF 等代谢组学技术，比较罗汉果种质间根、叶、果实代谢谱差异；在此基础上，综合分析代谢组与转录组数据，以揭示果实中糖苷类物质代谢规律。本研究将能全面了解不同种质罗汉果的植化多样性，阐明糖苷类物质代谢网络，为罗汉果野生资源的可持续开发利用及产业化种植提供理论指导。

罗汉果是葫芦科多年生藤本植物，原产于桂北地区，是一种重要的南方特产经济作物。植化分析表明罗汉果果实是富含三萜类糖苷物质，甜度高，低热量，可用作甜味剂，特别罗汉果甜V为蔗糖甜度250倍。但是主要活性甜味物质含量低，深入研究其积累模式是解决其成本高的有效途径。我们利用广西丰富的种质资源优势，采用同质园实验拟合野生与栽培种的生长曲线，进一步综合分析代谢组与转录组数据，全面了解不同罗汉果种质的植化多样性，阐明了罗汉果糖苷的代谢调控网络，主要结论如下：1）虽然野生、栽培与三倍体罗汉果生长曲线呈现相似方式，但生长曲线的波动趋势因品种和生长阶段不同而有差异。除了拉丹果在授粉后20天完成早期膨大及三倍体罗汉果比二部倍体罗汉果波动趋小外，大部分罗汉果的鲜果重量、横径、纵径、种子总重、果肉总重和果皮总重都在授粉后30天内增长迅速，表明授粉后30天内是果实膨大的关键时期。有趣的是，果实生长曲线与种子的曲线相似，而果肉在果实中的比重却呈相反趋势，从而反映出罗汉果果实的发育与种子发育有一定的相关性。2）开发了一种利用超高效液相色质谱仪联用同时鉴定5种罗汉果糖III和糖苷IV同分异体及7种糖苷（如罗汉果糖苷）方法。依据成熟果实中糖苷特有的变化方式，定量了680多种果肉代谢物，并划为9个类组；定量了320多种叶片代谢物，划入8个类组。代谢谱表明罗汉果苷V含量高的罗汉果一般Siamenoside I、Isomogroside V 和 11-O-mogroside V也相对丰富。一些代谢物在不同品种间差异比较大，可用作代谢标记区分长滩果、红毛果等不同类型罗汉果。而且，一些差异代谢物只出现特定组织中（比如片、雄花）。3）8个发育时间（授粉后10-80天）野生高（或低）糖苷罗汉果、高糖苷栽培种和三倍倍体罗汉果靶向代谢分析揭示罗汉果不同类型间糖苷动态差异方式。野生高糖苷型、三倍体、栽培和野生低糖苷型罗汉果果肉中罗汉果糖苷V积累分别始于授粉后50、50、60和70天。应用非靶标代谢分析果实发育过程中代谢物水平变化，定量1042个代谢物，划分至9个类组。不同罗汉果在膨大期、青果期和收获期代谢物有着明显差异方式，基于以上品种间差异，确定了影响罗汉果糖苷V合成的核心网络及潜在途径。本研究将能全面了解不同种质罗汉果的植化多样性，阐明糖苷类物质代谢网络，为罗汉果野生资源的可持续开发利用及产业化种植提供理论指导。