内生真菌对酿酒葡萄酚类物质的调控及其机制研究

The status of secondary metabolites (SM) can be shaped by environmental factors, and it is also one of the key stones to determine the quality and characteristics of wine grape. It is no doubt that there will be great theoretical and practical significance if the controllable, safety and long-term lasting methods can be continuously developed to control the SM of wine grape. At present, evidences have showed that fungal endophytes had taken part in the regulation process of SM in plants. However, if endophytic fungi are also able to influence the SM of wine grape and the influence degree and mechanisms still lying unknown. Therefore, the present project will use aseptic grapevine seedlings to reconstruct new grapevines containing different endophytic fungi or endophytic fungal communities. By analyzing the phenolic compounds (which significantly influence the quality and characters of wine grape) status within different endophytic fungus infected grapevines at different developmental stages, we try to verify that fungal endophytes did take part in the regulation process of SM in wine grape, as well as to prove the hypothesis that different endophytic fungus may have different SM regulation effects on grapevine. Simultaneously, by treating vine vitro somatic cells with endophytic fungus using dual culture and co-culture method, we further want to detect the rapid and short-term responses of grapevine cells in enzymatic activities and expression profiling of the phenolic metabolites regulating genes, to explore the possible SM regulation mechanisms of fungal endopytes to the host plants. The results will develop a new research field for wine grape quality and character controlling, and will greatly improve the technologies for characteristic wine grapevine management and regulation.

酿酒葡萄中的次生代谢产物（SM）状态具有环境可塑性，也是果实品质特征的决定性因素之一。不断发掘可控且安全长效的酿酒葡萄SM调控因子，无疑具有重大的理论和应用价值。较多研究表明内生真菌参与了植物SM的调控过程，而是否也以同样的方式影响酿酒葡萄的SM组成及其程度和机制期待一个系统的研究结果。本项目将采用无菌苗系统有选择性的构建含不同已知内生真菌的酿酒葡萄植株，集中监测与酿酒葡萄品质关系密切的酚类物质在不同处理材料中的短、中和长期状态，证明内生真菌参与了酿酒葡萄SM的调控和不同内生真菌调控SM的效应不同这一假说。同时用酿酒葡萄离体细胞与内生真菌相互作用后，分析葡萄细胞中酶活性及相关调控基因表达动态变化，探索葡萄细胞对内生真菌的快速和短期响应，阐明内生真菌调控酿酒葡萄SM的机制。研究成果将开启内生菌调控酿酒葡萄品质特征的新领域，并将在高品质特色酿酒葡萄精细化种植与调控方面发挥积极作用。

酿酒葡萄中的次生代谢产物（SM）状态具有环境可塑性，也是果实品质特征的决定性因素之一。作为酿酒葡萄关系密切的环境因子之一的内生真菌是否也参与了植物SM的调控过程并不明确。本研究通过系统的实验研究内生真菌对酿酒葡萄次生代谢的可能效应，主要取得以下几方面的研究成果：1）证明了内生真菌可以有效调控酿酒葡萄酚类及其它代谢产物，进而调控酿酒葡萄品质特征。内生真菌对酿酒葡萄代谢产物的调控作用可以在植株、器官和细胞不同层面起作用；2）内生真菌调控酿酒葡萄次生代谢物质的效应具有菌株/种属的特异性；3）诱导葡萄（宿主）细胞产生新的代谢产物是内生真菌影响葡萄酚类及其它次生代谢产物的机制之一，内生菌诱导宿主产生新代谢产物可能通过自代谢、共代谢和信号转导三条途径实现；4）内生真菌可以影响酿酒葡萄细胞花色素的含量和组成，内生真菌影响葡萄花色素含量和组成的主要机制是激活葡萄细胞中与花色素生物合成有关的转录因子家族基因如MYBA1，以及相关节点的结构基因表达；5）构建了基于离体无菌叶片和水琼脂培养法的内生真菌-葡萄互作研究的技术体系。该技术的意义在于i)检测不同内生真菌对葡萄叶片侵染并实现内共生化的能力；ii）检测不同内生真菌共生对葡萄叶片的伤害程度；iii）检测内生真菌与葡萄（叶片）间的代谢互作效应；iv）分析不同内生真菌在侵染某种葡萄（叶片）过程中的相互作用；6）以可培养内生真菌为例，阐明了内生真菌在不同品种、不同发育阶段叶片中的分布特征；阐明了葡萄植株不同部位的叶片和枝干中内生真菌的区域性分布格局，并探讨了叶片和枝干中内生真菌的来源、不同区域和器官间的传递等；7）构建了近200株内生真菌的资源库；8）发表学术论文6篇，其中SCI论文4篇；待刊出论文一篇，完成并投稿论文5篇。本项目的研究成果已达到预定目标并有所拓展，研究结果为进一步通过内生真菌改善酿酒葡萄品质特征奠定了必要的理论和物质，所形成的方法技术为同类型研究提供了参考。

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