假眼小绿叶蝉侵害诱导茶叶特有蜜果香成分diendiol I形成的机制研究

Herbivore-induced plant volatiles (HIPVs) play important roles in defense against stresses, and also can be used as quality components of agricultural plants. As an example, the famous oolong tea (Oriental Beauty), which is manufactured by the tea (Camellia sinensis) leaves infected by tea green leafhoppers (Empoasca vitis Göthe) with piercing-sucking mouthpart, has a unique aroma reminiscent of ripe fruit and honey. Our preliminary experiments proposed that the unique aroma was derived from 2, 6-dimethyl-3, 7-octadiene-2, 6-diol (diendiol I), which was induced by the attack of tea green leafhoppers. Interestingly, single mechanical damage and attack of other herbivores did not induce the formation of diendiol I in tea leaves, suggesting characteristic inducement mechanism of tea green leafhoppers. Currently, biosynthesis of diendiol I in plants and HIPVs-related elicitors of tea green leafhoppers are unknown. We have isolated, identified, and characterized in vitro functions of diendiol I synthetase and glycoside transferase. In addition, we tentatively proposed the occurrence of beta-glucosidase as a potential elicitor in tea green leafhoppers. This research proposal will further characterize the in vivo functions of diendiol I synthetase and glycoside transferase based on Agrobacterium-mediated transient expression and RNAi of protoplast system. Moreover, this research proposal will identify beta-glucosidase occurred in tea green leafhoppers, and validate if beta-glucosidase is an elicitor involved in formation of HIPVs using RNAi. This research proposal will provide evidences of biosynthesis of diendiol I in plants and occurrence of HIPVs-related elicitor in insects with piercing-sucking mouthpart for the first time. The results obtained within these studies will advance our understanding of formation of HIPVs, and also can be essential for the future utilization of stress-responses of plant secondary metabolism for improvement of quality components of agricultural products.

昆虫侵害诱导植物产生的挥发物质除了具有抵御外界胁迫作用，还可作为农业植物的品质成分。刺吸式口器昆虫-假眼小绿叶蝉（EG）取食茶叶制成的“东方美人茶”具有特有的蜜果香。我们前期工作表明此蜜果香来源于EG侵害茶叶时产生的香气diendiol I（二醇）。有趣的是单一机械损伤和其他昆虫侵害茶叶不能产生二醇，表明EG侵害诱导的特异性。目前二醇在植物中合成和EG中诱导挥发物形成的激发子尚未知。我们已分离、鉴定与体外功能验证了二醇合成酶和糖苷体转移酶；获得EG中存在潜在激发子葡萄糖苷酶的初步证据。本项目拟采用瞬时表达与原生质体的RNAi方法，进一步验证二醇合成酶和糖苷体转移酶的功能；鉴定EG中葡萄糖苷酶作为激发子的功能。本项目有望首次揭示二醇在植物中的生物合成，并获得刺吸式口器昆虫中存在诱导挥发物形成的激发子的直接证据，为将来利用植物对逆境胁迫的代谢应答机制应用于提高农产品的品质成分奠定理论基础。

昆虫侵害诱导植物产生的挥发物质除了具有抵御外界胁迫作用，还可作为农业植物的品质成分。刺吸式口器昆虫-茶小绿叶蝉取食茶叶制成的“东方美人茶”具有特有的香气。本项目主要阐明了茶小绿叶蝉侵害诱导茶叶重要单萜类香气形成机制，取得的主要研究结果包括： .（1）茶小绿叶蝉侵害可显著提高香气物质芳樟醇的形成与释放，其主要原因是茶小绿叶蝉侵害诱导芳樟醇合酶CsLIS1和CsLIS2基因表达水平上调。体外功能验证表明CsLIS1和CsLIS2可催化GPP形成S型芳樟醇。此外，通过机械模拟，发现连续机械损伤是影响茶小绿叶蝉侵害诱导芳樟醇形成和释放的关键因子。 .（2）茶小绿叶蝉侵害可显著提高香气物质香叶醇的形成与释放，然而茶叶的香叶醇合酶的酶活并没有被诱导升高。茶小绿叶蝉来源的香叶醇合酶可能是茶小绿叶蝉侵害茶叶引起香叶醇形成与释放的主要因子。在茶小绿叶蝉中发现了一个香叶基二磷酸合成酶EoTPS，体外酶活实验表明EoTPS可以催化GPP生成香叶醇。.（3）茶小绿叶蝉侵害和稳定同位素标记前体共处理茶叶，发现茶小绿叶蝉侵害可诱导芳樟醇转化形成二醇。二醇在加工过程中发生脱水反应，最终形成“东方美人茶”特有蜜果香成分-脱氢芳樟醇。有趣的是单一机械损伤（简单针刺叶片）和其他昆虫侵害如刺吸式口器的蚜虫、咀嚼式口器的茶尺蠖幼虫、茶卷叶蛾幼虫和神泽叶螨均不能诱使茶叶产生二醇，由此表明了茶小绿叶蝉侵害诱导二醇形成的特异性。.本研究结果一方面有助于深入理解“东方美人茶”特有蜜果香的形成机制，另一方面可为将来利用植物对逆境胁迫的代谢应答机制应用于提高农产品的品质成分提供一定的理论依据。.项目实施期间，已在相关领域的SCI期刊发表论文3篇（均已标注本项目资助），其中1篇论文入选ESI 1%高引论文和热点论文；获授权发明专利1件；参与制定企业标准2项；参与开发软件著作权2项。