茶树萜类糖苷香气前体的生物合成及其囊泡运输的机理与调控

In tea plants, accumulation of glycosidically bound aroma precursors is crucial for the aroma quality of a tea product. It has been recently revealed that in vitro glycosyltransferases CsGT1 and CsGT2 are capable to sequentially catalyze the formation of geranyl-primeveroside. Inhibition of plant cell exocytosis leads to a significant change in free linalool emission and glycosidically bound linalool accumulation. However, in planta, the catalytic function of the two glycosyl-transferases are unknown and little is known about vesicle transportation of aroma glycosides in plant cells. This proposal suggests that intobacco and tea hair roots, CsLIS/NES with known function as a linalool synthase and CsGT1 and -2 would be co-overexpressed for further characterizing the functions of glycosyltransferases individually, mutually. Secretory exocytosis and vacuolar accumulation of these glycosides will also be investigated after vesicle fusion with cytoplasmic or vacuolar membranes is regulated by suppressing or over-expressing VAMP72, VTI11 and VTI12 genes. Moreover, by targeting β-glycosidases to different subcellular compartments, cellular distribution and transportation routes and accumulation sites of terpenol glycosides will be identified. This work will significantly promote our understanding of biosynthesis, transport and accumulation of aroma glycosides in tea plants and definitely helpful to establish the novel approaches for tea aroma improvement.

茶鲜叶中萜类糖苷香气前体的积累对成品茶香气品质至关重要。新近发现，茶树糖基转移酶CsGT1和CsGT2在离体条件下可催化香叶醇等生成樱草糖苷，且萜类糖苷的积累可因囊泡的胞吐抑制而增加。然而，茶树活体中萜类香气物质的糖基化以及糖苷的胞内运输、积累机理几无所知。申请人拟以烟草和茶树发根为试材，围绕萜类糖苷代谢与积累，进行多层次、多节点关键基因的表达调控，以研究对萜类糖苷积累的影响。通过逐一或叠加表达CsGT1和CsGT2，研究其单一和协同催化的功能；通过在不同细胞器中β-糖苷水解酶的瞬时表达，研究萜类糖苷在细胞中的运输路径和积累部位；通过调节胞吐和液泡融合关键SNARE元件（VAMP72和VTI11/VTI12）的表达，探明囊泡运向胞外和液泡支流的调控对糖苷前体分泌与积累的影响；本研究将显著推进对萜类糖苷香气前体的生物合成及其囊泡运输机理的认识，有助于创立茶叶增香的代谢调控新技术。

茶鲜叶中萜类糖苷香气前体的积累对成品茶香气品质至关重要。新近发现，茶树糖基转移酶CsGT1和CsGT2在离体条件下可催化香叶醇等生成樱草糖苷，且萜类糖苷的积累可因囊泡的胞吐抑制而增加。然而，茶树活体中萜类香气物质的糖基化以及糖苷的胞内运输、积累机理几无所知。本项目研究结果表明在模式植物本氏烟中，对未做任何处理的叶片、注射缓冲液的叶片和过表达ObGES的叶片进行处理，发现在不加外源糖苷水解酶的情况下，检测不到芳樟醇以及香叶醇等萜类香气的生成。AtVTI12, CsVTI12, MtVAMP721e-RNAi分别和ObGES共过表达都使香叶醇等萜类物质的含量增加，说明VTI12的表达直接或者间接的影响了香叶醇等糖苷物质的积累。以本氏烟为研究材料进行荧光强度分析，结果发现COPⅡ组分与ObGES共表达后荧光强度，与单独表达COPⅡ组分并无显著差异。在本氏烟中共过量表达Sar1a+ObGES，Sec23a+ObGES，Sec24a+ObGES, Sec31a+ObGES并不能显著增加香叶基二糖苷的含量。茶树糖基转移酶（CsGT1, CsGT2）双元表达载体的构建与ObGES的共表达关系及其亚细胞定位分析表明，CsGT1-RFP定位在细胞质中，这与最初推测的在线粒体或叶绿体中不一致，但与其他物种的GT1蛋白定位保持一致；CsGT2-GFP的定位也在细胞质中，说明香叶醇的糖苷化是在细胞质中进行的，完成糖苷化之后再由囊泡运输到目的细胞器中储存。本研究将显著推进对萜类糖苷香气前体的生物合成及其囊泡运输机理的认识，有助于创立茶叶增香的代谢调控新技术。