AP2/ERF转录因子调控酿酒葡萄果实糖苷键合态单萜生物合成的机制研究

Monoterpenes are a group of terpenoid compounds with the most diverse composition and abundant content in grape berry. Glycosidically-bound monoterpenes are important aroma precursors of grape berries, and they potentially contribute to the floral and fruity attributes of the resultant wines. However, until now little is known about the regulation of glycosidically-bound monoterpene biosynthesis. Our previous studies have preliminary found that AP2/ERF transcription factor can up-regulate the expression of monoterpene glucosyltransferase genes. Based on this finding, the present research program will focus on the regulation mechanism of AP2/ERF family transcription factors in glycosidically-bound monoterpenes biosynthesis in wine grape berries. The differentially-expressed genes coding for AP2/ERF family and glycosidically-bound monoterpene biosynthetic pathway will be screened out by the transcriptome compariosn between different treatments. VvAP2/ERF candidate member(s) in relation to glycosidically-bound monoterpene biosynthesis will be identified according to the gene co-expression pattern. These transcription factor genes and the promoters of their target genes will be isolated from grapes. The transcriptional regulation of VvAP2/ERF gene(s) on its (their) traget genes involved in glycosidically-monoterpene synthesis will be further verified and confirmed by adopting DNA pull-down technique, yeast one-hybrid system, tobacco transient expression system as well as heterologous and homologous transgenic systems. The regulation mechanism will be elucidated. The research outcome will help to illustrate the regulation network of important aroma precursors biosynthesis, and provide new approaches for improving the potential aroma quality of wine grape berry and enhancing the floral/fruity notes of Chinese wines by means of biotechnology and viticulture practices.

单萜是葡萄果实中种类最多、含量最丰富的萜类物质。糖苷键合态单萜是果实中重要的香气前体物，对所酿葡萄酒花香和果香有潜在的重要贡献。然而，迄今关于糖苷态单萜生物合成调控的研究仍鲜有报道。我们前期研究初步证实：AP2/ERF家族转录因子可上调葡萄中单萜糖基转移酶基因的表达。本项目拟在此基础上，进一步利用转录组分析分离差异表达的AP2/ERF家族成员和糖苷态单萜生物合成相关的结构基因，通过基因共表达分析确定参与糖苷态单萜合成的VvAP2/ERF候选成员，利用DNA Pull-down、酵母单杂交、烟草瞬时表达以及异源和同源转基因等手段验证并明确可转录调控糖苷态单萜合成的VvAP2/ERF成员，阐明VvAP2/ERF对酿酒葡萄果实糖苷态单萜合成的调控机制。此项研究旨在解析葡萄重要香气前体物合成调控网络，并为应用生物技术或栽培手段改善酿酒葡萄潜在香气品质、解决我国葡萄酒果香欠佳的问题提供新的思路。

糖苷结合态单萜是葡萄果实中主要的单萜贮存形式，其含量在很大程度上决定了葡萄酒的花果香气质量，但关于其积累的调控机制仍知之甚少。针对我国葡萄酒普遍存在香气单薄的问题，本项目探究了AP2/ERF转录因子调控酿酒葡萄果实糖苷态单萜合成的机制，主要结果如下：.（1）构建了葡萄61种糖苷态香气物质的UPLC-QTOF二级质谱库，通过优化提取方法和色谱条件，建立了无需酶解直接测定糖苷态香气物质的方法。.（2）加权基因共表达网络分析表明VviGT14表达与单萜含量有极高关联度；通过DNA pull-down、酵母单杂交和双荧光素酶实验，鉴定到3个可直接激活VviGT14启动子活性的AP2/ERF家族转录因子VviERF003、VviERF2-like和VviRAP2-12以及1个抑制活性的VviWRKY40。.（3）VviERF003 、VviERF2-like、VviRAP2-12被证实均为核定位且具有转录激活活性；VviERF003与VviGT14的时空表达高度一致，VviERF2-like、VviRAP2-12与VviGT14在葡萄果实发育过程中也有相似表达趋势；VviERF003可与靶基因起始密码子上游256 bp区域结合，但它与已报道的其它植物中参与萜烯调控的ERF家族转录因子的亲缘关系较远；葡萄愈伤组织稳定过表达和毛葡萄叶片瞬时过表达分析均表明，VviERF003可上调愈伤组织中VviGT14和毛葡萄叶片中VqGT7的表达，增加糖苷态单萜含量；VviERF003可响应乙烯诱导但VviGT14不能。上述研究表明，葡萄果实成熟过程中VviERF003响应乙烯诱导，上调VviGT14的表达从而促进单萜的合成积累。 .（4）VviWRKY40也定位于细胞核中，但无转录激活活性；酵母单杂交、双荧光素酶和EMSA都证明其能够与VviGT14启动子的w-box结合并抑制其活性；葡萄悬浮细胞体系证实了VviWRKY40可下调VviGT14表达，且VviWRKY40表达受ABA抑制，而VviGT14表达则受ABA诱导；烟草瞬时过表达分析发现， VviWRKY40下调了NbGT表达并抑制叶片中里那醇从游离态向糖苷态转化。.该项目已发表论文6 篇，培养博士3名、硕士3名；项目成果已用于指导优质酿酒葡萄栽培；作为部分支撑材料，相关工作获得1项省部级科技进步奖一等奖（位列8/15）。