酿酒葡萄果实中ABA调控C13-降异戊二烯物质积累的分子机制

At present the problem of weak fruity exists commonly in domestic wines. C13-Norisoprenoids are major components responsible for floral fruity of wines. Based on our previous studies and the related reports, we infer that the accumulation of this group of aroma components should be closely related to the ripening time of grape berry. However, it remains unclear whether or by what mechanism abscisic acid (ABA) regulates the synthesis of C13-norisoprenoids since ABA is a key hormone triggering grape berry ripening. Thereafter, this project will be performed to explore this field. The effect of ABA on the accumulation of C13-norisoprenoids will be assessed by viticulture manipulation and spraying exogenous ABA, respectively, to alter berry ripening time. Based on transcriptome sequencing, the expression profiles of the genes relevant to a series of events including hormone biosynthesis, berry ripening regulation and norisoprenoid synthesis will be compared between ABA-treated and control berries. A gene co-expression network reflecting the interaction between genes will be constructed and the association relationship among ABA, berry ripening regulation and C13-norisoprenoid synthesis will be elucidated. Furthermore, studies will be made on the transcriptional regulation of critical genes expression in C13-norisoprenoid biosynthetic pathway by ABA or ripening-related transcriptional factors.Through the above research and studies, we will uncover the molecular mechanism of C13-norisoprenoid accumulation regulated by ABA. Our research findings will provide some scientific guidance for improving the accumulation of these floral fruity compounds in wine grape berries by viticulture manipulation to control berry ripening time.

当前我国葡萄酒普遍存在果香较弱的问题，而C13-降异戊二烯类物质是贡献葡萄酒花果香的重要组分。根据我们前期研究及相关报道，推断该类物质的积累与果实成熟进程密切相关。但ABA作为调控葡萄果实成熟的关键激素，是否调节或通过什么机制调节C13-降异戊二烯的合成尚不清晰。本项目拟在前期研究基础上，分别采用一些栽培调控措施和ABA喷施，改变果实成熟进程，研究ABA对C13-降异戊二烯物质积累的影响；基于转录组测序，着重比较ABA处理与对照间在激素合成、成熟调控和降异戊二烯合成等关联事件中相关基因表达谱的差异，构建关键基因表达调控网络，解析ABA、成熟调控和C13-降异戊二烯合成之间的关联性；深入探究ABA和成熟促进因子对C13-降异戊二烯合成关键酶基因表达的转录调控，从而揭示葡萄果实中ABA调控C13-降异戊二烯积累的分子机制，为通过栽培手段调节果实成熟进程、促进酿酒葡萄花果香物质积累提供科学依据。

C13-降异戊二烯是非芳香型葡萄和葡萄酒花果香气味的主要组分，但关于其合成调控机制仍知之甚少。本项目在前期研究基础上，探究了葡萄成熟激素ABA调控降异戊二烯生物合成的分子机制，主要研究结果如下：（1）在新疆干热产区连续3年的不同时期摘叶处理试验表明，葡萄果实内源ABA、油菜素内酯等成熟激素合成途径基因表达上调对应于降异戊二烯合成关键酶基因VvCCD4a 和VvCCD4b的表达下调，摘叶降低了成熟果实中降异戊二烯含量，而疏穗处理降低了转色期果实中ABA/IAA比，提高了大马士酮含量；（2）转色前喷施800-1000 mg/L ABA显著提升了转色期ABA/IAA水平，上调了ABA生物合成途径、成熟相关的信号途径及果实成熟相关基因的表达，加速成熟进程，但分别上调和下调了VvCCD4a和VvCCD4b表达，导致成熟果实降异戊二烯含量未有明显变化；（3）喷施100-200 mg/L NAA推迟并降低了ABA峰值，上调整个降异戊二烯合成途径基因的表达，显著提高了总的降异戊二烯尤其是大马士酮含量；值得注意的是，NAA处理使降异戊二烯合成中的VvLECY和VvZEP发生了差异可变剪切，其对应的基因显著上调，说明IAA在调控降异戊二烯合成中发挥更为重要作用。（4）通过k-means聚类分析发现WRKY家族转录因子与VvCCDs表达密切相关，其中WRKY40-like-1，WRKY24，WRKY70在NAA处理的葡萄果实发育后期（E-L36和E-L38）显著下调; （5）采用酵母单杂交和EMSA证实了WRKY4，WRKY20，WRKY24，WRKY32，WRKY40-like-1可与VvCCD4a、VvCCD4b和VvCCD1启动子互作；亚细胞定位确定它们均只存在细胞核中；酵母自激活实验确定WRKY20和WRKY24为转录激活因子，其余为转录抑制因子；采用烟草双萤光素酶实验证实WRKY24和WRKY70可显著抑制VvCCD1活性，WRKY40-like-1可显著抑制VvCCD4b活性；葡萄悬浮细胞瞬时表达初步映证了这一结果。综上，研究认为调控葡萄果实ABA/IAA比，可下调WRKY24和WRKY70表达从而促进降异戊二烯的积累。以项目成果作为理论依据之一研发了我国优质酿酒葡萄栽培管理技术已经得到应用示范，并在2017年获得了中国酒业协会技术发明一等奖。