紫薯糖基化修饰酶Ib3GGT对花青素修饰和富集的研究

The glycosylation of anthocyanidin influences anthocyanins stability in plant cells and is a key process leading to compound diversity and function. In contrast to Arabidopsis, purple sweet potato anthocyanidin 3-O-glucosides can be further modified to yield anthocyanidin 3-O-glucoside-2’’-O-glucoside (i.e. anthocyanidin 3-O-sophorosides) by the enzyme UDP-glucose:anthocyanidin 3- O- glucoside-2”- O- glucosyltransferase (3GGT). The aim of this study is to explore biological functions of purple sweet potato Ib3GGT on glycosylation, transport and compounds diversity of anthocyanins. Our previous study demonstrated that Ib3GGT was localized in endoplasmic reticulum and golgi apparatus and catalyzed the substrate anthocyanidin 3-O-glucosides into anthocyanidin 3-O-sophorosides. Thus, anthocyanin was modified by Ib3GGT before transported and accumulated in vacuole. Detection of anthocyanin autofluorescence in young leaf of RNAi and overexpression Ib3GGT transgenic plant revealed that anthocyanin accumulation in vacuole was affected of Ib3GGT level. In future study, we will look for the interaction proteins of Ib3GGT through yeast-two-hybrid and co-imunoprecipitation methods. Our objective is to elucidate the biological function of Ib3GGT in further anthocyanidin glycosylation. This work can provide the fundamental knowledge and new approaches for anthocyanin improvement in crops.

花青素糖基化修饰影响花青素在细胞中的稳定性。不同于拟南芥，紫薯以花青素-3-O-葡糖苷进一步糖基化形成花青素-3-O-葡糖苷-2”-O-葡糖苷(又称花青素-3-O-槐糖苷)为基础衍生出花青素的不同组分，解析催化该步骤的糖基转移酶Ib3GGT的生物学功能，可深入了解该酶对紫薯花青素糖基化修饰、转运及富集的影响。前期实验表明Ib3GGT可催化花青素-3-O-葡糖苷形成花青素-3-O-槐糖苷，且其定位在内质网及高尔基体中表明该酶是在花青素转运到液泡前进行糖基化修饰的。干扰及过表达Ib3GGT的转基因紫薯幼叶花青素自发荧光实验表明该酶影响到花青素在液泡中的富集程度。下一步将开展酵母双杂等实验寻找Ib3GGT与花青素转运及富集有关的互作蛋白、酵母中该酶功能的验证和转基因植株的分析等。本研究旨在阐明紫薯花青素进一步糖基化修饰与花青素转运及富集的联系，为解析花青素的富集机制及作物花青素改良提供理论依据。

花青素糖基化修饰影响花青素在细胞中的稳定性，不同的糖基转移酶负责不同类型的花青素糖基化修饰。本项目的研究发现紫薯糖基转移酶Ib3GGT催化花青素-3-O-葡糖苷进一步糖基化形成花青素-3-O-葡糖苷-2”-O-葡糖苷(又称花青素-3-O-槐糖苷)；不同于拟南芥中At3GGT进一步糖基化修饰形成花青素-3-O-葡糖苷-2”-O-木糖苷。进化分析也表明拟南芥和甘薯中的3GGT选择性的以UDP-木糖或UDP-葡萄糖为底物。蛋白晶体结构模型及点突变都表明Ib3GGT 138位点的Thr对供体底物UDP-葡萄糖的识别至关重要。在拟南芥的野生型及突变体ugt79b1中过表达Ib3GGT产生新的花青素组分花青素-3-O-槐糖苷。 Ib3GGT定位在细胞质而不是内质网，组织特异性表达关联到花青素的富集，并且受转录因子IbMYB1的调控。该课题首次揭示紫薯糖基化与模式植物拟南芥糖基化修饰方式的不同，138位点的Thr对选择性的识别UDP-葡萄糖为底物起了关键作用，从而对紫薯花青素的稳定性起到修饰作用。