杨梅果实原花青素合成及其转录调控机制研究

Proanthocyanidins are one of terminal products of flavone secondary metabolism in plants. They not only have a wide diversity of bioactivities such as antioxidant, cancer preventive and cardioprotective activities, but also are important factors influencing nutritional quality in fruits. Chinese bayberry fruit is rich in anthocyanins, phenolics, flavonoids and proanthocyanidins. However, the researches on its secondary metabolites were mainly focused on anthocyanin biosynthetic pathway and regulation, the information on biosynthesis of proanthocyanidins is quite limited. Recently, the applicant isolated two proanthocyanidins biosynthesis genes MrLAR and MrANR from bayberry fruit, suggesting that MrLAR and MrANR may be involved in proanthocyanidins biosynthesis coexist with anthocynians accumulation. It is still an open question concerning how pre- or post-harvest factors influences proanthocyanidins biosynthesis and the competitive regulation between biosynthesis of proanthocyanidins and anthoycanins in Chinese bayberry fruit. To verify this hypothesis, we will investigate the effect of pre-harvest factors on proanthocyanidins synthesis in four different colours cultivars of Chinese bayberries with plant physiological, chromatographic, and molecular biological (such as yeast two hybrid assay, GST Pull-Down assay, EMSA and LUC/REN transient expression assay) methods to reveal the mechanisms by which transcription factors regulate proanthocyanidins biosynthesis, and clarify the competitive regulation between biosynthesis of proanthocyanidins and anthoycanins. In addition, the effect of different storage temperatures and different light irradiations on proanthocyanidins synthesis in four cultivars of Chinese bayberries will be conducted to determine the role and transcriptional regulation mechanism of storage environmental conditions on the proanthocyanidins biosynthesis of harvested fruits during storage. The combined results will also provide molecular mechanism for transcriptional regulation of proanthocyanidins biosynthesis and finally improves the nutritional quality in postharvest fruits.

原花色素(PAs)是植物类黄酮次生代谢的末端产物之一，具有抗衰老、抗癌变、抗动脉硬化等功效，是影响果实营养品质的重要因素。目前有关杨梅果实中次生代谢的研究主要集中在花色苷的生物合成及调控机制上，而果实中PAs种类、含量和合成机制研究却鲜有报道。在前期研究中，申请人从杨梅果实中克隆到了杨梅PAs合成的关键基因MrLAR和MrANR，暗示杨梅果实除花色苷合成外，还存在PAs的合成，但二者的具体竞争机制及其调控方式并不清楚。为此，本项目以4种颜色差异显著的杨梅果实为实验材料，采用植物生理学理论、色/质谱和分子生物学(酵母双杂交、Pull-Down、EMSA和LUC/REN双荧光瞬时表达)手段，阐明杨梅果实PAs合成调控机制及其与花色苷合成途径的内在竞争调控机制；并根据果实采后的抗胁迫特性进一步揭示采后不同温度和光质处理对果实PAs合成的转录调控机制，明确杨梅果实PAs合成转录调控的分子机制。

原花色素（PAs）作为植物类黄酮次生代谢的末端产物之一，是影响果实营养品质的重要因素。目前有关杨梅果实中PAs种类、含量和合成机制研究却鲜有报道。本项目利用RT-PCR和RACE方法克隆得到了MrANR 和MrLAR基因，通过重组酶体外催化反应和烟草转基因过表达技术证实了MrANR和MrLAR是杨梅PAs合成途径关键基因。利用系统进化分析从RNA-Seq数据库中筛选出7个MYB转录因子，3个bHLH转录因子和3个WD40转录因子可以作为调控PAs合成的候选转录因子。“荸荠”果实成熟后期MrMYB308和MrMYB9表达量高于“水晶”，这与“荸荠”果实中含有较高的MrANR表达量和可溶原花色素含量相关。在“水晶”果实成熟过程中，MrMYBPA1表达与MrANR和MrLAR表达及可溶原花色素含量变化相一致。MrMYB308、MrMYB9和MrMYBPA1通过与MrEGL3或MrbHLH1形成复合体激活MrANR和MrLAR启动子以及上游类黄酮合成途径基因启动子，促进PAs合成。MrMYB3和MrMYB6基因被鉴定为是属于MYB第4亚组的负调控转录因子。在杨梅果实成熟过程中，MrMYB3和MrMYB6基因表达趋势与PAs含量变化及MrANR和MrLAR基因表达趋势相反。MrMYB6通过与MrbHLH1和MrWD40-1形成复合体以负调控PAs合成。本研究初步解析了MYB-bHLH-WD40转录复合体调控杨梅果实PAs合成的作用机制，为阐明杨梅果实PAs合成调控机制提供了一定的理论依据。