葡萄风信子R2R3-MYB转录抑制子调控花瓣呈色的分子机理

Anthocyanins, the most important pigments for flower coloration. The regulatory network of anthocyanin biosynthetic pathway is so complicated. It is a highlight in the world recently that R2R3-MYB repressors negatively regulate anthocyanin biosynthesis. In the earlier study, we found that it is the key reason for no pigmentation that the high expression of R2R3-MYB repressors in the white flowers of grape hyacinth. However, the molecular mechanism of regulating of petal pigmentation by R2R3-MYB repressors is not yet clear. In this study, we will select grape hyacinths as the materials. Then, we will clone R2R3-MYB repressors using the RACE technique. The relationship between the transcriptional expression pattern of R2R3-MYB repressors, R2R3-MYB activators and flavonoid biosynthetic genes, the phenotype of petal color and the anthocyanin substances in the petals will be illuminated. Function of R2R3-MYB repressors when overexpress in transgenic petunia and grape hyacinth will be studied. The interaction and regulation relationships between R2R3-MYB repressors, R2R3-MYB activators and the key anthocyanin biosynthetic gene protein will be analyzed by Yeast two-hybrid (Y2H) experiment and the dual luciferase assay. This research aims to illuminate the fine regulatory network of anthocyanin metabolism in grape hyacinth and provide specific transcriptional gene resources and new theoretical supports for artificial regulation of anthocyanin biosynthesis in grape hyacinths and other ornamental monocotyledonous plants.

花青素苷是观赏植物花瓣呈色的重要色素之一，其合成调控网络极其复杂。R2R3-MYB抑制子负调控花青素苷合成是目前国际研究的热点。课题组前期研究发现R2R3-MYB抑制子在白色葡萄风信子花中的高表达是造成其无色花型的关键，但其调控的分子机理尚不清楚。本项目将以葡萄风信子为试材，RACE克隆花青素苷R2R3-MYB抑制子；分析R2R3-MYB抑制子、激活子和结构基因的表达与花色表型、花瓣中花青素苷物质间的关联性；在矮牵牛和葡萄风信子中研究R2R3-MYB抑制子调控花瓣呈色的功能；分析R2R3-MYB抑制子与激活子、花青素苷关键结构蛋白之间的互作和调控关系，阐明葡萄风信子R2R3-MYB抑制子协同激活子MBW精细调控花青素苷合成和花瓣呈色的分子机理。本项目将完善葡萄风信子花色代谢精细调控网络，为人工调控葡萄风信子及其它单子叶观赏植物花青素苷的生物合成和花色分子育种提供特异基因资源和新的理论依据。

花青苷是单子叶植物葡萄风信子花色的主要色素类型。前期研究表明葡萄风信子花青素苷合成主要由R2R3-MYB激活子(MaMybA和MaAN2)及MabHLH1调控，而MYB抑制子协同正向调节因子精细调控花青素苷合成的分子机理尚不清楚。本项目通过基因筛选克隆、转录表达分析、基因功能及转录调控等分析，探讨了葡萄风信子MYB抑制子响应花发育信号协同激活子精细调控花青素苷合成和花瓣呈色的分子机理。结果表明：①克隆获得了三条R2R3-MYB抑制子基因MaMYB4、MaMYB4-1、MaMYB4-2序列，表达谱分析显示它们与花青素苷的积累关系不甚密切，转基因烟草花色表型变化也不甚明显，推测这类MaMYB4抑制子可能主要参与苯丙烷早期途径的调控，在花青素合成途径发挥着非主要的功能。②克隆获得了R3-MYB抑制子MaMYBx基因序列；qPCR显示在葡萄风信子花发育过程中MaMYBx的表达模式与MaMybA和MaAN2相似，且与花青苷的积累呈正相关；双荧光素酶实验结果显示MaMybA单独或者MaMybA/MaAN2协同辅助因子MabHLH1可激活MaMYBx转录表达；MaMYBx可与MaMybA/MaAN2竞争MabHLH1抑制自身及MaMybA的启动子活性；MaMYBx可与MaMybA/MaAN2竞争MabHLH1抑制花青苷合成关键基因MaDFR和MaANS启动子活性和花青苷积累；在模式植物烟草中过表达MaMYBx可抑制烟草内源花青苷后期合成基因及关键调控基因的表达，从而大大抑制了花瓣中色素的积累，使得花色变浅。总之，本研究阐明了葡萄风信子MaMYBx通过与MaAN2和MaMybA竞争ＭabHLH1精细调控花青素苷合成及花瓣呈色的分子机理，进一步完善了葡萄风信子花色代谢的精细调控网络，为人工调控葡萄风信子及其它单子叶观赏植物中花青素苷的生物合成提供特异基因资源和新的理论依据。