石蒜属植物生物碱对花色苷呈色影响机制的研究

Lycoris Herb. belongs to Amaryllidaceae family of perennial plants, and well known for their ornamental and medical values, therefore the interest of plant breeding is high. With pharmacological activities ,alkaloids have also been discovered in petal cells, and their content was first detected the positive correlation with flower color from red– violet/blue /white – yellow. By inference, alkaloids can increase vacuolar pH to change color and stability of anthocyanins. Moreover, little knowledge of vacuolar pH in petal cells of Lycoris species as well as the structures characters of alkaloids is available. In this study, we want to verify regular correlation with vacuolar pH, anthocyanins and alkaloids in petal cells of different species of Lycoris, by confocal laser scanning microscopy and liquid chromatography-mass spectrometry techniques, and evaluate effect of absorption, acid-base equilibrium constants and the activation energy of anthocyanin by different alkaloids in vitro.It would clarify that pKa value of alkaloid is the key factors to affect the coloration of anthocyanin, and explores a new way to find Lycoris species with high content of alkaloids through flower color, and cultivate new varieties of colors by regulating the alkaloids.

石蒜科石蒜属植物为著名的药赏两用多年生植物，因此新品种的选育受到广泛关注。石蒜花瓣中也含有药用活性生物碱成分，并且首次发现红花-紫/蓝/白花-黄花中生物碱含量呈上升趋势，推论生物碱提高了液泡pH值而改变了花色苷的呈色和稳定性。然而，石蒜花瓣细胞液泡pH值和不同结构生物碱影响差异还没有报道。本课题采用共聚焦激光扫描显微镜和液质联用仪技术，验证不同石蒜属植物花瓣细胞液泡pH值、花色苷和生物碱相互联系的动态变化规律，并在体外测定不同结构的生物碱对花色苷吸收值、酸碱平衡常数和活化能的影响，阐明生物碱pKa值是影响花色苷呈色的关键因素，为从花色来寻找高生物碱含量的药用石蒜资源，以及通过调控生物碱来培育花色新品种探索新途径。

石蒜科石蒜属植物为著名的药赏两用多年生植物，因此新品种的选育受到广泛关注。前期研究证明花色与生物碱含量具有相关性。课题建立了石蒜花瓣活细胞色素与生物碱的定位方法，证明石蒜花瓣中花色苷和生物碱都存在于液泡中。通过BCECF-AM染色和共聚焦激光显微镜技术测定荧光强度从而分析石蒜花瓣活细胞pH值，液质联用仪技术定性定量分析了石蒜生物碱和花色苷成分，证明花瓣液泡的pH值橙色花-黄花-白花-红花递减，总生物碱含量递减。总结了石蒜生物碱的质谱裂解规律，可以应用于含有苄基苯乙胺、四氢异喹啉和吡咯烷类生物碱的植物鉴定。测定了花色苷的其他共色素黄酮和金属离子，Fe的含量与红色矢车菊素桑布双糖苷正相关。pH差异法体外测定不同结构的生物碱对矢车菊素桑布双糖苷吸收值、酸碱平衡常数和活化能的影响，证明8种结构类型的生物碱对花色苷的影响有差异，其中文殊兰碱型生物碱结构中含有N-乙基桥，pka13-14，能提高矢车菊素桑布双糖苷活化能，增加稳定性，从而在红色和蓝紫色花色中具有稳定调节作用。对变色花瓣进行研究，同一花瓣的红色区域的pH值低于白色区域，蓝紫色区域的pH值高于黄色区域，蓝紫色区域的生物碱含量较高，并且已发现的18种石蒜科生物碱骨架中，文殊兰碱型生物碱在pH高的区域含量较高，进一步证明体内文殊兰碱型生物碱对花色苷存在共色作用。后期工作将解决培养文殊兰碱型生物碱和矢车菊素桑布双糖苷的混合单晶的难题，从分子水平上解释相互作用的结合位点。之后研究文殊兰碱型生物碱生物调控基因和蛋白，为从花色来寻找高生物碱含量的药用石蒜资源，以及通过调控生物碱来培育花色新品种探索新途径。