环境因子对藏红花柱头发育过程中藏红花苷积累的调控机制研究

Crocus sativus is characterized by its three red stigmas, whose main medicinal components are crocins, a kind of apocarotenoids. The content of crocins in Crocus sativus from different sources is in the range of 2-35%. The previous studies show that environment factors such as drought stress and temperature and the soluble sugar of the corm have a large effect on crocins content, however the regulatory mechanism is not clear by now. Our preliminary experiments have confirmed that drought stress can enhance the expression of CsZCD with the accumulative increase of crocins content. We speculate that the environment factors may influence the accumulation of crocins through regulating the expression of the key enzyme genes in the crocins biosynthesis pathway. During the progress of the stigmas development, we will cultivate Crocus sativus under different conditions (such as drought stress, temperature and illumination). Meanwhile the contents of β-carotene, zeaxanthin, crocins and picrocrocin in the style and stigma will be determined using HPLC-DAD-ESI-MSn. Similarly, the content of the soluble sugar in the corm will be also determined. In the meantime, the gene expression of CsPSY, CsPDS, CsLCYβ, CsβCH, CsZCD, CsCCD will be detected using RT-PCR. This study will clarify the molecular mechanism of environment factors on the accumulation of crocins, and will find the key enzymes and steps in the crocin biosynthesis pathway. This can provide the basis for the future research to increase the quality of Crocus sativus.

藏红花仅以雌蕊上部三根柱头入药，主要药效成分为藏红花苷类，一种脱辅基类胡萝卜素；不同来源其含量在2-35%。前期研究表明，干旱胁迫与温度等环境因子及种球中可溶性糖对藏红花苷含量影响较大，但机制尚不明确。预实验初步证实干旱胁迫可增强藏红花苷生物合成途径中CsZCD基因表达量，并伴随藏红花苷积累增加；推测干旱胁迫等环境因子可能通过调控藏红花苷生物合成途径系列关键酶基因表达，影响藏红花苷形成、积累。本项目拟在柱头发育过程中，设置不同干旱胁迫、温度和光照等培育条件，同步应用液质联用等技术测定花柱中中间产物β-胡萝卜素和玉米黄素，终产物3种藏红花苷和苦藏花素及种球中可溶性糖含量的动态变化；RT-PCR定量检测6种关键酶基因表达量差异；进行Logistic拟合建模。阐明环境因子影响藏红花苷积累的分子机制，确定藏红花苷合成途径中关键酶及积累瓶颈，对优化藏红花柱头培育环境条件，提高藏红花质量具有重要意义。

藏红花仅以柱头入药，产量极低，柱头药用成分主要为含藏红花苷等脱辅基类胡萝卜素类化合物。本项目主要研究了藏红花培育方式（国内“二段式”室内培育开花，与葡萄套种仿原产地室外培育开花）和环境因子（温度，湿度，光照）对藏红花柱头发育过程中藏红花苷等次生代谢产物积累的调控规律和调控机制，挖掘了促进柱头脱辅基类胡萝卜素积累的关键环境因子。不同条件下柱头发育过程中藏红花苷等积累均呈倒“V”变化规律。与葡萄套种室外培育开花，种球经高温处理，干旱胁迫和黑暗条件下更有利于柱头中脱辅基类胡萝卜素类成分合成与，尤其是干旱胁迫和无光照条件。并分析了柱头发育过程中脱辅基类胡萝卜素生物合成途径各基因的表达规律，揭示了藏红花苷积累呈倒“V”变化的机制，柱头中脱辅基类胡萝卜素含量变化与基因表达变化趋势一致，含量上升阶段生物合成通路基因表达基本上调，含量下降阶段通路上基因表达基本下调，尤其是CCD2下降幅度较大。揭示了与葡萄套种室外培育开花，种球经高温处理，干旱胁迫和黑暗条件提高柱头成分合成的机制，是由于这些条件更能促进生物合成途径中CCD2的表达，CCD2可能是调控藏红花苷等生物合成和积累的关键酶。为进一步以CCD2为标志调控外源和内源因子协同促进柱头中脱辅基类胡萝卜素积累量，提高藏红花质量提供了基础。本项目还以入血成分（苦藏花素以原型入血，藏红花苷以代谢产物入血）为指标建立了HPLC结合UV-Vis一测多评质量评价方法，相较于《中国药典》方法，实现了1个对照品完成藏红花苷类和苦藏花素的含量测定，降低了检测成本，并能全面评价藏红花的质量。本项目为第一标注发表论文6篇，取得一定研究进展，达到预期研究目标，为藏红花代谢调控提供了研究资料。