参与NO信号途径介导UVB诱发银杏黄酮合成的小RNA研究

Regulation of secondary metabolism of medical plants by environmental factors plays important roles in formation of curative quality of the traditional Chinese medicine. However, the mechanisms involved in the regulation of secondary metabolism of medical plants by environmental factors are still largely unknown so far. Biosynthesis of flavonoids in plants is widely influenced by solar UVB, which is, therefore, considered to be a ideal model to investigate the mechanisms of the regulation of secondary metabolism of medical plants by environmental factors. In previous studies, we found that NO signaling triggered by NIA1 gene play a key role in mediating UVB-induced flavonoid biosynthesis in plants. However, the other signaling members of the pathway are still unknown. Recently, microRNAs have been reported to be involved in responding the biotic and abiotic stresses and in regulating secondary metabolism of medical plants. However, it is still unclear that whether microRNAs could mediate UVB-induced flavonoid biosynthesis through the NO-dependent signaling pathway. In this project, we plan to discover the microRNAs that mediate UVB-induced flavonoid biosynthesis via the same signaling pathway as NO and explore the relationship between microRNAs and NO in mediating UVB-induced flavonoid accumulation of Ginkgo biloba cells using the strategy of gain and loss of functions and deep sequencing technology. The results will be of great importance for understanding the molecular mechanisms in the regulation of secondary metabolism of medical plants by environmental factors.

环境因子对药用植物次生代谢的调控作用是中药材疗效品质形成的重要基础。然而，目前对环境因子调控药用植物次生代谢的机制尚不十分清楚。太阳光UVB对植物黄酮合成具有广泛调控作用，是研究环境因子调控植物次生代谢机制的重要模型。我们前期研究发现，由NIA1基因触发的NO迸发是介导UVB诱发植物黄酮合成的重要信号转导途径，但是目前对该途径的其它信号转导成员所知甚少。最近研究表明，小RNA可以响应外界UVB刺激并参与药用植物次生代谢的调控作用。然而，目前尚不清楚小RNA与NO是否可以通过相同信号通路介导UVB诱发药用植物黄酮合成。本项目拟以银杏细胞为材料，采用靶基因定向匹配分析、体外验证及细胞内功能试验等手段发掘与NO通过相同通路介导UVB诱发银杏黄酮合成的小RNA，解析其与NO在介导UVB诱发银杏黄酮合成中的信号转导关系。研究结果对深入理解UVB调控药用植物黄酮合成的信号转导机制具有重要意义。

细胞信号转导作用是介导UVB等环境因子诱发植物次生产物合成的重要机制，阐明药用植物细胞中介导次生活性产物合成的信号转导通路是研究环境因子影响中药材疗效品质形成机制的基本科学问题。前期研究表明，依赖nia1的NO信号途径是介导环境因子调控药用植物次生代谢的重要机制，但是目前对该途径的成员所知甚少。小RNA广泛参与植物生长发育等多种生命活动，但是目前尚不清楚小RNA是否参与药用植物次生代谢信号转导过程。本项目采用高通量测序的方法检测到UVB处理下银杏细胞中16个差异表达的小RNA。通过对差异表达小RNA靶基因预测发现了两种可能靶向nia1基因的小RNA：miR397a、miR5020b，并通过双荧光素酶活性分析对miR397a和miR5020b靶向作用nia1基因进行体外生化验证，结果表明miR397a对nia1具有调控作用，而miR5020b对nia1无直接调控作用。构建了miR397a过表达载体和转基因银杏细胞系，对miR397a靶向nia1进一步进行了体内验证。在此基础上建立了UVB通过抑制miR397a上调nia1表达、激活胞内NO信号促进黄酮合成的作用模式。分析了UVB处理后的银杏细胞和经过NO信号淬灭剂预处理的银杏细胞小RNA差异表达谱，发现了8个可能作用于NO下游响应UVB胁迫的小RNA。靶基因预测结果表明，其中miR166a和miR837可能分别靶向黄酮生物合成基因FLS2和FBM。双荧光素酶体外分析和过表达miR166a和miR837的体内实验结果进一步证实了上述推测，说明UVB可以通过NO下调miR166a和miR837表达，解除其对FLS2和FBM的抑制作用，激活黄酮生物合成途径，促进银杏黄酮积累。综合上述实验结果提出了miR397a、miR166a和miR837分别通过作用于NO上、下游介导UVB诱发银杏黄酮合成的作用模型，验证了项目的研究设想。