灯盏花转录因子EbP1调控灯盏乙素生物合成的分子机制研究

The scutellarin is core active ingredient at Erigeron breviscapus which is one of the most important developed and folk characteristic medicinal plants in Yunnan Province. There is great theoretical and practical significance to study the scutellarin biosynthesis and its regulation at Erigeron breviscapus. Based on the previous transcriptome dataset, two EbP1 genes encoding the R2R3-MYB transcription factors were identified and isolated, the multiple alignment and phylogenetic analysis demonstrated that two gene was highly homologues with maize P1 (R2R3-MYB transcription factors), which was provided to regulate the flavonoids biosynthesis. We suppose that two of EbP1 genes might involve in scutellarin biosynthesis regulation. However, little is known about the regulation of the scutellarin biosynthesis pathway in Erigeron breviscapus. In order to verify the hypothesis and to illuminate the molecular mechanisms, the following work will be carried out: (1) EbP1 transgenic plants will be detected and their scutellarin content will be elevated. (2) These two EbP1 genes expression pattern and transcriptional regulation activity will be performed and analyzed. (3) The downstream target genes and interaction proteins involved in EbP1 genes will be indetified. The results could expound that the molecular mechanisms of regulating scutellarin biosynthesis, and be helpful for deeply understanding the regulatory mechanism of scutellarin biosynthesis. The results also will provide a theoretical basis and guidance for the molecular breeding in E. breviscapus.

作为云南重点发展的特色道地药材，灯盏花核心药效成分灯盏乙素的生物合成及其调控研究具有重要的理论和实践意义。前期根据转录组测序结果，已分离克隆了灯盏花中2个编码R2R3-MYB转录因子的EbP1基因，分析表明与已知调控黄酮类化合物生物合成的ZmP1基因高度同源。据此推测2个EbP1基因参与了灯盏花中灯盏乙素的生物合成调控，但灯盏乙素生物合成调控分子机制还知之甚少。为验证该假说并分析EbP1介导的调控机制，基于前期研究基础拟开展以下工作：(1)EbP1转基因植株的分子检测及其灯盏乙素含量分析；(2)EbP1基因的表达和转录调控活性分析；(3)EbP1下游靶基因的调控鉴定及互作蛋白的筛选。研究结果将揭示灯盏乙素生物合成调控的分子机制，加深类黄酮生物合成途径调控机理的认识，为灯盏花分子育种提供新的理论依据和指导。

灯盏乙素是灯盏花的核心药效成分，本研究在灯盏花基因组中进行了R2R3-EbMYB基因家族的成员鉴定，并对其表达特性和激素响应进行了初步分析，利用转基因植物材料，明确了EbP1在灯盏乙素生物合成中的功能和作用机制。主要研究结果如下：在灯盏花全基因组范围内鉴定到108个R2R3型MYB基因，分布于9条染色体上。R2R3型EbMYB蛋白均含有关键保守结构域，根据系统进化关系分为30个亚家族。基于转录组测序分析结果表明，各MYB基因在灯盏花不同组织和外源激素ABA、SA、GA处理不同时间，呈现差异表达。亚细胞定位结果显示EbP1基因定位在细胞核。酵母自激活实验表明EbP1具有自激活活性，并明确转录自激活的结构域为靠近C端的片段。通过EMSA和双荧光素酶报告分析证实，EbP1可以直接结合CHS和CHI等基因的启动子并上调其转录。转录组和代谢组分析EbP1过表达烟草株系，结果表明差异代谢物主要富集在黄酮和黄烷酮生物合成、类黄酮生物合成等代谢通路中。类黄酮合成通路中的关键基因在过表达EbP1烟草中的表达量显著上调，说明EbP1可以通过直接激活类黄酮合成路径关键酶基因的表达，从而促进灯盏乙素的生物合成。本研究丰富了EbP1的分子调控网络，为灯盏花分子育种提供了优质的基因资源。