LjMYB1响应干旱胁迫调控金银花类黄酮合成的分子机理研究

Environmental factors are one of the main factors in the formation of famous region drugs. The research on the growth and development of Lonicera japonica and the quality of medicinal materials is quite clear. However, there are few reports on the mechanism of metabolic regulation, which seriously restricts the study on the geoherbalism of Lonicera japonica. In the previous study, we found a MYB family key transcription factor LjMYB1, which may be involved in the positive regulation of the biosynthesis of flavonoids in Lonicera japonica by drought stress. This project will focus on the epigenetic mechanisms of LjMYB1 and its role in flavonoid biosynthesis. At first, with analysis of the flavonoid content and the DNA methylation, histone methylation and acetylation levels of the control and treatment with DNA methylation inhibitors, demethylation inhibitors and histone modification inhibitor, we will determine the relationship between the epigenetics and flavonoid content. Secondly, using subcellular localization, transcriptional activation, EMSA and fixed point mutation technology, we will reveal its transcript function. We also will initially revealed the function of the gene by analysis of the expression level of LjMYB1 and key genes which were related to flavonoid biosynthesis in overexpression and RNAi transgenic Lonicera japonica. Thirdly, we will resolve the mechanisms of LjMYB1 in the regulation of flavonoid biosynthesis by CHIP-sequencing and EMSA. Finally, these findings will reveal the molecular mechanism of ecological factors on flavonoid biosynthesis and lay foundation of regulating cultivation measures to stabilize the quality of Lonicera japonica.

环境因子是道地药材形成的主要因素之一。干旱胁迫对金银花药材品质的影响已比较清楚，然而关于其代谢调控机理的报道却很少，这严重制约了对金银花道地性的研究。申请者在前期研究中发现一个MYB家族关键转录因子LjMYB1，可能参与了干旱胁迫对金银花类黄酮生物合成的正调控。本研究拟在获得金银花MYB转录因子LjMYB1的基础上，开展：①干旱胁迫下LjMYB1的表观遗传修饰及其表达水平的变化。②LjMYB1基因功能分析，包括：LjMYB1的亚细胞定位、转录激活活性；过表达和RNAi转基因金银花的获得及类黄酮生物合成相关基因表达分析，初步揭示该基因的功能。③解析LjMYB1作用机制，包括：利用染色质免疫沉淀测序法，结合凝胶阻滞技术，发掘、验证LjMYB1直接结合的下游靶基因。最终揭示干旱胁迫影响类黄酮生物合成的分子机理，为调控栽培措施、稳定金银花药材质量提供理论依据和技术支撑。

山东道地药材金银花为忍冬科忍冬属植物忍冬的干燥花蕾或初开的花，药用历史悠久，为常用大宗中药材之一。类黄酮是金银花主要活性成分之一，其中木犀草苷已被列入《中国药典》。受环境影响，金银花类黄酮含量差异较大。挖掘调控金银花类黄酮生物合成的关键基因，了解其应答调控机制，对培育金银花新种质、稳定金银花质量、指导金银花规范化生产都具有重要意义。本项目主要开展了4 个方面的研究：①分析对照及干旱胁迫处理不同时间金银花中Ljmyb的表达、DNA甲基化水平，确定了Ljmyb的表观遗传修饰与其表达变化的动态调控关系；分析对照处理、DNA甲基化抑制剂处理下，金银花株系耐旱性变化及类黄酮含量的变化，初步确定了Ljmyb的表观遗传修饰的确影响类黄酮的生物合成。②构建Ljmyb与GFP融合载体，洋葱表皮中瞬时表达，共聚焦显微镜显示GFP蛋白定位于整个细胞，而LjMYB3-GFP定位于细胞质和细胞核，LjMYB8-GFP和LjMYB63-GFP都定位于细胞核；利用酵母单杂交系统，证实Ljmyb具有转录激活活性。③构建Ljmyb基因过表达载体，转化金银花。各转基因株系中LjMYB的表达水平差异显著，其中株系2和3表达水平最高，显著高于对照和其他株系。各转基因株系中均有绿原酸积累。其中含量最高的为1.43μg/g FW, 最低为0.82μg/g FW, 均高于野生型。说明LjMYB基因的上调表达促进了金银花中绿原酸的生物合成。④分析了Ljmyb过表达对拟南芥抗旱能力及黄酮物质生物合成的影响。干旱胁迫下，转基因拟南芥的发芽率高于野生型拟南芥，其中LjMYB63系列的发芽率最高。转基因株系的总黄酮含量高于野生型，其中LjMYB63-Lines的总黄酮含量最高，是野生型的2.2倍。LjMYB3转录因子可以促进CHS，FLS基因的表达，从而增加黄酮醇类化合物的含量。LjMYB8和LjMYB63可以促进CHS、CHI基因的表达，增加下游相关产物的含量，从而提高原花青素、花青素类化合物含量。总之，通过项目实施，我们初步揭示了干旱胁迫影响金银花类黄酮生物合成的分子机理，为调控栽培措施、稳定金银花药材质量提供理论依据和技术支撑。