土壤因子镧促进丹参有效物质积累的信号转导和分子机制研究

To clarify the mechanism of quality formation of traditonal Chinese medicine (TCM) is the core content of resources science of TCM. However,the most studies were focused on macroscopic description of the ecological factors and active constituents, which can't elucidate the environmental mechanism of TCM quality formation clearly. Our study shows that lanthanum (La) in soil is an important ecological factor of the formation of the quality of S. miltiorrhiza , which promotes the accumulation of active ingredients in Salvia miltiorrhiza, and significantly enhances the gene expression of key enzyme in the synthesis. Taking the above results as an opportunity ,the theory and methods of signal transduction and molecular biology will be introduced and the thinking "La→signaling molecules and the mechanism of cross and talk→gene expression→the accumulation of active ingredient" will be used in this project. Firstly, the characteristics of absorbing La in different parts and different growth stages of S. miltiorrhiza will be clarified and the effects of La on the accumulation of active ingredient will be studied. Secondly, the signaling molecules involved in La-induced active ingredient biosynthesis and their cross and talk mechanism will be explored. Finally, the effects of La on the expression of key enzyme in diterpenoid quinones biosynthesis and the mechanism of hormesis effect between La, gene expression and active ingredient will be explored. The mechanisms of La-induced active ingredient biosynthesis in S. miltiorrhiza will be clarified from the angle of signal transduction and gene expression, which will provide foundation for revealing the mechanism of environment influencing the quality of S. miltiorrhiza, and provide a theoretical basis for stability and improvement of the quality of S. miltiorrhiza.

阐明药材质量形成的环境机制是中药资源学研究的核心内容。目前大多数研究集中在药效成分与生态因子的宏观描述上，距离阐明生态因子影响药材质量形成的机制还相差甚远。申请人前期研究发现，土壤中稀土镧与丹参中有效物质的含量呈显著正相关，而且能显著提高其合成中关键酶基因的表达，是影响丹参质量的重要土壤因子。本项目拟以此为契机，引进细胞信号转导和分子生物学的理论和方法，沿着"镧→信号分子及应答机制→基因表达→有效物质积累"的思路，首先，探讨丹参对镧的吸收特点及镧对丹参有效物质积累的影响；其次，揭示镧促进丹参有效物质积累的信号分子及应答机制；最后，阐述丹参有效物质合成中关键酶基因对镧的响应特点以及镧与基因表达、成分之间的Hormesis效应及机制。本项目将从信号和分子水平阐述土壤因子镧促进丹参有效物质积累的机制，为揭示丹参质量形成的环境机制奠定基础，为生产中丹参栽培土壤的选择和施肥方案的确定提供理论依据。

阐明药材质量形成的环境机制是中药资源学研究的核心内容。目前大多数研究集中在药效成分与生态因子的宏观描述上，距离阐明生态因子影响药材质量形成的机制还相差甚远。本项目采用细胞信号转导和分子生物学的理论和方法，沿着“镧→信号分子及应答机制→基因表达→有效物质积累”的思路，研究了LaCl3对丹参毛状根活性成分和关键酶基因表达的影响，在此基础上借鉴“Loss and gain”策略，利用信号分子淬灭剂和合成酶抑制剂确定介导镧诱导丹参次生代谢途径的信号分子及其转导机制。主要研究结果包括：①明确了LaCl3诱导对酚酸类成分及其关键酶基因的影响及其“剂量”效应关系。LaCl3诱导对迷迭香酸和丹酚酸B表现为显著的“抛物线”效应，即低浓度促进和高浓度抑制，0.01 mmol•L-1为迷迭香酸和丹酚酸B的最佳诱导浓度。LaCl3诱导对关键酶基因C4H、4CL、TAT和HPPR均表现出显著性影响，并且基因表达变化规律与活性成分变化相一致。②明确了LaCl3诱导对丹参酮类成分及其关键酶基因的影响及其“剂量”效应关系。LaCl3诱导对二氢丹参酮和隐丹参酮表现为随着诱导子浓度升高，活性成分含量升高；而对丹参酮Ⅰ和丹参酮ⅡA表现为显著的低浓度促进和高浓度抑制效应。LaCl3诱导对关键酶基因FPPS和GPPS表现出显著性影响，FPPS对诱导子响应表现为随着诱导子浓度升高，基因表达水平升高，而GPPS表现为显著的低浓度促进和高浓度抑制现象。③明确丹参活性成分和关键酶基因之间的相关性。4CL与迷迭香酸和丹酚酸B呈显著正相关（P<0.05），FPPS与二氢丹参酮和隐丹参酮呈显著正相关（P<0.05），4CL和FPPS可考虑作为今后研究丹参活性成分质量差异重点的靶基因。④筛选并验证了JA是介导LaCl3诱导丹参次生代谢途径的信号分子。⑤LaCl3对活性成分、关键酶基因和信号分子的诱导结果符合“Hormesis”效应。本项目将从信号和分子水平阐述土壤因子镧促进丹参有效物质积累的机制，为揭示丹参质量形成的环境机制奠定基础，为生产中丹参栽培土壤的选择和施肥方案的确定提供理论依据。