基于目标成分"敲出"的地黄化感自毒物质筛选研究

Continuous monoculture problem in medicinal plant culture has became a core problem that hinders the sustanible development of industry chain of traditional Chinese Medicine. Previews researchs indicate that autotoxicity is origin of continuous monoculture disease of Rehmannia glutinosa. In order to indicate the structure and interaction of autotoxic substances, Rehmannia cultivars "wen-85-5" and its rhizosphere soil are the tested material in this research and a strategy based on target constituents knock-out proposed for allelochemical screening. Hence, contributes to provide theory basis and technical support to indicate mechanism of continuous monoculture problems in the production of Rehmannia glutinosa. HPLC-MS were employed to analyze the chemical constituents of effective fractions, and determine target compounds by comparing effective fraction chromatograph of water extraction from rhizosphere soil of Rehmannia glutinosa with that of the same fraction from blank soil. By Using the information of molecular ion peak, fragment ions and their fragmentation patterns, the structure of target compounds was inferred, which guides the isolation and purification of target constituent from water extraction by macroporous resin, LH - 20 gel, ODS or other chromatography. Pot experiments (exogenous adding obtained target constituents or its negative control into the pots for simulated continuous cropping condition) are carried out to test allelopathy effect of obtained target constituents on Rehmannia glutinosa. Thus, the growth and biochemical indexes can be determined and then index of harm can be calculated. Main allelochemical that lead to continuous monoculture problems of Rehmannia glutinosa can be screening with data analysis. Identify allelochemical structure by IR, NMR and MS data analysis. To test the validity of these allelochemical, Pot experiments (exogenous adding allelochemical into the pots) are carried out again and research signal transduction, regulation of gene expression and physiological effects of Rehmannia glutinosa caused by allelochemical.

药用植物的连作障碍问题，已经严重制约了大中药产业链的可持续发展。研究证明，化感自毒作用是地黄连作障碍发生的源头。为了阐明地黄化感自毒物质的种类、结构和作用方式，本研究拟以HPLC-MS技术为依托，以"温85-5"地黄品种和地黄根区土壤为供试材料，通过构建目标成分的敲出技术体系，对化感自毒物质进行筛选，为揭示地黄连作障碍发生机制提供理论依据和技术支持。通过根区和空白土壤水提液指纹图谱的对比分析，确定目标成分，利用其分子离子峰和离子碎片裂解规律初步推断结构；在此基础上采用树脂、凝胶、反相色谱等技术，建立从土壤水提液中"敲出"目标成分的方法；采用盆栽法，对根区土壤水提液、目标成分及其阴性对照液进行连作障碍效应分析；通过生理指标测试、连作障碍率计算和数据分析，找到在地黄连作过程中起主导作用的化感自毒物质或物质群；鉴定化感物质结构并从信号转导、基因调控、植物反应等几个方面着手对其化感自毒效应进行验证。

本项目以广泛种植的地黄品种“温85-5”为试验对象，围绕地黄根区土壤中的化感物质及其连作障碍效应展开，自2013至今，针对地黄根区土壤中的化感物质鉴定、目标化感物质分离、连作障碍效应研究方法等方面进行了较为深入的研究。通过研究建立了地黄根区土壤水提液中的化学成分指纹图谱，并对连作土壤中的特征峰进行了HPLC-MS鉴定，初步鉴定出阿魏酸、对羟基苯甲酸、对羟基苯乙酸、对羟基苯乙醇、香豆酸、香草酸、丁香酸、咖啡酸、梓醇、地黄苷D、水苏糖、毛蕊花糖苷等特征性化学成分；建立了根区土壤水提液中的目标化学成分及成分群“敲出”技术体系；模拟连作地黄盆栽试验，对地黄根区土壤水提液中不同萃取部位、不同化感物质及化感物质群、酚酸类物质单体以及“敲出”酚酸类物质后根区土壤提取液进行了连作障碍效应分析，发现不同萃取部位的化感效应差异较大，且存在拮抗效应，确定了酸酸类成分为在地黄连作过程中起主导作用的化感自毒物质；研究了1-6年不同连作年限地黄生理生态效应及根区土壤中微生态变化情况，发现连作3年左右的土壤微生物种类较少，真菌数量较多，细菌真菌比最低，随着连作年限的增加，连作地黄根际土壤的细菌真菌比又有所上升，根际微生态环境有所改善；并对不同药用植物间套作对地黄连作障碍的消减效应进行了研究，发现地黄/大蒜的地黄的地上地下部分生长发育和生物量积累均优于重茬地黄；对连作障碍的试验方法进行了探索和改良，建立了快速浓缩土壤水提液的方法，为进一步的研究奠定了基础。项目进行期间发表论文3篇，培养硕士研究生5人。