逆境胁迫对茅苍术药效成分合成积累的影响及分子调控机制

Atractylodes lancea (Thunb.) DC. is a kind of perennial herb in Compositae and is famous in Jiangsu province. Due to the multiple atrocious weather of genuine producing areas，the variation of A. lancea's effective components accumulation is relatively large，and its quality is uneven. The key of manufacturing procedure of A. lancea is how to ensure its high and uniform quality. During the process of A. lancea's quality forming， numbers of environmental stresses impact the synthesis and accumulation of its effective components. However，the influence and mechanism of adversity stress on the synthesis and accumulation of A. lancea's effective components is still unclear.This affects its quality improvement.The main medical components of A. lancea are sesquiterpenoids and the biosynthetic pathway has been elucidating. The expression of its key enzyme genes has been compared and analyzed；meanwhile，the conservative fragments of its key enzyme genes have been cloned. In the project，based on the previous studies，the influence of adversity stress on synthesis， accumulation of the medical components and genetic variation will be studied by analyzing the expression of medical components，key enzyme gene ，physiological and biochemical indices as well as medical components under adversity stresses.The mechanisms of tolerance，influence and molecular control mechanism of adversity stress on synthesis and accumulation of A. lancea's medical components will be revealed thereby.Our research will provide the basic information for exploring the quality forming process，the cause of genuine medicinal materials， and breeding and cultivation of A. lancea.This will provide guidance to the study of adaptation mechanism to the ecological environment and the quality theory of the herbs.

茅苍术为菊科植物，为江苏著名道地药材。由于道地产区多发恶劣气候，造成药效成分的积累变异较大，药材质量参差不齐。如何在药材生产过程中保证茅苍术的优质和质量均一已成为茅苍术生产中的关键问题。诸多生态环境胁迫均可影响茅苍术品质的形成，然而逆境胁迫对茅苍术药效成分的合成积累影响及其机制如何尚未阐明，从而影响了茅苍术药材品质的提升工程。倍半萜类为茅苍术主要药效成分，其生源途径已基本阐明，我们已克隆了其关键酶基因保守区片段，并比较分析了关键酶基因的表达。本项目拟在此基础上通过对茅苍术在逆境胁迫情况下药效成分关键酶基因表达、生理生化指标及药效成分的分析，探讨逆境胁迫对茅苍术药效成分合成积累、遗传变异的影响，以揭示其对茅苍术抗逆机制、药效成分合成积累的影响及分子调控机制，从而为探索茅苍术药材品质形成过程、道地药材的成因及育种栽培提供基础资料，对研究植物类药材生态环境适应机制及品质理论具有示范性和指导作用。

在茅苍术品质形成过程中，诸多生态因子均可影响茅苍术次生代谢物的合成与积累。课题组运用生理生化指标测定技术、RACE技术、qRT-PCR、HPLC、LC-MS、GC-MS、ICP-MS等方法对茅苍术在不同逆境胁迫下生理生化指标、关键酶基因表达及其药效成分积累等方面进行了研究。结果表明，逆境胁迫会影响茅苍术生理生化响应，药效成分的关键酶基因表达，从而影响茅苍术的品质形成。具体结果如下：水涝前期适当胁迫有助于果后与休眠期茅苍术根茎FPPS、HMGR基因表达，超过24 h时茅苍术各项生理机能开始衰退；干旱胁迫初期可通过自身调节缓解伤害，建立了茅苍术正常和干旱胁迫下根茎及叶的转录组数据库，筛选出12个干旱候选基因及内参基因UBQ2和EF-1α；酸雨胁迫初期茅苍术生理生化指标均有所回升，pH≤3.0时两种关键酶基因表达及药效成分的积累均受到抑制；适当的温、光胁迫有利于茅苍术有效成分积累，但超过其耐受力则会严重抑制茅苍术叶片FPPS、HMGR基因的表达量；铜胁迫浓度为100 mg/kg 时可促进茅苍术的光合荧光能力、挥发油得率及FPPS表达，而铅胁迫并无促进作用，茅苍术根茎中β-桉叶醇、苍术酮含量和关键酶基因表达量大致上与铜、铅浓度呈负相关，且两者胁迫浓度超过300 mg/kg 时各生理生化指标均呈下降趋势；抗氧化酶活性及光合等指标随汞、镉浓度呈先升后降趋势，当其浓度分别为10 mg/kg、5 mg/kg时达最大值，茅苍术根茎中三种有效成分含量大致与汞、镉胁迫浓度呈负相关，与关键酶基因表达量呈正相关；外源性水杨酸可有效缓解汞、镉胁迫对苍术光合机构带来的损伤，其中浓度为1 mmol/L时效果最佳；5~10mmol/L外源氯化钙可有效缓解高温伤害。此外，课题组利用RACE技术克隆获得 AlFPPS 和AlHMGR基因的全长 cDNA，初步证明AlFPPS 基因是茅苍术倍半萜类成分生物合成途径中重要调控位点，建立了茅苍术根茎及叶的转录组学平台。研究结果为探索茅苍术药材品质形成过程、道地药材的成因及育种栽培提供基础资料，对研究植物药材生态环境适应机制、品种变迁规律及品质理论具有示范性和指导作用。