可培养内生菌与七叶一枝花品质的相关性及促进机理研究

Research on the promotion mechanism of endophytes on Chinese traditional medicine plants is one of the keys for improvement of the medicinal quality of host plants. In order to resolve the quality degradation of artificially cultivated Paris polyphylla Smith var. chinensis, this project used culturable endophytes of P. polyphylla as research subject to develop a series of research. Firstly, we comprehensively and systematically investigated the diversity, community structure, distributing characteristics of culturable endophytes isolated from the roots, stems and leaves of P. polyphylla, respectively, that collected from 3 main producing areas in China, as well as polyphyllins content of all plant tissue samples. Then, we analyzed the influence of geographic locations and histological types of P. polyphylla on population and distribution of their culturable endophytes. Positively correlated culturable endophytes were screened through using correlation analysis between endophyte populations and the polyphyllins content of host plant samples. Furthermore, the candidate symbiotic promoting strains were determined by co-culture systematic analysis of positively correlated endophytes and P. polyphylla tissue culture seedlings. The promotion mechanism of the candidate endophytes on the growth and medicinal qualities of P. polyphylla was primarily clarified through analysis of the growth indexes, physiological indexes, polyphyllins contents and polyphyllins-related gene expression characters of tissue culture seedlings. These research results would provide theoretical evidences for guarantee and promotion of the medicinal quality of P. polyphylla using endophytic biotechnology.

研究内生菌对中药品质的影响及其促进机理，是利用内生菌改良中药品质的关键。针对七叶一枝花人工栽培过程中品质退化的问题，本项目以七叶一枝花可培养内生菌为研究对象，通过全面系统地考察3个产地七叶一枝花的根、茎、叶3种组织中可培养内生菌的多样性、菌群结构、分布特征以及重楼皂苷含量，分析地理位置和组织类型对可培养内生菌组成和分布的影响，以及内生菌与重楼皂苷含量的相关性及主要相关菌群，筛选与重楼皂苷含量呈正相关的内生菌；并通过内生菌与七叶一枝花共培养体系筛选促生菌株，进一步通过检测目标促生菌对七叶一枝花生长指标、生理指标、重楼皂苷含量以及重楼皂苷合成相关基因表达的影响，揭示目标内生菌促进七叶一枝花生长和药用品质的机制，从而为利用内生菌保障与提升七叶一枝花药材品质提供理论支持。

针对七叶一枝花人工栽培过程中重楼皂苷含量下降、品质退化的问题，本项目围绕内生菌与七叶一枝花品质的相关性和内生菌对其生长和品质的影响及机制开展工作。从3个主产地人工种植七叶一枝花样品的根、茎、叶组织分离获得内生细菌9451株和内生真菌797株；基于其16SrDNA序列和18SrDNA序列分析揭示了可培养内生菌的多样性，发现地理位置和组织类型明显影响可培养内生菌组成和分布。通过检测上述植物样品的重楼皂苷含量及其与可培养内生菌的相关性，筛选到与重楼皂苷含量呈正相关的主要内生菌类群。采用高通量测序技术分析这些植物样品的总内生菌的多样性及其与重楼皂苷含量的相关性，并通过与可培养内生菌的比较分析，发掘了潜在的关键内生菌资源。通过筛选获得具显著促生能力的内生菌49株，完成了12株内生菌的自身促生长特性分析。在人工共培养体系检测了6株促生菌对七叶一枝花组培苗生长指标的影响。分别在自然共培养体系（盆栽试验）和大田小区试验条件检测了10株促生菌和4株促生菌组合对七叶一枝花组培苗和三年生七叶一枝花栽培苗的生长指标、生理指标、重楼皂苷含量以及重楼皂苷合成相关基因表达的影响，初步阐明了这些促生菌及促生菌组合促进七叶一枝花生长和药用品质的机制。检测了6株促生菌对黄芪幼苗生长和黄芪皂苷合成相关基因表达的影响，初步表明本项目所得促生菌也可用于黄芪生长和品质的提高。另外，在本项目资助下，采用高通量测序技术分析了人工种植不同年份的千层塔的内生菌多样性，拓展了药用植物内生菌研究资源。本项目已发表论文11篇，授权发明专利8项，申请发明专利2项，培养已毕业硕士研究生9人。本项目的完成为利用内生菌保障和提升七叶一枝花药材品质提供了菌种资源和科学依据。