荧光标记真菌PL21在滇重楼体内的内生定殖及提质机理研究

Paris polyphylla var. yunnanensis rhizome is a traditional Chinese medicine. Its medicinal active ingredient is steroidal saponins, which has anti-cancer, anti-inflammatory, hemostatic, analgesic and sedative effects. However, the current research on polyphyllins metabolic pathways and molecular biology is scare. This project will focus on the study of fungi strain PC21, which can promote the accumulation of saponins of P. polyphylla var. yunnanensis during our past studies. The study will get the construction of PC21 GFP transformants for inoculating P. polyphylla var. yunnanensis plantlets. Then we will observe the strain infecting process during the colonization, distribution, and the change of saponin content of roots during the inoculation process, so as to determine the critical period of PC21 infection. At the same time, we will carry on the gene expression analysis druing PC21 inoculating in P. polyphylla var. yunnanensis, in order to find the key genes related to the promotion of steroidal saponins in P. polyphylla var. yunnanensis. This project uses the integration of structural botany, microbiology and molecular biology techniques to study corresponding relation between fungal infection and saponin content. The relationship between fungal infection and changes of saponin content will provide the firstly experimental data and scientific basis for the medicinal quality formation, which will also promote the study of genuine theory from the perspective of endophytic fungi.

滇重楼根茎是我国传统中药，其药用活性成分甾体皂苷具有抗癌、镇痛、镇静、抗炎、止血等作用，当前对其甾体皂苷合成途径的分子生物学研究基本处于空白。本项目拟在已建立的滇重楼组培苗技术基础上，以项目组前期发现的能促进滇重楼皂苷积累的真菌PC21菌株为实验材料，通过构建真菌PC21的GFP转化子并接种滇重楼组培苗的方式，观察该菌株在滇重楼中的侵染、定殖、分布规律，并观测植株根茎在真菌侵染过程中的皂苷含量变化，从而确定PC21侵染的关键时期；同时通过基因表达技术分析PC21侵染对滇重楼皂苷合成相关基因表达水平的影响，寻找滇重楼响应PC21侵染、促进甾体皂苷积累的关键基因。本项目通过整合结构植物学、微生物学和分子生物学等技术手段，在组织与细胞水平上分析真菌侵染与皂苷含量变化的对应关系，为揭示真菌影响中药材品质机制提供科学依据，为从内生真菌角度阐释药材道地性形成进行有益的探索。

本项目研究了前期分离到的真菌菌株在滇重楼中的侵染、定殖、分布规律，并观测植株根茎在真菌侵染过程中的皂苷含量变化，同时通过基因表达技术分析侵染对滇重楼皂苷合成相关基因表达水平的影响。为揭示真菌影响中药材品质机制提供科学依据，为从内生真菌角度阐释药材道地性形成进行有益的探索。.项目将促生菌单一回接至滇重楼植株，不同共生菌对滇重楼幼苗生长均有不同程度的促进作用，以PL21对株高、根长、根重、叶长、叶宽、地上鲜重的促进作用最显著；能有效刺激滇重楼幼苗对重楼皂苷的合成，显著增加滇重楼总皂苷含量。所有处理都不同程度的提高了皂苷合成通路基因的表达。项目还研究了共生菌抗病特性，促生组和拮抗组植株生理指标均有不同程度的增加，对病原菌接种促生拮抗菌后的病情指数和抑制率进行分析，接种促生拮抗菌对疫霉起拮抗作用，菌株处理组的生长及抗性相关基因均表达上调。项目还进一步研究了共生菌互作对滇重楼生长及甾体皂苷积累的影响，中度干旱下双接菌与更有利于滇重楼体内激素含量的增加；营养元素的吸收、平衡；总皂苷含量增加，并且中度干旱下接种还不同程度的上调某个或多个甾体皂苷合成路径上关键基因和UGTs基因的表达，从而达到刺激滇重楼根状茎合成并积累甾体皂苷的促进作用。双接菌与适当的土壤干旱结合影响滇重楼的重楼皂苷含量的积累，提高滇重楼的品质。