基于内生真菌群落结构地域性差异的铁皮石斛产地分子标签研究

With the development in planting technique, as well as the effects of big market and great circulation, introduction and transplantation of some Chinese traditional medicines are booming rapidly. This weakened the regional characteristics of medicinal materials from different cultivation areas and then leaded to the deficient of effectiveness in locality identification by present technology. As a natural part of plant microecology, endophytes are closely related to the ecological factors such as soil microbial, showing its regional characteristics in community structure. In the early study on Dendrobium officinale, we found that, compared to traditional means of endophytes single cultivation method, metagenomics can provide abundant species information, accurately reflect the community structures of the endophytic fungus in Dendrobium officinale from different cultivation areas, thus meet the requirement in distinguishing the real regional differences of endophytic fungus community structure. Therefore, we put forward the idea of the origin label of Dendrobium officinale based on the regional differences in community structure of endophytic fungus. Via comparing the difference in community structure of the endophytic fungus in Dendrobium officinale from different cultivation areas during the different phases, such as seedling breeding, soil cultivation, harvesting and processing, we hope to make clear the process and regularity in the construction of community structure, and elucidate the causes of difference in the community structure of endophytic fungus and the relationship with ecological factors such as the soil microbe and clarify the mechanism about the formation of regional characteristics. Select the endophytic fungus with stable and obvious regional characteristics as origin identification indicator, and construct the origin molecular label based on their characteristic gene sequences. Establish evaluation mechanism and method of the identification of cultivation area, and verify the reliability and stability of the origin molecular label. The development of origin molecular label will provide a new technology and method for the origin distinction and genuine identification of medicinal materials.

随着种植技术的发展，部分中药材引种栽培发展迅速，原有地域性栽培模式被打破，药材地域特征被弱化，现有产地鉴别技术存在区分度下降问题。内生真菌作为植物微生态的组成部分，既与环境微生物紧密关联又独具特性，其群落结构存在地域性差异。我们在前期研究中发现，较之传统的纯培养手段，宏基因技术能更全面、准确地反映各产地铁皮石斛内生真菌的群落结构特征并揭示其差异性分布规律。据此，我们提出基于内生真菌群落结构地域性差异的产地标签研究思路，利用宏基因数据比较不同产地铁皮石斛从种苗到采收加工各时期内生真菌群落结构的差异及其与环境微生物的关系，阐明群落结构构建过程和规律，分析地域性差异的形成机制，筛选具有显著、稳定的差异性分布菌株作为产地指示菌，利用其特征性核苷酸序列构建铁皮石斛产地分子标签，建立评判机制和方法并验证其可靠性、稳定性。产地分子标签的发展将为中药材原产地区分及道地性鉴别提供新的技术手段和方法。

我国幅员辽阔，环境多样，不同产地出产的中药材品质良莠不齐。在长期的生产实践中，逐渐形成“道地药材”概念，对优质中药材冠以产地标签。这种将“产地标签”作为中药材质量控制手段的理念和方法在中医药学发展进程中历经检验，行之有效。随着经济与社会的发展，道地产区已难以满足现代市场体系对中药材的巨大需求，部分中药材引种栽培发展迅速，原有地域性栽培模式被打破，药材地域性特征弱化，现有产地鉴别技术区分度下降。筛选新的产地间差异评价指标来满足中药材产地区分和鉴别需要具有重要的现实意义。. 内生真菌作为植物微生态的组成部分，既与环境微生物紧密关联又独具特性。本课题用宏基因测序技术比较了浙江、云南和四川不同产地人工栽培铁皮石斛各年份、各季节田苗及市售品中内生真菌群落结构信息，阐明了铁皮石斛内生真菌群落结构的构建过程及其与环境微生物的关系。结果表明，铁皮石斛在大规模异地引种栽培的背景下，不同产地铁皮石斛种苗来源、栽培基质以及栽培方法趋同，但具有地域特性的灌溉用水和空气等环境因素在铁皮石斛内生真菌群落结构的构建中仍能形成地域性差异并可据此对铁皮石斛各类市售品进行产地区分。铁皮石斛市售干品由于经历高温干燥等加工过程，内生真菌群落结构发生显著变化，但地域性差异仍足以进行产地区分。本课题综合考察内生真菌物种出现概率与丰度两个因素筛选出了可用于产地区分的指示菌20株，利用其核苷酸序列构建了分别适用于铁皮石斛鲜品和干品的两套产地分子标签，建立了基于内生真菌群落结构地域性差异的铁皮石斛产地分子标签的技术体系和应用方法。. 本研究证实了环境条件差异造成的环境微生物群落结构差异可以传递到植物内生真菌群落结构中，使得内生真菌群落结构被打上环境的烙印，并可用于中药材产地区分，解决大规模引种栽培背景下中药材产地间区分度下降问题，为中药材原产地区分及道地性鉴别提供创新性的技术手段，将在中药材的质量控制中发挥重要作用。