构树-内生真菌在逆境适应中的互作机制及抗肿瘤次生代谢成分

Our previous researches on the medicinal plant of Broussonetia papyrifera and its fruits (Chushizi ) indicate that the extracts of Broussonetia papyrifera and total Chushizi alkaloids have significant anti-tumor activities, and the plant has widely intra-specific variation and good adaptation to divergent environments with high metal resistance. The symbiotic system of Broussonetia papyrifera and its endophytic fungi changes both in resistance and second metabolism under special circumstances. An endophytic fungus GB48 with significant metal Zinc resistance, along with another endophytic fungus Z1,G7 with anti-tumor activities have been isolated in our lab from the symbiotic system. What is the interaction mechanism of the adversity acclimation of endophytic fungi and Broussonetia papyrifera? In addition, under special eco-circumstances, how are the secondary metabolites changing? In order to illustrate the endophytic fungi mechanism in the adaptation of Broussonetia papyrifera to divergent environments under heavy metal press, to make clear the production of their second metabolism under heavy Zn circumstances. In this project, the populations under heavy metal stress and normal population are to be investigated. The endophytic fungi of Broussonetia papyrifera will be isolated and identified with morphology and molecular biological methods. The heavy metal resistance will be screened by dilution plate method, solid-plate and liquid culture methods. Artificial inoculations, potted plant methods and molecular biological methods are to be used to investigate the interactions and correlations between endophytic fungi and host plant. The antitumor activities of the endophytic fungi will be screened in vitro. The constituents of the bioactive endophyte fermentation are to be isolated and identified and take homologous comparison. This research may provide new approach both for the phyto-remediation of heavy–metal-polluted soil and antitumor candidates from endophyte under special eco-environment.

我们前期对药用植物构树及其果实（中药楮实子）研究中发现，构树提取物、楮实子总碱良好抗肿瘤活性，构树存在广泛种内变异现象，具有较强逆境适应性和重金属耐性。特殊生境下构树-内生真菌共生体系在抗性及次级代谢方面均发生明显变化。从中分得到的内生真菌BP-Z1，G7具有抗肿瘤活性，GB-48具有锌抗性。构树-内生真菌在重金属胁迫下逆境适应的互作机制是什么？特境下次级代谢有何变化规律？为了阐明共生体系逆境适应的形态与分子机制，探讨锌胁迫下内生真菌与构树次级代谢规律，本项目拟对锌胁迫构树-内生真菌进行系统分离鉴定，采用平板稀释法初筛、固体平板和液体培养液复筛获得抗性菌株。通过人工接种、共培养体系及分子标记研究内生真菌与宿主逆境适应的互作机制。采用体外抗肿瘤活性筛选菌株，寻找抗肿瘤活性次生代谢产物，并与宿主同源比较。构树-内生真菌有望成为重金属污染土壤的生态修复及抗肿瘤活性成分的中药新资源。

构树Broussonetia papyrifera Vent.为桑科重要的经济树种和药用植物，也是生态树种，分布广，适应性强。构树存在广泛种内变异现象，具有较强逆境适应性和重金属耐性，部分内生真菌代谢产物具有较好的抗肿瘤活性。为进一步挖掘特殊生境内生真菌资源，探讨构树与内生真菌之间的相互作用机制，本项目对构树-内生真菌在逆境适应中的互作机制及抗肿瘤次生代谢成分进行了研究。通过对锌胁迫下构树内生真菌进行系统分离鉴定，并采用宏基因组方法对铅锌逆境构树内生真菌多样性及分布规律进行研究，包括OTU聚类分析、Alpha、Beta多样性分析、菌群差异分析等结果表明，构树内生真菌具有菌群结构多样性，并存在组织间差异。采用平板稀释法初筛、固体平板和液体培养液复筛获得抗性菌株并保藏于中国典型培养物保藏中心。通过形态、微形态以及分子标记方法，结合生理生化分析研究内生真菌与宿主逆境适应的互作机制，其对Pb2+、Zn2+抗性机制与SOD/GSH/APX活性有关,并发生相关基因的差异表达。采用体外抗肿瘤活性筛选，获得抗肿瘤活性内生真菌链格孢菌Alternaria alternata、燕麦镰刀菌Gibbe rellaavenacea等菌株，其主要次生代谢产物有链格孢素A、C、麦角甾-4,6,8 (14),22-四烯-3-酮、东莨菪素以及脂肪酸等成分，可作为活性天然产物的新来源。构树内生真菌复合体系能够提高逆境适应能力，可为重金属污染土壤的生态修复提供新途径。