越南槐根内生真菌生态功能及其与宿主药材品质相关性研究

Guangxi is traditional geo-authentic-producing area of Sophora tonkinensis radix from Sophora tonkinensis Gagnep. with the threat of its resource exhaustion in China, however, Cultivated Sophora tonkinensis radix et rhizome with low biomass and active ingredient can't meet market demand. Give full play to ecological functions of endophytic fungi (EF) from the S.tonkinensis roots promoting the growth and accumulation of bioactive compounds in host plants can improve the growth and crud drug quality of host medicinal plants. Sixteen EF ecological function isolates (EFEI) were isolated from S.tonkinensis roots collected from three different localities in Guangxi. Four isolates could enhance the roots growth in S.tonkinensis. Twelve isolates exhibited much stronger antimicrobial activity than their host. These suggested that EFEI may play a very important ecological role in host plant micro-ecosystems. To full excavate the EFEI resource from S.tonkinensis roots, in this research, we will isolate EF from S.tonkinensis roots collected from different localities in Guangxi, and identify EF species based on morphological characters and molecular characters, and screen for the target EF by study the effects of EF- host symbiosis on the growth, active ingredients and bioefficacy in S.tonkinensis roots, and further investigate the effects of EFEI elicitor on the growth and biosynthesis of active ingredients in S.tonkinensis hairy roots. The objective of this study is to clear understanding of the EF biodiversity in the S.tonkinensis roots, and obtain EFEI, and find out the material basis of EFEI influencing on the active ingredient biosynthesis in S.tonkinensis hairy roots. It is hopeful to provide experimental evidences and scientific basis for revealing the mechanisms of EFEI influence on the quality of S.tonkinensis.

广西是山豆根道地产区,基源植物越南槐野生资源濒临灭绝。栽培越南槐根生长缓慢、有效成分含量低，难以满足市场需求，充分挖掘其内生真菌（EF）促进宿主生长、生物活性成分积累的生态功能，可促进宿主生长、提高宿主药材品质。前期从广西3个产地越南槐根筛选获得16个EF生态功能菌株（EFEI），4株能显著促进根生长，12株抑菌活性比宿主强，发现EFEI在宿主微生态系统中可能扮演重要生态角色。为了全面发掘越南槐根EFEI资源，本研究拟全面采集广西各产地野生越南槐根，从中分离纯化EF并结合形态学和分子生物学对其进行分类鉴定，通过“EF-宿主”共生体探讨EF对宿主根生长、活性成分及生物药效的影响，深入探讨EFEI诱导子对宿主毛状根生长及活性成分生物合成的影响，目的在于明确广西越南槐根EF的物种和分布、筛选获得EFEI、探明EFEI作用的物质基础，为揭示EFEI影响宿主药材品质及其机制提供实验基础和科学依据.

针对广西道地药材山豆根的基源植物越南槐野生资源濒临灭绝，而栽培越南槐根生长缓慢 、有效成分含量低的问题，本项目围绕内生真菌对越南槐根生长和药材品质的影响及机制开展工作。从10个道地产地野生越南槐根系分离获得内生真菌583株，基于形态学特征和ITS序列分析揭示了可培养内生真菌的多样性，发现地理区域和土壤理化性质明显影响内生真菌组成和分布。通过菌株促生理化特性筛选获得促生潜力菌株10株；采用TLC、HPLC技术和UPLC-MS技术筛选获得产苦参碱菌株2株；采用琼脂块法、平板对峙法、菌丝生长法和微量肉汤稀释法筛选获得强抗菌活性菌株13株。该25株活性菌株分别接种越南槐建立共培养体系， 从中确定11株为内生真菌生态功能菌株（EFFI），其中5、2、8株分别能有效提高越南槐生物量、根系苦参碱和氧化苦参碱（指标成分）含量、根提取物生物学活性。将能促生和提高指标成分的7株EFFI诱导子及其诱导子组分分别培养越南槐，确定了EFFI诱导子特别是其蛋白-多糖部位（PEF）能显著影响越南槐根生长、根指标成分生物合成。通过转录组学研究诱导子PEF对越南槐根转录水平的影响，通过其基因表达谱分析探明促生的PEF能上调生长激素合成相关基因，而促指标成分合成的PEF则能上调免疫系统相关基因，初步阐释了越南槐EFFI诱导子对越南槐根系作用的物质基础及可能的分子机制。另外，在本项目资助下，从越南槐根系分离鉴定了内生细菌425株， Burkholderia cenocepacia ETR-B22挥发性活性成分具强的抗病功能，靖西野生越南槐茎、种子内生真菌组均具有强的抑菌功能，越南槐根系内生真菌Arthromyces ramosus同源菌株生成的过氧化物酶（ARP）具有高的催化效率，能快速降解环境抗生素，拓展了越南槐内生菌研究资源和生物学功能；本项目已发表论文6篇，培养已毕业硕士研究生1人。本项目的完成为利用内生菌及其生态功能菌株改善栽培越南槐药材品质提供了菌种资源和科学依据。