药用牡丹内生细菌抗菌抗肿瘤活性次生代谢产物的研究

Polyketides（PKs) and nonribosomal peptides (NRPs) secondary metabolites with diverse bioactivities are produced by plant endophytes. The polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) gene-based molecular approach can be applied to efficient screening of endophyte-derived novel bioactive natural products. The root of medicinal tree peony contains many kinds of pharmacological compounds, such as anti-microbial, anti-inflammatory and anti-tumor activities compound. Preliminary results indicated that many endophytic bacteria existed in tree peony root, and the nonribosomal peptide compounds produced by some bacteria exhibited inhibitory activity on mycelium growth of peony pathogenic fungi. In this study, endophytic bacteria will be isolated further from medicinal tree peony root. The novel bioactive peptide and polyketide-producing capability of endophytic bacteria will be assessed based on approach of polymerase chain reaction (PCR) screening of function gene associated with the PKS and NRPS. The transcriptional expression levels of PKS and NRPS gene will be studied to determine the optimal fermentation conditions of the aimed strains. The bioactive compounds will be isolated and purified from the active strains by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods. The structures of these compounds will be elucidated by means of spectroscopic methods, and the compounds will be evaluated for their antimicrobial and antitumor activities. The microorganism resources and new natural active compounds with particular function and potential application will be obtained through this research. This study will lay the foundation for the correlation between microbial functional gene and microbial secondary metabolites.

植物内生菌产生的聚酮类和非核糖体肽类次生代谢产物具有多种生物活性，基于聚酮合成酶（PKS）和非核糖体多肽合成酶（NRPS）功能基因定向筛选，预测内生菌产生新的次生代谢产物的能力，成为发现新颖天然活性物质的重要策略之一。药用牡丹根皮具抗菌、消炎和抗肿瘤等多种生物活性，前期研究表明牡丹根皮中存在大量的内生细菌，部分菌株的非核糖体肽类化合物对牡丹病原真菌具有较好的抑制作用。在前期工作基础上，继续对药用牡丹根皮中的内生细菌进行分离，采用功能基因定向筛选技术探讨内生菌株合成新型聚酮类和非核糖体肽类化合物的能力；根据功能基因转录组表达水平优化目标菌株发酵条件；利用现代色谱和波谱技术，分离、纯化并鉴定活性代谢产物的结构；生物活性追踪其抗菌抗肿瘤活性。研究结果将为抗菌抗肿瘤药物提供产生菌及其结构新颖的候选化合物，并为探讨微生物功能基因与微生物次生代谢产物间的相关性研究奠定基础。

植物内生菌在与植物共进化过程中，可产生丰富多样的次生代谢产物。药用牡丹品种‘凤丹’（Paeonia ostii）根皮具抗菌、消炎和抗肿瘤等多种生物活性，其根部组织中内生细菌的种群多样性，以及内生细菌产生的次生代谢产物的多样性和新颖性，能够为药用牡丹内生细菌次生代谢产物的开发利用提供科学依据。本项目采集‘凤丹’根部组织，分离获得内生细菌124株，其中4株内生细菌对牡丹病原真菌抑菌效果显著，经鉴定4株内生细菌均为芽胞杆菌属（Bacillus）细菌。利用PCR扩增的方法筛选‘凤丹’根部内生细菌中包含PKS和NRPS功能基因的菌株，其中13株内生细菌含有PKS功能基因，5株内生细菌含有NRPS功能基因，PKS功能基因主要与抗生素bacillaene的生物合成密切相关，NRPS基因片段主要与脂肽类物质的生物合成密切相关，表明‘凤丹’根部内生细菌中具有丰富的PKS和NRPS序列片段，且与聚酮类化合物与非核糖体多肽类次生代谢产物的合成具有显著的同源性。荧光定量PCR分析目标菌株Md1-33、Md2-17和Md2-35在与牡丹病原菌对峙培养过程中，其表面活性素、丰原素和伊草枯菌素基因的表达量，结果发现丰原素和伊草枯菌素基因被诱导表达，表明丰原素和伊草枯菌素脂肽类物质在抑制牡丹病原真菌生长的过程中起主要作用。酸沉淀和甲醇抽提获得3株目标菌株Md1-33、Md2-17和Md2-35的脂肽类粗提物，Sephadex LH-20凝胶层析柱分离脂肽类物质粗提物，收集组份，测定各组份对牡丹病原真菌的抑菌活性，基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析具有抑菌活性成分的脂肽类物质，获得对牡丹病原真菌具有抑菌活性的化合物Bacillomycin D和Iturin B。本研究初步探明了微生物功能基因组与微生物次生代谢产物间的相关性；通过基因筛选-化学筛选和活性追踪相结合的手段，获得了目标菌株中具有抗菌活性的化合物Bacillomycin D和Iturin B，为具有抗菌活性的生物农药的研发提供了资源。