广西北部湾红树林根际放线菌生物多样性及其次生代谢产物的研究

Mangrove forests are unparalleled productive ecosystems of intertidal coasts in tropical and subtropical coastal districts. The ecosystems exhibit characteristics "high salinity, extreme tides, strong winds, high temperature and muddy, anaerobic soils". Nowdays, more and more reports imply that actinomycetes population play important ecological roles considered as one of the major group of the unique habitat. The mangrove environments has become an invaluable source for discovering novel actinomycetes hence new chemical diversity. So, a well-defined biodiversity and taxonomic study of actinomycetes is valuable to understand actinomycetes from the mangroves environment.Actinobacteria play an important role in life cycles in the cycling of organic matter in the soil ecosystem. Actinomycetes maintain a distinguished role due to their diversity and proven ability to produce novel compounds, so the discovery of new actinomycetes, especially of rare actinomycetes, is becoming increasingly important. Many studies have been done to isolate and identify novel actinomycetes by conventional Petri dish plating. Indeed, the tranditional methods are steadily out of action, which is likely due to repeated isolation and screening of the identical actinomycetes, the rediscovery of ordinary antibiotics from cultivable species is a major obstacle for antibiotic discovery. The rRNA and metagenomice approaches promised to provide access to the uncultivated species while bypassing the problem of uncultivability. Significant departures from traditional protocols were clearly in order, and indeed, the novel methods substantially diverged from the cultivation convention by adopting single-cell and high-throughtput strategies,better mimicking the habital and increasing the length of incubation. New method- isolation procedure and molecular biology technique bring new hope to get new actinomycetes. The distribution of bioactive actinomycetes showed the corelation between the nature and source of the environmental samples. The highest ratio isolates showing anti-tumor and anti-infective were got from rhizosphere soil samples. These findings are not only appealing but also will advise next bioprospecting strategies..The present study was to combine two kinds strategies to observe the biodiversity and chemdiversity of the actinomycetes from mangrove forests rhizosphere soil in Guangxi Beibu Gulf. We will use an in situ cultivation such as diffusion chamber,Ichip and microbial trap that bypassed the difficulties of replicating the natural environment inherent in traditional Petri dish-based approaches and a tranditional isolation and characterization protocols to recover and identify diverse actinomycetes. At last,we will to demonstrate their activities in a number of biological screening assays. Representative isolates found to be novel were fully characterized by chemotaxonomic, morphological and molecular systematic methods.

海洋放线菌的多样性与其次生代谢产物的化学多样性是密切相关的，所以海洋放线菌已经成为人类寻找具有生物活性新化合物不可替代的资源。地处亚热带的广西北部湾海域是迄今保护最好的红树林生态系统之一，孕含丰富的根际放线菌资源。本申请拟采用原位培养、实验室培养和免培养的方法，对广西北部湾海域红树林根际放线菌的生物多样性进行研究。优化原位培养与实验室培养方法，旨在尽可能多的分离放线菌菌株包括稀有放线菌，初步建立菌种资源库。进一步通过波谱手段与BOX-PCR方法排除重复菌株后，利用16SrDNA序列进行分子聚类和系统发育分析，全面了解该地区放线菌资源的多样性。对于新种进行多相分类鉴定。在生物活性的指导下，利用理化性质及波谱学方法对分离的单体化合物进行结构解析，初步研究放线菌次生代谢产物的化学多样性。对及时开发和保护广西北部湾海域海洋放线菌资源具有重要意义。

海洋放线菌广泛存在于海洋无生命的基质（如海水、海底沉积物等）以及海洋动植物（如红树林等）的体内和体表，海洋具备特殊的生境造就了海洋放线菌独特的次生代谢途径，人们已经从海洋放线菌中获得了多种多样的次生代谢产物。本课题采用原位培养装置，埋于根际土壤中俘获放线菌，选用15种培养基并利用平板涂布法对4个地点的原位培养样品进行分离纯化，同时基于16srDNA 基因序列进行系统发育分析，共分离得到113株放线菌。同孙承航课题组合作红树植物内生放线菌从不同植物包括秋茄，白骨壤，红海榄，木榄，桐花树，海莲，海桑，角果木，拉关木，老鼠簕，卤蕨，美国大红树，木果楝，阿吉木，厚藤和阔苞菊等的各个组织，包括根、茎、叶、树皮、花、胚珠、果实等中分离到分布在放线菌纲下9个目22个科下的47个菌属菌株，北仑河共分离到菌株200 株为本实验室独立完成，放线菌S3Af-1经多相分类为丙酸杆菌科(Propionibacteriaceae）小月菌属（Microlunatus）新种，菌株S3cf-2经多相分类研究为小单孢菌科(Micromonosporaceae)中的库奇游动菌属（Couchioplanes）新种，已经公开发表，初步建立了菌种资源库。从钦州茅尾海红树林土壤中筛选分离出一株降解羽毛角蛋白产活性多肽的菌株弗氏链霉菌（Streptomyces fradiae UMBR 0070已经专利菌种保藏。采用单因素和响应面分析的方法，对弗氏链霉菌降解羽毛产抗氧化物活性的发酵条件进行优化。结果表明，在接种量为13 %，羽毛含量8 %和发酵时间23 d最优化条件下，抗氧化性、酶活和多肽含量分别达到274.06 µg/mL、480.12 U/mL和1.58 mg/mL，其中多肽含量和抗氧化性相关系数为（R2=0.9797）。从自建的放线菌菌种库筛选共有23株菌株的发酵液或乙酸乙酯萃取液对香蕉枯萎病病原菌F. oxysporumTR4 或F. oxysporumP1的菌丝体生长或孢子萌发具有拮抗活性，其中菌株UMBR 0029和UMBR 0133的菌株发酵液和乙酸乙酯萃取液都对F. oxysporumP1的孢子萌发具有明显抑制活性，这两个菌株具有开发成为农用抗生素生产菌的潜力。