【作者】 黄惠琴；

【导师】 鲍时翔；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 近年来耐甲氧西林金黄色葡萄球菌（MRSA）的分离率不断升高,临床上可供选择的药物极少,MRSA已成为难以治疗的“超级细菌”,极大地危害着人类健康,人们必须不断寻找新的药源。新药的开发已突破陆生资源的束缚,拓展到生态和物种远比陆地生境复杂多样的海洋,独特的海洋环境使海洋微生物在物种、基因组成和生态功能上呈现较高的多样性,被誉为天然药物的资源“宝库”,目前海洋微生物已成为世界各国开发新型药物的热点领域。 本文从海洋放线菌的分离、抗MRSA菌株的筛选、菌株的鉴定、发酵条件的优化、活性物质的分离纯化及结构鉴定等几个方面进行了系统研究,结果如下: 采用多种分离培养基与抑制剂,从红树林沉积物中选择性分离放线菌,实验结果表明,以0.005%重铬酸钾为抑制剂的高氏一号合成培养基上生长的放线菌种类最多,且细菌、真菌数量少,最适于分离放线菌。以该培养基作为分离培养基,先后从海南、湛江和北海沿海采集样品75份,采用平板稀释法共分离获得放线菌395株,通过纸片扩散法筛选得到43株有抗MRSA活性的放线菌,其中菌株A115、A209和AM105活性最强且遗传稳定,选择这三个菌株作为目标菌株进行下一步研究。 通过对菌株A115、A209和AM105的形态特征、培养特征、生理生化特征、16SrDNA序列测定及其系统发育分析的研究,鉴定菌株A115为白浅灰链霉菌海洋变种S.albogriseolus var.marine,A209为生金链霉菌S.aureofaciens。AM105与Micromonospora floridensis DSM43907、Micromonospora matsumotoense DSM44100的亲缘关系最近,DNA-DNA杂交率分别为73.5%和53.6%,结果表明AM105应归入M.floridensis。鉴于菌株AM105产生利福霉素,且在形态学、生理生化方面与M.floridensis DSM43907存在较大差异,鉴定放线菌AM105为佛州小单孢菌利福霉素亚种Micromonospora floridensis subsp.rifamycetica,这是佛州小单孢菌产生利福霉素的首次报道。AM105的16S rDNA在GenBank的登录号为AY561829。 研究明确了菌株AM105的最佳发酵工艺和培养基组成。实验考察了碳源、氮源、无机盐、pH值及接种量等对AM105产抑菌活性物质的影响,并通过正交实验对培养基碳氮源进行了优化。确定其最佳培养基组成及发酵条件为:葡萄糖0.5%,淀粉1.5%,大豆粉1%,酵母膏0.5%,K₂HPO₄ 0.025%,用50%的天然陈海水与50%的蒸馏水配更多还原

【Abstract】 Methicillin-resistant Staphylococcus aureus （MRSA） which is resistant to most antibiotics is increasing rapidly and has become the "super-bacteria" in the clinic due to absense of effective drugs, which badly endangers the health of human. It is essential to find novel drugs for us. Marine environment is being exploited for new drugs with unique structure and excellent bioactivity. Marine microorganisms with biodiversities on species, genotypes and ecological functions have become the resource treasury of natural drugs, thereby they have turned to the hotspot of new drug research in the world.In this study, research was carried out on following aspects: isolation of marine actinomycete, screening and identification of anti-MRSA strains, optimizing fermentation condition and medium, separation and purification of active compounds and structure identification of the active compound AM105-Ⅱ and so on.Employing several culture media and inhibitors, marine actinomycetes were isolated from mangrove sediment by the method of dilution. The results showed that Gause No.1 medium containing 0.005% K₂Cr₃O₇ was the best one for marine actinomycete isolation. Utilizing the aforesaid medium, a total of 395 actinomycetes isolates were separated from 75 marine samples, which were collected from near-shore of Hainan Island, Zhangjiang and Beihai. 43 active actinomycetes isolates were obtained by the method of Kirby Bauer. From them 3 strains with prominent anti-MRSA bioactivity and genetic stabilization were selected, named A115, A209 and AM105.By studying the morphology, cultural characteristics, physiological and biochemical properties, 16S rDNA and phylogenetic tree, A115 was proposed as Streptomyces albogriseolus var. marine, A209 as Streptomyces aureofaciens and AM105 was closely related to Micromonospora floridensis DSM43907 and Micromonospora matsumotoense DSM44100. Strain AM105 exhibited levels of DNA-DNA relatedness of 73.5% and 53.6% to M. floridensis DSM43907 and M. matsumotoense DSM44100 respectively. On the basis of DNA-DNA relatedness and the character of producing Rifamycin, strain AM105 is proposed the name as Micromonospora floridensis subsp. rifamycetica. Strain AM105 is the first discovery of M. floridensis that produces Rifamycin. The GenBank accession number for the 16S rDNA gene sequence of strain AM 105 is AY561829.The best optimal liquid fermentation condition and medium component was更多还原