【作者】 易志伟；

【导师】 王风平；

【作者基本信息】 国家海洋局第三海洋研究所 ， 海洋生物学， 2010， 硕士

【摘要】 深海环境占地球表面积的49%,被认为是目前地球上物种资源最丰富的地区。深海微生物处于高压、低温(部分区域如热液口为高温)、厌氧、极端pH、高盐浓度、高金属浓度、无光照、寡营养、高卤素等特殊极端环境中。由于其生态环境的特殊性,决定了深海微生物具有一些特异的代谢途径和遗传背景,导致深海微生物次生代谢产物具有化学结构奇特、新颖,生物活性多样等特点。特殊结构天然产物,对于增强药物的特异性和有效性、减少毒副作用和防止耐药性等均至关重要,对发现药物先导化合物、研究药物作用新靶点、新机理等具有重要科学意义。自20世纪60年代以来,海洋天然产物开发越来越受人关注,分离自海洋的化合物总数和新化合物数量都快速增长,2007年有报道的海洋的新天然产物数量为961个,较2006年数量增长24%。海洋微生物来源的天然产物开发成为热点,其化合物增长速度远高于海洋天然产物中比重最大的海绵动物来源化合物的增长速度。2007年来自海洋微生物的化合物数量较1965至2005年平均数量增长了5倍,比2006年增长了60%以上。本文利用中国大洋一号20航次采集的深海沉积物样品进行了真菌分离鉴定,获得110株深海来源真菌,这些菌分属于15个属。其中曲霉和青霉最多,共为55株,占50%,且重复较多,只分布于10个种。利用CCK8法及琼脂扩散法对其中的100株深海来源真菌进行了抗肿瘤及抗菌活性检测,发现深海来源真菌具有良好的生物活性,细胞毒阳性率占49%,26%的菌株具有明显抗大肠杆菌效果,23%明显抗枯草,5%抗白假丝酵母,21%抗金黄色葡萄球菌。生物活性与培养基成分及筛选用肿瘤细胞株类型相关。对具有生物活性的20株深海来源真菌发酵粗提物进行了HPLC指纹图谱初步分析,发现其HPLC指纹图谱与培养基成分相关。该研究为后期分离鉴定深海来源真菌产生的生物活性化合物奠定了良好基础。细菌Shewanella piezotolerans wp3为一株分离自西太平洋1914米深海沉积物中的一株革兰氏阴性菌。通过检测WP3和分离自同一深海位点的两株深海耐嗜压菌Shewanella psychrophila WP2次生代谢产物的抗肿瘤活性,研究了温度、压力、嗜菌体对WP3次生代谢产物抗肿瘤活性的影响。发现WP3次生代谢产物具有良好抗肿瘤活性,低温因素可提高其抗肿瘤活性,在最适温度情况下,压力因素可降低其抗肿瘤活性,而在低温情况下,压力因素对其抗肿瘤活性无影响,嗜菌体对抗肿瘤活性影响不大。HPLC指纹图谱分析发现不同温度条件下,其次生代谢产物指纹图谱有变化。结合细胞毒活性分析结果,通过色谱分离、质谱、核磁共振技术,对WP3发酵浸膏进行了化合物的分离鉴定,目前已经解析了两个单体化合物,但该两个化合物不具有细胞毒活性,后期还需进一步分离鉴定发酵浸膏中具有抗肿瘤活性的次生代谢产物,探讨细胞毒活性化合物与WP3深海环境适应性之间的可能关系。更多还原

【Abstract】 Deep-sea environment, accounting for 49% of the Earth’s surface, is considered the present species of the planet’s most resource-rich areas. Deep-sea micro-organisms live in the high-pressure, low temperature (some regions in high-temperature such as hydrothermal mouth), anaerobic, extreme pH, high salt concentration, high metal concentrations, no light, oligotrophic, high-halogen and other special extreme enviroment. Because of its environment specificity, deep-sea microbes have some specific metabolic pathways and genetic background, their secondary metabolites have peculiar, novel chemical structures, and diverse characteristics. Special structure of natural products are very important to enhance drugs’specificity and effectiveness, reduce drugs’side effects, prevent drugs’resistance, they also have important scientific significance on lead compounds discovery and drug study of new targets, new mechanism.Since the 60s’20th century, the concern in the development of marine natural products is growing,961 new compounds were isolated from marine in 2007, there was an increase of 24% from the number of compounds reported for 2006. Marine microbial natural products development became a hot spot, its growth rate is much higher than sponge which was the most large part of marine natural compounds. In 2007 the number of compounds from marine organisms got five-fold increase to the average number from 1965 to 2005, above 60% increase to 2006.In our study,deep sea sedimental samples collected by the 20th voyage of Chinese Ocean No.1 ship were used to isolate and identify deep sea originated fungus and 110 strains have been obtained. These strains belonged to 15 genus,50% of them,55 stains, are Penicillium or Aspergillus. These 55 stains just belonged to 10 different species. Antitumor and antibacterial activity analysis of second metabolites of 100 fungi were performed by CCK-8 method and agar diffusion method. Results showed that deep sea originated fungi possess good antitumor and antibacterial activity,49% strains indicate cytotoxical activities,26% stains showed significant anti Escherichia coli activities and 32% against Bacillus subtilis,5% against Candida albicans,21% inhibited Staphlococcus aureus growth. the bioactivity related to culture medium and tumor cell type. HPLC analysis of 20 fungus second metabolism product fingerprints indicated map changes with culture medium. These results could be useful to next isolation of bioactive compounds from deep sea originated fungus second metabolism product.The deep-sea bacterium Shewanella piezotolerans WP3 and Shewanella psychrophila WP2 used in this study was isolated from deep-sea sediments of 1914m depth in west Pacific. Cytotoxic activity analysis of second metabolism products of them were detected through CCK-8 method. Study on impacts of temperature, pressure and phage on cytotoxic activity of Shewanella piezotolerans WP3 second metabolism product were also conducted. Results showed that Shewanella piezotolerans wp3 second metabolism product had strong cytotoxic activity to tumor cells, and low temperature could enhance this activity. Pressure could reduce cytotoxic activity at most optimal temperature and had almost no effect on this activity at low temperature on the contrary. Meanwhile, phage had no effect on cytotoxic activity. HPLC analysis of Shewanella piezotolerans WP3 second metabolism product fingerprints indicated map changes at different temperature.Based on the analysis of cytotoxic activity, by chromatographic separation, mass spectrometry, nuclear magnetic resonance technology, two compounds of wp3 fermentation extract have been isolated and identified, but the two compounds showed no cytotoxic activity, further work will focus on the active compound isolation of WP3 secondary metabolites to explore the possible relationship between the compounds and the environmental adaptability of WP3.更多还原