【作者】 方海环；

【导师】 吴雪昌；

【作者基本信息】 浙江大学 ， 微生物学， 2010， 硕士

【摘要】 芽孢杆菌是一类重要的药源性微生物,能产生丰富的具有人类医药应用价值或动植物病害防治功能的次级代谢产物。为了探索天目山国家自然保护区土壤中具有产次级代谢产物潜能的芽孢杆菌多样性,并期望从分离株中发现新种及新抗生素,本研究从天目山采集土壤样品,采用纯培养的方法,分离获得芽孢杆菌。利用特异性引物扩增法,筛选出基因组中具有编码非核糖体肽合成酶(NRPS)基因或Ⅰ型聚酮合酶(PKSI)基因的菌株。对筛选出的菌株进行抗菌活性和代谢产物表面活性物质的检测,并进行基于16S rDNA序列的系统发育分析,以期挑选出具有较大研究价值的菌株进行进一步的研究。通过分离与筛选,从天目山土壤中共得到35株产芽孢细菌,其中14个菌株的基因组中具有NRPS或PKSI基因。这14株菌株中有9株菌对金黄色葡萄球菌,大肠杆菌或白色念珠菌表现出不同程度的拮抗作用,7个菌株的代谢产物中检测出了表面活性物质。经16S rDNA序列分析,在这14株菌株中,6株属于芽孢杆菌属,6株属于类芽孢杆菌属,2株属于短芽孢杆菌属,其中有4个菌株为潜在新种。对潜在新种菌株B5进行多相分类学研究,最终确定其为芽孢杆菌新种,命名Bacillus tianmuensis.本研究结果表明,天目山自然保护区土壤中拥有较为丰富的具芽孢细菌产抗微生物多样性。运用基于编码非核糖体肽合成酶(NRPS)基因与Ⅰ型聚酮合酶(PKSI)基因的特异性引物扩增法、并结合抗菌活性及系统发育分析的方法集成,是研究筛选产抗菌株,尤其是筛选潜在产抗新菌株,从而发现产抗新种与新活性化合物的针对性强、较快速的有效方法。此外,芽孢杆菌新种Bacillus tianmuensis的发现,不仅进一步丰富了芽孢杆菌种群,也为探索潜在的新的生物活性化合物提供了新资源。更多还原

【Abstract】 Strains of Bacillus produce a wide range of second metabolites with great pharmaceutical importance, are important sources of antibiotic. With the aim to study the diversity of antagonistic Bacillus from soil of Tianmu Mountain National Natural Reserve and to discover new species and new antibiotics, Bacillus strains were isolated by culture-dependent method from soil samples collected from Tianmu Mountain. The isolated strains were screened for nonribosomal peptide synthetase (NRPS) and typeⅠpolyketide synthase (PKSⅠ) gene by using specific primer PCR method. The selected strains were then detected for antimicrobial activities and surface active substance, and subjected to phylogenetic analysis based on 16S rDNA sequences, with the expectation to obtain strains of considerable research value for further investigation.Total 35 strains were isolated form soil of Tianmu Mountain. Of them,14 strains possessed NRPS or PKSⅠgene. Among the 14 strains,9 strains showed different antimicrobial activities against to Staphylococcs aureus, Escherichia coli or Candida albicans, and 7 strains could produce surface active substance. Phylogenetic analysis based on the 16S rDNA sequences revealed that 6 of the 14 strains belonged to the genus of Bacillus,6 of the 14 strains belonged to the genus of Paenibacillus and 2 of the 14 strains belonged to the genus of Brevibacillus. Among the 14 strains,4 strains were novel species candidates. One of the novel species candidate strain B5 was subsequently subjected to a polyphasic taxonomic analysis and was classified to be a novel Bacillus species, for which the name Bacillus tianmuensis was proposed.The results of this paper showed that there were diverse antagonistic endospore-forming bacteria existed in the soil of Tianmu Mountain. The screening method based on the specific primer PCR of NRPS or PKSⅠgene combining with antimicrobial activity detection and phylogenetic analysis was a well targeted, rapid and effective method for screening antibiotic producing strains, especially the new strains with potential antibiotic production ability, and leading to the discovery of new antibiotic producing strains and new active metabolites. Moreover, the discovery of new species Bacillus tianmuensis, not only enriched the species of Bacillus, but also provided a good resource for exploring new potential biological active substance.更多还原