【作者】 陈伟峰；

【导师】 吴雪昌；

【作者基本信息】 浙江大学 ， 微生物学， 2008， 硕士

【摘要】 本文从天目山自然保护区分10条路径采集土样,对20份土样进行微生物系统分离和筛选,共获得霉菌145株,细菌65株。以Ⅰ型聚酮合成酶(PKSⅠ)基因和非核糖体肽合成酶(NRPS)基因这两个抗性相关基因为筛选标记进行筛选,并研究抗菌活性,并用16S rDNA和蛋白编码基因(gyrA和gyrB)序列分析相结合,确定它们的系统发育地位。共得到基因组中含有PKSⅠ基因或NRPS基因的细菌18株,其中8株检测到抗菌活性。筛选到的细菌全部鉴定到属,其中8株鉴定到种,3株(B5、B10、B27)可能是尚未被鉴定的菌株。选取有抑菌能力且可能为新菌株的B27进行形态观察、培养条件、生理生化分析和16S rDNA系统发育分析,并研究了其NRPS基因序列。从一系列特征的分析得出,B27是一个未被鉴定的类芽孢杆菌属(Paenibaeillus)菌株。由其NRPS基因部分序列预测其产生的抗性物质可能属于环脂肽类,并给出了进一步研究的建议。结果表明,天目山自然保护区的原始环境中有着丰富的微生物资源。以抗性相关基因PKSⅠ和NRPS基因为筛选标记,并在分子水平上进行菌株快速鉴定,具有与前人报道方法不同的较好的筛选效果。为最终挑选到的几个有理论与应用价值的菌株之深入研究提供了重要基础。更多还原

【Abstract】 Soil samples were collected from 10 different routes in Tianmu Mountain Natural Reserve, and 20 of them were studied for microbe diversity. 145 mildews and 65 bacteria were isolated. All bacterial stains were directed selected with the standard whether there were polyketide synthases I (PKS I ) or nonribosomal peptide synthetases (NRPS) genes in there genomes. The selected stains were tested for antimicrobial activities. There phylogenetic relationships were obtained with sequences analysis of 16S rDNA, gyrA and gyrB. Eighteen strains with PKS I or NRPS genes in there genomes were selected, and eight of them have inhibitory activities towards test stains. All eighteen strains were identified to genus level, and ten of them were identified to species, another three have some differences with those stains in public database, and they may have not been studied or identified. B27 which have remarkable resistant activities was studied further, and the phenotype, cultivation situation, physiological and biochemical characteristics, and phylogenesis was tested in detail.On the basis of these results, B27 was identified as a member of genus Paenibacillus. However, there are differences between B27 and other Paenibacillus stains in 16S rDNA sequences and physiological and biochemical characteristics, thus B27 was considered as a new strain of genus Paenibacillus. Its partial NRPS gene sequence indicated that its antimicrobial products might be cyclo-lipopeptide. All there results showed that Tianmu Mountain Natural Reserve has a great storage of microbe resources. Directed selection with PKS I and NRPS genes as the marker, which is followed with fast identification on molecular level, is a effective way of isolation. Ultimately, several valuable stains were picked out, the stain B27 is a good example.更多还原