【作者】 李垚；

【导师】 向文胜；

【作者基本信息】 东北农业大学 ， 微生物学， 2017， 硕士

【摘要】 近年来科学家们在探寻新型抗生素的筛选途径时,发现昆虫内生菌能够产生许多种类多样的次生代谢产物,尤其那些能够帮助宿主抵御病原菌入侵的放线菌,其中一些具有新颖的结构并表现出较好的生物活性。由于这些化合物能够存在于真核宿主体内,且不危害宿主,所以它们很有可能对人类也没有毒副作用。因此这些昆虫内生放线菌具有很大产生人类药用的抗生素潜力,所以近年来昆虫内共生放线菌受到越来越多科学家的关注,昆虫内生放线菌也将是持续地发现新抗生素的重要领域之一,这对于对抗日益增多的抗药病原菌和新型疾病具有重要意义。本研究以实验室筛选得到的30株蚂蚁内生菌为材料,通过培养特征观察、抗菌活性测试和16S rRNA序列测序结果分析,发现两株潜在的放线菌新种3H-GS17~T和2H-TWYE14~T和一株具有良好抗菌活性放线菌1H-GS2,对它们分别进行了系统的分类学鉴定和活性物质的分离鉴定,结果如下:(1)16S rRNA分析结果显示菌株3H-GS17~T是珊瑚放线菌属的一株菌,与Actinocorallia glomerata JCM 9376T(98.13%)和Actinocorallia longicatena JCM 9377~T(97.64%)的16S rRNA序列相似性最高。化学分类学特征也与Actinocorallia的特征一致。而DNA–DNA杂交结果和表型分析结果显示菌株3H-GS17~T明显不同于与它相近的菌株,进一步的实验证明菌株3H-GS17~T和珊瑚放线菌属的其他菌株都不相同。因此确定菌株3H-GS17~T是珊瑚放线菌属的一个新种,命名为Actinocorallia lasicapitis,典型菌株是3H-GS17~T(=DSM 100595T=CGMCC4.7282T)。(2)16S rRNA分析结果显示2H-TWYE14~T是链霉菌属的一株菌,与Streptomyces niveus NRRL 2466T(98.84%)的16S rRNA序列相似性最高。gyr B基因分析结果同样显示2H-TWYE14~T属于链霉菌属。化学分类学特征也与链霉菌属的其他菌株相一致。而2H-TWYE14~T与S.niveus NRRL 2466T之间的DNA–DNA杂交结果和表型分析结果显示2H-TWYE14~T明显不同于S.niveus NRRL 2466T。因此确定菌株2H-TWYE14~T是链霉菌菌属的一个新种,命名为Streptomyces camponoticapitis,典型菌株是2H-TWYE14~T(=DSM 100523T=CGMCC 4.7275T)。(3)活体生物活性检测显示菌株1H-GS2对部分测试真菌具有较好的生物活性,将菌株进行发酵,发酵产物同样显示良好的真菌抑制活性。对发酵产物中的化合物进行分离纯化后,利用现代光谱学进行结构分析鉴定,最终得到化合物白诺菌素(Albonoursin)。更多还原

【Abstract】 Recently,scientists findings indicated that endophytic organisms could produce a large variety of secondary metabolites,and several novel compounds with antimicrobial activity had been isolated from mutualistic bacteria,particularly those that protect their host against pathogens.Defensive bacterial symbionts of eukaryotes constitute especially promising candidates for the production of antibiotics with potential applicability in human medicine,because the compounds involved should not harm the eukaryotic host and are therefore also less likely to have harmful side effects on humans.So,in recent years,more and more attention has been paid to the insect symbiotic actinomycetes,and further research on protective symbioses will undoubtedly continue to yield novel antibiotics that might prove valuable in the arms race against increasingly resistant human pathogens.In this study,30 strains of endosymbiotic bacteria were selected as materials,cultivative morphological characteristics,biological activity and 16 S rRNA gene sequence similarity studies showed that the strain 3H-GS17 T and 2H-TWYE14 T maybe a novel species belonged to Actinocorallia and Streptomyces,respectively,and 1H-GS2 had good bioactivity in resisting fungi,so,the new speices are identified in a polyphasic approach and the bioactive constituents were isolated and identified from these symbionts actinobacteria.The results are as follows:(1)16S rRNA gene sequence similarity studies showed that strain 3H-GS17 T belongs to the genus Actinocorallia with high sequence similarities to Actinocorallia glomerata JCM 9376T(98.13%)and Actinocorallia longicatena JCM 9377~T(97.64%).The chemotaxonomic properties of strain 3H-GS17 T are also consistent with those members of the genus Actinocorallia.A combination of DNA–DNA hybridization experiments and phenotypic tests were carried out between strain 3H-GS17 T and its closely related strains,which further clarified their relatedness and demonstrated that 3H-GS17 T could be distinguished from these strains.Therefore,the strain is concluded to represent a novel species of the genus Actinocorallia,for which the name Actinocorallia lasicapitis sp.nov.is proposed.The type strain is 3H-GS17~T(=DSM100595T=CGMCC 4.7282T).(2)16S rRNA gene sequence analysis showed that strain 2H-TWYE14 T belongs to the genus Streptomyces with the highest sequence similarity to Streptomyces niveus NRRL 2466T(98.84%).gyr B gene analysis also indicated that strain 2H-TWYE14 T should be assigned to the genus Streptomyces.The chemotaxonomic properties of strain 2H-TWYE14 T are consistent with those members of the genus Streptomyces.A combination of DNA–DNA hybridization experiment and phenotypic tests were carried out between strain 2H-TWYE14 T and its phylogenetically closely related strain S.niveus JCM 4251 T,which further clarified their relatedness and demonstrated that2H-TWYE14 T could be distinguished from this strain.Therefore,it is concluded that strain2H-TWYE14 T can be classified as representing a novel species of the genus Streptomyces,for which the name Streptomyces camponoticapitis sp.nov.is proposed.The type strain is2H-TWYE14~T(=DSM 100523T=CGMCC 4.7275T).(3)The bioactivity of isolated endosymbiotic actinobacteria was performed with using an in vitro screening technique,one strain with good biological activity were found from these endosymbiotic actinobacteria.The fermentation products of strain 1H-GS2 showed good antifungal activity.After separation and purification of the fermentation products,one known compounds,albonoursin,was isolated and identified from 1H-GS2.更多还原