【作者】 夏占峰；

【导师】 张利莉； 关统伟；

【作者基本信息】 塔里木大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 盐环境中的放线菌不仅能产生多种抗生素,还能产生多种耐盐胞外酶,其中耐盐淀粉酶、耐盐核酸酶、耐盐蛋白酶等有广阔的应用前景。盐环境中的放线菌产生的四氢嘧啶和甜菜碱是高效的保护剂,又是酶类、核酸、细胞膜的稳定剂,在酶技术、医药工业和环境保护领域有重要的应用价值。研究盐环境中的放线菌多样性,能促进放线菌资源开发,有利于新物种和新产物的发现。位于新疆吐鲁番盆地南部的艾丁湖具有低海拔、高盐度的特点。本研究采集艾丁湖沉积物样品,应用基于16S rDNA基因序列系统发育分析的免培养方法和选择性分离培养方法揭示放线菌多样性,以期获得艾丁湖放线菌物种资源,为开发利用提供技术支撑和菌种支持。(1)利用放线菌特异性引物扩增16S rDNA基因,构建16S rDNA基因克隆文库,对文库中的克隆片段进行RFLP(Restriction Fragment Length Polymorphism,限制性片段长度多态性)分析。研究分析表明:273个克隆分布于放线菌纲(Actinobacteria)的三个亚纲中,即放线菌亚纲(Actinobacteridae 208个)、酸微菌亚纲(Acidimicrobidae 13个)和红色杆菌亚纲(Rubrobacteridae 52个)。优势放线菌为放线菌亚纲微球菌科的罗斯氏菌属(Rothia),占克隆总数的37%。有45.8%的序列与已有效发表菌株的序列相似性小于97%,部分序列形成了几个独立的进化分支,可能代表着放线菌新的类群。(2)采用不同盐浓度的9种选择性培养基分离放线菌,共得到55株放线菌,分布在放线菌目的6个亚目中,以链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)为主要类群。还分离到一些放线多孢菌属(Actinopolyspora)、拟无枝酸菌属(Amycolatopsis)、疣孢菌属(Verrucosispora)、糖多孢菌属(Saccharopolyspora)、糖单孢菌属(Saccharomonospora)和微球菌属(Micrococcus)的稀有放线菌。有3株放线菌的16S rDNA基因序列与已有效发表菌株的序列最大相似度小于97%,为潜在的放线菌新分类单元。(3)对2株为潜在新种的放线菌进行了多相分类,结果表明,菌株TRM46004为Streptoalloteichus属的新种,菌株TRM46012为Streptomyces属的新种。综合分析免培养和分离培养结果,表明艾丁湖放线菌具有较高的多样性,并蕴藏着多种新类型,有进一步研究价值。更多还原

【Abstract】 Actinomycetes from salt environment is well known for its remarkable antibiotic producing ability and the potential capacity to produce salt tolerant extracellular enzyme such as amylase, nuclease and proteinase. The betaine produced by salt tolerant actinobacteria is efficient preservative and stabilizer, which has potential application in the fields of medical industry, biological chemistry and environmental protection. Studies on the actinobacterial diversity in salt environment would promote the development and utilization of actinomycetes resources. This study aims at investigating the diversity of actinobacteria in Aiding Lake, a hypersaline lake and the lowest land point in China. The diversity of actinobacteria in the sediment from Aiding Lake was investigated by culture-independent method based on phylogenetic analysis of 16S rDNA gene sequences and selective isolation method.Specific primers were used to amplify the actinobacterial 16S rDNA gene, and corresponding clone libraries were constructed for the sediment samples. Different clones selected on the basis of HaeIII digestion patterns were sequenced. Nine selective media with different salinities were used to isolate actinobacteria from the sediment samples. The analysis of 16S rDNA gene sequences showed that 273 clone sequences belonged to subclasses Actinobacteridae (208), Acidimicrobidae (13) and Rubrobacteridae (52), respectively. The dominant actinobacteria was genus Rothia, which accounted for 37% of total clones. The similarity between 45.8% of 273 detected sequences and published sequences were less than 97%, which might represent novel taxa. Some sequences, which formed several distinct clades in phylogenetic tree may represent novel taxonomical groups of Actinobacteria.55 strains were isolated by different selective media. They were belonged to 6 suborders of the order Actinomycetales, of which Streptomyces and Nocardiopsis were the dominant groups. Some rare actinomycetes were isolated, in genus of Actinopolyspora, Amycolatopsis, Verrucosispora, Saccharopolyspora, Micrococcus and Saccharomonospora. 3 potential novel species were obtained.Aiding lake harbors abundant actinobacteria, including large number of unknown actinobacterial groups, is worth while for further study.更多还原