【作者】 纪建达；

【导师】 吴灶和；

【作者基本信息】 广东海洋大学 ， 海洋生物学， 2010， 硕士

【摘要】 浮游细菌在水生生态系统中分布广,数量巨大,具有丰富的遗传多样性和适应复杂环境的机制,其群落组成和结构的变化在生态系统中具有重要的功能。本论文基于培养与不依赖于培养的方法,对湖光岩采集来的水样分别通过两类通用培养基的纯系培养,结合BOX-PCR带型分析技术,以及通过构建了环境水样总DNA的10个主要微生物门类的文库,阐明了这一典型玛珥湖水层中的古菌多样性和浮游细菌多样性及其季节上的变化。我们选取了湖光岩湖体的中心点水层(水体最深位点之一)作为研究对象的典型区域,分别在夏季和冬季各采集了1m、5m及13m的水样进行浮游细菌多样性的分析。首先,通过R2A培养基和过滤湖水配制的琼脂培养基培养分离各水层浮游细菌,共获得了340株细菌。通过碱裂解法提取DNA,并进行BOX-PCR反应及手动带型分析筛选差异条带的菌株;以PCR扩增获取筛选的细菌16S rRNA基因用于测序分析。RDP数据分析结果显示,340株菌株分属于变形菌门、放线菌门、厚壁菌门、拟杆菌门等几类湖泊中常出现的主要门类。其中变形菌门虽然跟其他各类细菌一样,在不同季节、水层及培养基上占有的比例不一致,但一般都是处于优势地位,占据着总细菌数的76%以上。另外,拟杆菌在分析的结果上,只出现在冬季的样品中,其数量占总细菌数不大。纯系菌株的16S rRNA基因经BLAST比对后的结果显示,纯系中的各类细菌16S rRNA的同源性大多大于90%,除了湖水配置的培养基分离的夏季水样中Shui5-7、Shui5-12、Shui13-6、Shui13-11和冬季水样中dS1-1、dS5-26,以及R2A培养基上分离的夏季水样中R13-5、R13-7和冬季水样中dR1-11,dR1-21,它们的同源性都不大于90%,可能代表至今尚未认知的新类群。其次,在上述基于培养的方法研究浮游细菌多样性的基础上,进一步通过不依赖培养的方法,通过10个门类的通用引物对构建了湖光岩水体中古菌、细菌、放线菌、厚壁菌、蓝细菌、浮霉菌、拟杆菌、α-变形菌、β-变形菌和γ-变形菌16S rRNA基因的10个克隆文库。这一方面揭示出了与纯系分离结果不一样的特点,比如在结构上与纯系分离菌株相比,古菌和浮霉菌基本没在两类通用培养基上分离获得,而仅有在构建的文库中出现;另一方面也克隆出了相当一部分对应科属的新序列。这类序列在不同的文库中,比例不同,分别为:占古菌的6/30,占细菌的8/30,占放线菌的2/30,占厚壁菌的2/30,占浮霉菌的23/30,占拟杆菌的29/30,蓝细菌占3/30,占α-变形菌的8/30,占β-变形菌的11/31,占γ-变形菌的27/31。更多还原

【Abstract】 Numerous planktonic bacteria are widely distributed in aquatic ecosystems. They have high genetic diversity and the capacity of living in almost complex environment on earth. Their community structures play an important role in ecosystem cycles. Here we based on the culture-independent and culture-dependent methods, aiming to study the planktonic bacterial diversity in a typical Maar lake-Hugangyan and furthermore, address its seasonal changes.Our work incledes the analysis of bacteria isolations, culturing by two types of common medium, and the construction of 10 gene clone libraries, being constructed with the lake’s main microbial communities. As for the analysis of isolations, we use the BOX-PCR technique to type the various ones.First, we chose the center vertical watermass of Lake Huguangyan, one of the deepest site, as its typical region and investigated the microbial diversities on 1m, 5m and 13m water layers, respectively, in summer and winter. First of all, 340 bacterial strains were obtained by culturing on R2A medium and agar medium that were prepared by filtered lake water and agar. Then,for the analysis of BOX-PCR we extracted the DNA from each strain by alkaline lysis. Next, strains were distinguished by manual screening on different bands. Later, RDP data analysis showed that 340 strains belonged to Proteobacteria, Actinobacteria, Firmicutes, Bacteroides, which are the main microbial categories in lakes. We found that the proportion of types of bacteria was inconsistent in different seasons, water levels and mediums. But at the same time Proteobacteria is constantly as a superior, occupying more than 76% of the total number of bacteria. In addition, the results of the analysis reveal that Bacteroides was only found in winter samples. The data by BLAST comparison of 16S rRNA genes showed that various types of bacterial 16S rRNA homology are above 90%, excepting the strains of the summer sample from agar medium and R2A medium:Shui5-7, Shui5-12, Shui13-6, Shui13-11, R13-5, R13-7 and dS1-1, dS5-26, dR1-11, dR1-21 in winter. They are not exceeding 90% homology, which may represent a new species in the correspondent categories.Secondly, in addition to the analysis of bacterial diversity by culture-dependent methods, we also employed culture-independent methods to study the diversity. By using 10 categories of universal primers, we constructed 10 clone libraries of 16S rRNA, including Archaea, Bacteria, Actinomycetes, Firmicutes, Cyanobacteria, Bacteroides, Planctomycetes,α-Proteobacteria,β-Proteobacteria andγ-Proteobacteria. The results revealed different characteristics with the above culturing results. And sequences of Archaea and Planctomycetes were only cloned in these libraries. Besides, there are some more new sequences of other categories from the libraries. The value is 6/30 in Archaea, 8/30 in Bacteria, 2/30 in Actinomycetes, 2/30 in Firmicutes, 23/30 in Planctomycetes, 29/30 in Bacteroides, 3/30 in Cyanobacteria, 8/30 inα-Proteobacteria, 11/31 inβ-Proteobacteria, 27/31 inγ-Proteobacteria, respectively.更多还原