DGGE 指纹图谱分析太湖富营养化水体中细菌群落结构的变化

DGGE 指纹图谱分析太湖富营养化水体中细菌群落结构的变化 摘 要

太湖作为我国第三大淡水湖泊，其富营养化程度日益加深。太湖水 体水质不断变差，生态系统遭到严重破坏。在自然界中，微生物之间及 其与环境之间有着特定的关系，彼此影响，相互依存。因此，研究环境中的微生物组成对于富营养化湖泊的生物监测和生物修复具有重要意义。淡水生境中存在大量不可培养的细菌，使得传统的培养技术无法对细菌群落结构的多样性进行深入而全面的研究，而分子生物学技术的发展则为此方面研究开辟了新的路径。为了探究严重富营养化水体的细菌群落结构组成，本文运用PCR-DGGE 方法对太湖水体的细菌群落结构进行了分析。

本文采集太湖不同地点(梅梁湖、东太湖和南部沿岸区)、不同月份(06年4 至9 月)的表层水样，提取细菌总DNA，采用引物357F-GC-clamp和518R 进行PCR 扩增。实验结果表明，PCR 扩增产物的特异性会对DGGE 实验产生至关重要的影响，包括选择适合的引物及其浓度、适当的模板浓度，确定初始退火温度和总循环数。变性剂梯度被确定为40%～55%。DGGE 图谱的初步分析结果表明，太湖水样中的菌种丰富度非常高，并且不同取样时间和不同取样地点的DGGE 带谱在条带的数量、位置及条带的亮度上均存在较大的差异。Quantity One 软件包定量分析表明，太湖水样的细菌群落结构组成主要分为四种类型，即冬季期、水华发生期、水华暴发期和严

重水华期。梅梁湖作为太湖流域富营养化最严重的区域，其细菌群落结构较东太湖和南部沿岸区存在明显的差别。17 条典型条带的测序结果显示，序列属于放线菌、 α、β、γ -变形杆菌、拟杆菌、热微菌和蓝细菌。值得注意的是，3 条序列的最相似菌为来自梅梁湖克隆文库

分析中得到的序列，且相似度均大于98%。系统进化分析表明大部分序列属于被广泛定义的公认的淡水细菌群落。本文还运用了主成分分析和典型相应分析两种统计学方法。主成分分析结果证明了太湖水体中细菌群落结构存在显著的季节性变化。典型相应分析结果表明温度对于细菌群落组成的相关程度是最大的。此外，氮、磷、碳三类元素的含量同细菌群落结构的关联度也较大。

关键词： 细菌群落结构，变性梯度凝胶电泳，16S rRNA，太湖，

统计学分析

CHANGES IN BACTERIAL COMMUNITY STRUCTURE OF A HYPERTROPHIC FRESHWATER LAKE TAIHU, DETERMINED BY DENATURING GRADIENT GEL ELECTROPHORESIS FINGERPRINT ABSTRACT

Lake Taihu, the third-largest lake in People?s Republic of China, is atypical shallow freshwater lake. Due to agricultural intensification and excessive exploitation, its trophic status was increasingly getting aggravated.Water quality was

getting worse and the ecosystem was declining. In nature,there are extraordinary relationships among microorganisms and between microorganisms and environments. Due to the lack of cultured bacteria in aquatic ecosystems, the diversity of bacterial communities has not been extensively studied. However, developments in molecular biological technology bring a new path for scientists to investigate bacterial communities. In order to describe a complete picture of the structure of bacterial communities in a hypertrophic freshwater lake, PCR-DGGE method was applied to study water samples from Lake Taihu.

Water samples were collected with different locations (Eastern, southernand northern part of Lake Taihu) and different months (From April to September in 2006). After DNA extraction, the DNA was used as template to amplify 16S rRNA gene with universal primers 357F-GC-clamp and 518r.

Results indicated that nonspecific amplification could have a bad influence to DGGE process. The factors, which might lead to a bad amplification,

included the type of primers and their concentrations, concentration of template, annealing temperature and total cycles. The gradient of denaturant was finally confirmed as 40%-55%. Analysis of DGGE fingerprint revealed that richness of bacteria in Lake Taihu was relatively high. Distinct differences were detected in fingerprint at band numbers, band patterns and densities with different sampling sites and different sampling months.Quantitative analyses by Quantity One software package showed that there were four major types of bacterial community structure in Lake Taihu,including winter period, beginning period, happening period and heavy period. As an important hypertrophic part of Lake Taihu, Meliang Bay was found differently to other two locations in bacterial community composition. Seventeen typical bands were excised and sequenced. The sequences obtained were affiliated with Actinobacteria, α-, β-, γ-Proteobacteria, Bacteriodetes, Thermomicrobia and Cyanobacteria. Remarkably, the most relative representatives of three sequences came from a previous study based on clone library method

to investigate bacterial community composition of Meiliang Bay. The similarity was above 98%. According to phylogenetic analysis, these sequences could be grouped to previous putative freshwater clusters. Two statistical methods, principal component analysis (PCA) and canonical correspondence analysis (CCA), were carried out for further analysis. Seasonal changes of bacterial community structure in Lake Taihu could be confirmed based on PCA. Results of CCA indicated that bacterial community structure of Lake Taihu was primarily associated with temperature, followed by nitrogen, phosphate and carbon.

KEY WORDS ： bacterial community structure ，

denaturing gradient gel electrophoresis，16S rRNA，Lake Taihu，statistical analysis

第一章概 述

1.1 太湖流域现状

太湖是我国第三大淡水湖泊，位于长江下游沪、宁、杭三角带中心，地处北纬 30°55?～31°34?，东经119°53?～120°36?，流域面积达3165万km3，人口约为3770万，拥有大小城市38座，人口密度达1030人/km3，是全国人口最稠密、工农业生产最发达的地区之一，城市化水平占全国之首。太湖流域经济发达，GDP

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