Studies on development of a molecular method for analyzing microbial community structure of chemolithotrophic ammonia oxidizers by quantifying abundance of phylogenetic groups

通过量化系统发育群的丰度来分析化学营养型氨氧化剂微生物群落结构的分子方法的发展研究

• 批准号: 14380249
• 负责人: SUWA Yuichi
• 金额: $ 9.6万
• 依托单位: National Institute of Advanced Industrial Science and Technology (AIST)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380249/
• 关键词: chemolithotrophic ammonia-oxidizing; community structure analysis; real time PCR; 16S rDNA; 16SrDNA; ammonia monobxygenase; ammonia monooxygenase

Aim of the present study is to develop a novel molecular method for analyzing microbial community structure in environments, targeting aerobic chemolithotrophic ammonia-oxidizing bacteria(AOB), by quantifying each phylogenetic group comprising a community. AOB play a pivotal role in the global nitrogen cycle, and their monophyletic nature makes them especially suitable for the application of molecular methods. Since their poor growth, community structure of AOB has been analyzed by molecular methods. Recently, PCR has been introduced for quantifying AOB in environmental samples and its potential and reliability has been examined.In this study, real time PCR method, one of quantitative PCR methods which potentially allow to precisely estimating number of target cells in a sample, was applied to enumerate AOB cells, and was examined if the method can properly estimate AOB. A couple of primer sets which target different genes specific for AOB, and template DNA preparations extracted from several AOB strains and isolates which have relatively diverse phylogenetic positions were employed. Every combination of primer set and strain/isolate provided a standard curve for quantification, and its correlation was very good. However, standard curves obtained from different strains/isolates did not overlap, which would result in incorrect estimations of the exact target numbers when amplifying DNA from mixed communities, depending on the stain selected for creating a standard curve.We examined the factors affect the shape of standard curve, and elucidated the mechanism of incorrect estimation of the target genes. Based on the results, we specified the combination of types of primer set and template DNA necessary for correct estimation. Based on the results, realistic strategies for expressing abundance of each phylogenetic group of AOB in a community using available techniques were also discussed.

本研究的目的是开发一种新的分子方法，通过量化构成群落的每个系统发育群体，以好氧化学营养型氨氧化细菌（AOB）为目标，分析环境中的微生物群落结构。 AOB 在全球氮循环中发挥着关键作用，其单系性质使其特别适合分子方法的应用。由于AOB生长不良，人们通过分子方法对AOB的群落结构进行了分析。最近，PCR已被引入对环境样品中的AOB进行定量，并对其潜力和可靠性进行了检验。在本研究中，实时PCR方法（一种可能允许精确估计样品中目标细胞数量的定量PCR方法）被应用于计数AOB细胞，并检查该方法是否可以正确估计AOB。使用了针对 AOB 特异性不同基因的一对引物组，以及从具有相对不同系统发育位置的几种 AOB 菌株和分离株中提取的模板 DNA 制剂。引物组和菌株/分离株的每种组合都提供了定量的标准曲线，并且其相关性非常好。然而，从不同菌株/分离株获得的标准曲线并不重叠，这会导致在从混合群落扩增 DNA 时，根据创建标准曲线所选择的染色剂，错误估计准确的目标基因数量。我们检查了影响标准曲线形状的因素，并阐明了错误估计目标基因的机制。根据结果​​，我们指定了正确估计所需的引物组类型和模板DNA的组合。根据结果​​，还讨论了使用现有技术表达群落中 AOB 的每个系统发育群体丰度的现实策略。