采用变性梯度凝胶电泳研究双孢蘑菇培养料后发酵过程中的细菌群落结构  

Bacterial communities in the phaseⅡ of Agaricus bisporus compost by denaturing gradient gel electrophoresis

作　　者：何丽鸿[1,2] 陈明杰[1] 潘迎捷[3] 

机构地区：[1]农业部应用真菌资源与利用重点开放实验室,上海市食用菌工程技术研究中心,上海市农业遗传育种重点实验室,上海市农业科学院食用菌研究所,上海201106 [2]南京农业大学生命科学院微生物系,南京210095 [3]上海水产大学,上海200090

出　　处：《微生物学报》2009年第2期227-232,共6页Acta Microbiologica Sinica

基　　金：农业部公益性行业科研专项项目(nyhyzx07-008)~~

摘　　要：【目的】对双孢蘑菇培养料"后发酵"期间内的细菌群落结构进行了初步的研究,希望利用现代分子生态学的方法能快速、准确地对培养料发酵过程中的微生物群落结构进行动态的检测。【方法】采用变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE),对取自双孢蘑菇培养料"后发酵"不同时期的七份样品的特异性扩增细菌16S rDNA的V3可变区进行分析。【结果】双孢蘑菇培养料中的细菌组成要远远丰富于人们用传统的分离方法所获得的类群,"空气调节"时期的培养料中不仅检测到了前人用经典培养方法获得的Bacillus属嗜热细菌,还发现了一些未曾报道的Bacilli门的成员,如Trichococcus属、Planococcus属和Caryophanon属,以及γ-Proteobacteria亚纲。而在"后发酵"刚开始和即将结束的培养料中分别检测到了Thermus thermophilus和α-Proteobacteria亚纲的细菌,这些是近年来利用分子生物学方法在各类高温堆肥中发现的新类群;【结论】发现双孢蘑菇培养料"后发酵"过程中细菌的群落结构发生了明显的变化,在双孢蘑菇培养料"后发酵"过程中还发现了大量目前尚未获得培养的细菌类群。[Objective]We studied the bacterial community during Agaricus bisporus composting phase Ⅱ, to develop a quick and accurate method by modern molecular ecology technique for dynamic inspection on bacterial community during A. bisporus composting. [Methods] We selected seven A. bisporus compost samples from different stages of phase Ⅱ. We used PCR to amplify the V3 regions of the 16S ribosomal DNA from those samples. We analyzed PCR products by denaturing gradient gel electrophoresis. [Results] The denaturing gradient gel electrophoresis profile showed a dramatic change of bacterial communities which related to the process of phase Ⅱ of A. bisporus compost. DNA sequencing of the dominant bands of denaturing gradient gel electrophoresis profile indicated that the bacterial diversity in the phase Ⅱ of A. bisporus compost was much higher than that studied by traditional method cultured-based approach. We found not only the genus of Bacillus, commonly believed to dominate high temperature compost, but also some new groups belonged to the class of Bacilli such as Trichococcus, Planococcus, Caryophanon and even the γ-subgroup of Proteobacteria. At the same time, some sequences were related to the genus of Thermus thermophilus and α-subdivision of Proteobacteria which were only recently reported in the hot compost. Several sequences display extremely low similarity with the cultivated species in the GenBank, indicating high diversity of uncultivated bacteria in the process of A. bisporus composting. [Conclusion] We found distinctly changes on bacterial communities during A. bisporus composting phase Ⅱ by denaturing gradient gel electrophoresis profile. Also we found a lot of presently uncultured bacteria species during A. bisporus composting. The results indicted that molecular ecology methods broaden and deepen our knowledge about the environmental microbes, and also shown the potential application for A. bisporus compost evaluation.

关 键 词：双孢蘑菇培养料后发酵 细菌群落结构 PCR-DGGE 

分 类 号：S646.1[农业科学—蔬菜学]