Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils
Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils
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DOI：
10.1016/s0168-6496(04)00002-9
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发表时间：
2004-03-15
影响因子：
4.2
通讯作者：
Steinberg, C
中科院分区：
文献类型：
--
作者：
Edel-Hermann, W;Dreumont, C;Steinberg, C
监测农业和环境干扰下土壤中真菌群落的结构和动态是目前的一个挑战。在这项研究中，末端限制性片段长度多态性（T-RFLP）指纹技术的快速比较真菌群落结构的发展。利用10种限制性内切酶模拟了小亚基（SSU）核糖体RNA（rRNA）基因不同区域的末端限制性片段多态性，并利用真菌特异性引物对3种不同土壤进行了分析。用引物nu-SSU-0817-5'和荧光标记引物nu-SSU-1536- 3'对SSU rRNA基因的3'末端进行聚合酶链反应扩增，并用BamHI和MboI消化扩增子，发现这是最佳的，并用于标准化T-RFLP程序。将毛细管凝胶电泳检测到的末端限制性片段的数量和强度进行对应分析。根据土壤真菌群落的结构，对三种理化性质不同的土壤进行了区分。用堆肥或粪肥对两种土壤的真菌群落结构的影响进行了评估，证实了该方法的重现性和灵敏度。未修正和修正的土壤样品之间检测到的社区结构的变化。在这两种土壤中，不同的有机改良剂施加的移动。此外，同一种有机改良剂对两种土壤真菌群落结构的影响也不同。指纹图谱法为研究各种扰动对土壤真菌群落的影响提供了一种快速的工具。(C)2004年，欧洲微生物学会联合会。Elsevier B. V.出版，保留所有权利。
Monitoring the structure and dynamics of fungal communities in soils under agricultural and environmental disturbances is currently a challenge. In this study, a terminal restriction fragment length polymorphism (T-RFLP) fingerprinting method was developed for the rapid comparison of fungal community structures. The terminal restriction fragment polymorphism of different regions of the small-subunit (SSU) ribosomal RNA (rRNA) gene was simulated by sequence comparison using 10 restriction enzymes, and analyzed among three different soils using fungal-specific primers. Polymerase chain reaction amplification of the 3' end of the SSU rRNA gene with the primer nu-SSU-0817-5' and with the fluorescently labelled primer nu-SSU-1536-3', and digestion of the amplicons with AluI and MboI were found to be optimal and were used in a standardized T-RFLP procedure. Both the number and the intensity of terminal restriction fragments detected by capillary gel electrophoresis were integrated in correspondence analyses. Three soils with contrasting physicochemical properties were differentiated according to the structure of their fungal communities. Assessment of the impact on the fungal community structure of the amendment of two soils with compost or manure confirmed the reproducibility and the sensitivity of the method. Shifts in the community structure were detected between non-amended and amended soil samples. In both soils, the shift differed with the organic amendment applied. In addition, the fungal community structures of the two soils were affected in a different way by the same organic amendment. The fingerprinting method provides a rapid tool to investigate the effect of various perturbations on the fungal communities in soils. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.