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首页> 外文期刊>Applied Microbiology and Biotechnology >A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil
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A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil

机译：酪蛋白从Andosols中提取RNA的新方法及其在土壤中amoA基因表达研究中的应用

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摘要

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.
机译：缺乏从土壤中提取RNA的通用方法阻碍了与土壤硝化有关的研究的进展，这是氮循环中的重要一步。从土壤的某些类型的土壤中提取RNA尤其困难，因为土壤被RNA吸附，因此在日本占主导地位的农业土壤中为Andosols（火山灰土壤）。为了从这些具有挑战性的土壤中获得RNA来研究涉及硝化作用的细菌，我们开发了一种用于基因表达分析的土壤RNA提取方法。将高压灭菌的酪蛋白添加到RNA提取缓冲液中以从土壤中回收RNA，并从八种类型的理化特性明显不同的农业土壤中成功提取了高质量的RNA。为了检测细菌氨单加氧酶亚基A基因（amoA）的转录本，在与硫酸铵孵育期间，从两种不同类型的Andosols中共提取了细菌基因组DNA和信使RNA。在土壤微观世界中对amoA的聚合酶链反应-变性梯度凝胶电泳和逆转录聚合酶链反应-变性梯度凝胶电泳分析显示，经多巴胺处理后，这些土壤中仅表达了与多形亚硝基螺菌相似性最高的amoA。硫酸铵，尽管在所检查的土壤微观世界中存在多个amoA基因。

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