mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil
mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil
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DOI：
10.1128/aem.69.4.1928-1935.2003
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发表时间：
2003-04-01
影响因子：
4.4
通讯作者：
Zeyer, J
中科院分区：
文献类型：
--
作者：
Bürgmann, H;Widmer, F;Zeyer, J
土壤微生物群落的多样性和固氮生物种群个体成员固氮活性的关联性阻碍了对土壤中自生固氮生物的研究。我们开发了一种分子方法，允许分析土壤中的nifH mRNA表达的固氮活性和细菌生长的测定平行。本研究以棕色固氮菌（Azotobacter vinelandii）为模型菌，以棕色固氮菌为碳源，提供氮限制条件，同时添加NH 4 NO3抑制固氮。土壤RNA提取进行了一个新的优化的直接提取协议，产生非降解总RNA。RNA提取物是高纯度的，无DNA污染，并允许高灵敏度和特异性检测nifH mRNA的逆转录-PCR。通过PCR扩增逆转录的nifH mRNA片段，用A. vinelandii特异性nifH引物。这种新的方法揭示了nifH基因表达与土壤中（r（2）= 0.72）和液体培养中（r（2）= 0.84）的整体固氮活性正相关，因此是直接研究土壤环境中基因表达特异性调控的有力工具。
The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which served as the carbon source and provided nitrogen-limited conditions, while amendments of NH4NO3 were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r(2) = 0.72) and in liquid culture (r(2) = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.