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首页> 外文期刊>Applied and Environmental Microbiology >Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA
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Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

机译：反转录PCR扩增mRNA后河口沉积物中反硝化表达基因的检测与多样性

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摘要

The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.
机译：通过反转录（RT）-PCR分析从两个沉积物样品中提取的mRNA，分析了编码两个硝酸还原酶（narG和napA），两个亚硝酸还原酶（nirS和nirK）和一氧化二氮还原酶（nosZ）的五个反硝化基因的表达在科恩河河口（英国）获得的，该河口接受了大量的氮，反硝化率也很高。在分析基因表达之前，通过从提取的DNA进行PCR扩增，确认了两个沉积物样品中所有五个基因的存在。仅检测到nirS和nosZ mRNA。直接检测nirS作为RT-PCR扩增产物，并且在进行Southern杂交后检测到nosZ。这表明在采样时至少有nirS和nosZ基因在沉积物中活跃表达。克隆扩增的nirS RT-PCR产物并通过测序进行分析，并将其与来自相同沉积物的分离物中的扩增的nirS基因序列进行比较。观察到高多样性的nirS序列。检索到的大多数克隆nirS序列都对一个位点或另一个位点具有特异性，这突显了所分析的两种沉积物中参与反硝化作用的细菌群落组成的差异。

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