mRNA Extraction and Reverse Transcription-PCR Protocol for Detection of nifH Gene Expression by Azotobacter vinelandii in Soil

Abstract

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 sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH 4 NO 3 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.

A reduced fraction of plant N derived from atmospheric N (%Ndfa) and reduced rhizobial nifH gene numbers indicate a lower capacity for nitrogen fixation in nodules of white clover exposed to long-term CO<sub>2</sub> enrichment

“…2d). We did not measure nitrogen fixation per se in this experiment, but evidence for a positive relationship between expression of nifH genes and fixation (Bürgmann et al, 2003;Resendis-Antonio et al, 2011;Zehr et al, 2007) implies reduced BNF under eCO 2 , which is consistent with the earlier finding of a lower %Ndfa at eCO 2 . It is generally thought that eCO 2 will result in increased N fixation unless the supply of nutrients (particularly P) is inadequate, in which case the effect on BNF is removed (Edwards et al, 2006;Rogers et al, 2009;.…”

“…This paper describes both the initial study and the subsequent molecular analyses which focussed on rhizobial nifH genes and their transcripts (mRNA). The nifH gene codes for nitrogenase reductase and thus acts as a marker for BNF (Bürgmann et al, 2003); changes in transcript number represent changes in the capacity to fix nitrogen implying a positive relationship between nifH expression and BNF activity, and this has indeed been shown to be the case for free-living diazotrophs in soil (Bürgmann et al, 2003) and marine (Zehr et al, 2007) environments and symbiotic rhizobia in nodules (ResendisAntonio et al, 2011).…”

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

“…In recent years, nifH has been one of the most important functional genes used when studying structural and functional diversity in numerous environments including diverse soil habitats (Bürgmann et al 2003;Demba Diallo et al 2008;Knauth et al 2005;Poly et al 2001;Wu et al 2009). In the present study, the nifH gene was used as a molecular marker for studying the diazotrophic diversity and abundance in the rice paddy in an area with intensive farming practise, including two growth seasons a year.…”

Biological nitrogen fixation and nifH gene expression in dry beans (Phaseolus vulgaris L.)

“…Therefore, the detection of nifH amplified from mRNA indicates the presence of N 2 -fixing bacteria as well as evidence of N 2 -fixation in plants (Young 1992). Several approaches such as reverse transcriptase polymerase chain reaction (RT-PCR) (Bu¨rgmann et al 2003), gene hybridization (Neufeld et al 2001) and real-time PCR with SYBR Green-I (You et al 2005) have been suggested to quantify nifH gene abundance in environmental samples or pure culture cDNA. However, RT followed by the PCR is a highly sensitive procedure for analysis of mRNA expression as it allows quantification of small changes in gene expression (Pfaffi 2001).…”