Quantification of homogentisate-1,2-dioxygenase expression in a fungus degrading ethylbenzene

A quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) assay was utilized to quantify the expression of ElHDO in the fungus Exophiala lecanii-corni during the biodegradation of ethylbenzene and other volatile organic pollutants. The assay was applied to measure the impact of pollutant mixtures on ElHDO expression relative to that of a housekeeping gene ( 18S rRNA). Three compounds were tested in mixtures with ethylbenzene: methyl propyl ketone, phenylacetate and o-xylene. These chemicals repressed, induced, or had no effect on ethylbenzene degradation, respectively. The results demonstrate that the gene target expression value ( T N) is a useful parameter for evaluating the effect of pollutant mixtures on gene expression. T N was found to reflect macroscopic changes in ethylbenzene utilization rates although these two parameters were not related in a linear fashion for all compounds. The assay was log-linear over 5 orders of magnitude of RNA concentration and reproducible between samples (the largest T N standard deviation was 20%). The comparative qRT-PCR assay used in this research represents a viable alternative to absolute quantification methods to monitor in situ fungal gene expression in natural and engineered environmental systems.