Novel intron-containing luciferase genes for quantitative analysis of mRNA levels in transient gene expression assays

Abstract

Firefly luciferase (Fluc) is commonly used as a sensitive reporter for transient gene expression assays in plants.Although the protein level of Fluc can easily be quantified, it is difficult to quantify the mature mRNA level of the reporter gene in these assays. The mRNA level can be measured using quantitative reverse-transcription PCR (qRT-PCR), but the reaction often amplifies both the target mature mRNA and contaminating DNA or unspliced pre-mRNA, thus providing inaccurate results. To address this problem, we constructed a series of novel Fluc genes containing intronic sequences measuring 315 bp or longer. Here, we show that the contaminating DNA and unspliced mRNA were not PCR-amplified due to presence of these introns. Moreover, the intron-containing Fluc gene conferred superior Fluc activity to the original intron-less Fluc gene. We propose that the novel Fluc genes reported here can be used to quantitate both protein and functional mRNA levels in transient gene expression assays in plants.

Overexpression of a <i>CO</i> homologue disrupts the rhythmic expression of clock gene <i>LgLHYH1</i> in <i>Lemna gibba</i>

“…Tiny floating plant bodies, rapid growth rates, and strictly controllable aseptic culture conditions make these Lemna plants experimentally attractive (Hillman 1961a). Various techniques to monitor luciferase activities have been developed in plants to allow quantitative analyses of gene expression and time-series data collection (Hayakawa et al 2012;Muranaka et al 2013;Watanabe et al 2011). A transient gene expression system to monitor circadian expression of bioluminesent reporters has also been developed in Lemna plants (Miwa et al 2006;Muranaka et al 2013).…”