LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo

Abstract

(M.R., B.W.)Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra-and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore, LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC1. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo.

RALFL34 regulates formative cell divisions in Arabidopsis pericycle during lateral root initiation

“…The pRALFL34::LUC was generated as follows: the 400bp RALFL34 promoter fragment was PCR amplified using the RALFL34 forward (CAACTGGACCCATCCGAA) and reverse primer (CGGCGATTGTTGGGGGA) and cloned into the pGem-T-easy vector. After sequencing confirmation, the 416bp RAFL34 promoter fragment was released from the pGem ® -T Easy vector with restriction enzymes Pst I and Nco I and then cloned into the LucTrap vector ( Calderon-Villalobos et al , 2006 ; Lau et al , 2011 ; De Smet et al , 2013 ). The sequence of the final construct was confirmed.…”

A cyclic nucleotide sensitive promoter reporter system suitable for bacteria and plant cells

“…The A. thaliana OPTX gene has been annotated as a member of the plant oligopeptide transporter family (although it was originally annotated as NTL1 ( LOW AFFINITY NITRATE TRANSPORTER )) and is predicted to be a membrane bound transporter of small peptides. Promoter fragments of approximately 1000 bp of SOS3 , CHX21 and OPTX were amplified and cloned into the plant luciferase vector pLucTrap3(GW) [ 30 ] to create p SOS3 : LUC , p CHX21 : LUC and p OPTX : LUC (Figure 2 A and 2 C).…”

Auxin dynamics: the dazzling complexity of a small molecule’s message

“…The time lag between transcriptional induction and actual protein availability has been assessed by a luciferase-based reporter assay for one of the very early auxin-inducible genes, GH3.2, to be 30 to 45 min (Calderon-Villalobos et al 2006). Therefore, auxin-induced responses within the Wrst 30 min require signaling components other than the TRANSPORT INHIBITOR RESPONSE1 (TIR1)-dependent signaling pathway described below.…”