Nanoluciferase-based transient expression system for plant gene regulation studies

Overview

This research proposes a transient expression system that leverages Nanoluciferase (NLUC) reporter fusions to investigate gene expression regulation in plants, with a focus on potato species. Current transient Agrobacterium-mediated transformation methods in plants are limited by low transformation efficiency and weak reporter signals, making it difficult to quantify gene expression dynamics accurately over time. By using the exceptionally bright NLUC reporter gene as both a transcriptional and translational fusion partner, this approach enables more sensitive, longer-duration tracking of promoter activity and protein expression in vivo. The system is particularly suited for studying dynamic processes such as circadian clock gene regulation under light/dark cycles.

Technical specifications

Key features:

• Uses a plant codon-optimized canonical Nanoluciferase (NLUC) as a protein-level reporter, with genes (including promoter, exons, and introns but excluding the stop codon) cloned in-frame upstream of NLUC
• Employs a destabilized NLUC-PEST variant as a transcriptional reporter for promoter activity analysis
• Leverages Agrobacterium tumefaciens-mediated transient transformation of potato leaf tissue
• Constructs are imaged using a CCD camera system for non-invasive signal detection
• Builds on prior validation of the GH3 promoter's oscillatory activity using firefly luciferase in stably transformed potato plants
• Prioritizes putative circadian clock genes displaying high protein dynamics for translational fusion testing

Technology readiness level

The system is at an early-to-mid validation stage. The research team has demonstrated prior expertise working with canonical NLUC in algae using both transcriptional and translational fusions, and has shown that Agrobacterium-mediated transient transformation with firefly luciferase is feasible in potato leaves, though signal intensity is currently insufficient for extended time-course studies. Future validation efforts will focus on demonstrating that NLUC-based constructs produce strong, trackable signals over multi-day periods in transiently transformed potato species, enabling accurate measurement of dynamic RNA and protein expression patterns under light/dark cycles.