Generation of a Highly Efficient and Chemically Inducible Gene Expression System.

Abstract

Background: Chemically inducible gene expression systems provide precise control over the temporal and spatial expression of genes. They are powerful tools for analyzing gene function during plant development and can effectively avoid the issues associated with transgene expression driven by constitutive promoters. The alc gene expression system derived from Aspergillus nidulans is one of the most promising chemically inducible expression systems in plants, owing to its significant potential for both basic research and agricultural applications. Previous studies have demonstrated the utility of the alc system in several dicot species, but systematic optimisation of promoter architecture and induction methodology in Arabidopsis remains comparatively limited.

Methods: We employed a multi-level study design, combining Agrobacterium-mediated genetic transformation, GUS histochemical staining, and GUS quantitative assays. First, different types of alc gene expression systems were constructed through codon optimization of the GUS and alcR genes, and by incorporating the CaMV 35S minimal promoter (min35S), the TMV omega sequence, the Kozak sequence, and specific binding sites for the AlcR transcription factor. Second, these distinct alc expression systems were individually transformed into Arabidopsis thaliana to obtain single T-DNA insertion homozygous transgenic plants. Third, ethanol was applied via root drench, foliar spray, or vapor induction, and the performance of different alc systems was evaluated based on GUS protein expression, to determine the optimal induction conditions and identify the most efficient ethanol-inducible gene expression system.

Results: Four alc systems (Ves1-Ves4) were successfully developed. GUS gene expression driven by these systems was precisely regulated by ethanol and was dependent on induction time and the method of induction. Among the induction methods, ethanol vapor was the most effective, followed by root drench, whereas foliar spray was the least effective. Furthermore, Ves1 was identified as the most efficient alc expression system, Ves2 as an efficient alc expression system, Ves3 as a usable alc expression system, and Ves4 as potentially defective with limited practical application value.

Conclusions: This work lays a foundation for further research on the efficient alc system and the exploration of its molecular mechanism underlying high expression efficiency. The most efficient Ves1 and the highly efficient Ves2 provide a clear basis for selection in subsequent applied research, and they are expected to play a greater role in plant functional genomics and agricultural biotechnology.