Combination of the endogenous promoter-intron significantly improves salt and drought tolerance conferred by TdSHN1 transcription factor in transgenic tobacco

Recent years have witnessed a renewed interest in introns as a tool to increase gene expression. We previously isolated TdSHN1 gene encoding a transcription factor in durum wheat. Here we show that TdSHN1 intron contains many CT-stretches and the motif CGATT known to be important for IME. When subjected to bioinformatics analysis using IMEter software, TdSHN1 intron obtained a score of 17.04 which indicates that it can moderately enhance gene expression. TdSHN1 gene including its intron was placed under the control of TdSHN1 endogenous salt and drought-inducible promoter or the constitutive 35S promoter and transferred into tobacco. Transgenic lines were obtained and designated gD (with 35S promoter) and PI (with native promoter). A third construct was also used in which intron-less cDNA was driven by the 35S promoter (cD lines). Results showed that, gD lines exhibited lower stomatal density than cD lines. When subjected to drought and salt stresses, gD lines outperformed intron-less cD lines and WT. Indeed, gD lines exhibited longer roots, higher biomass production, retained more chlorophyll, produced less ROS and MDA and had higher antioxidant activity. qRT-PCR analysis revealed that gD lines had higher TdSHN1 expression levels than cD lines. In addition, expression of ROS-scavengering, stress-related and wax biosynthesis tobacco genes was higher in gD lines compared to cD lines and WT. Interestingly, under stress conditions, PI transgenic lines showed higher TdSHN1 expression levels and outperformed gD lines. These results suggest that TdSHN1 intron enhances gene expression when used alone or in combination with TdSHN1 endogenous promoter.