pCAMBIA 1302 vector

As functional proteins, dehydrins are present in many seeds and vegetable tissues that mature under adverse environmental conditions. However, the regulation of dehydrin expression remains unclear. In this study, a novel drought stress-related bHLH transcription factor TabHLH49 was isolated from a wheat cDNA library treated with drought and cold stress using the yeast one-hybrid system. TabHLH49 protein has a typical conserved bHLH domain and is a Myc-type bHLH transcription factor. TabHLH49 was detected in the nucleus of tobacco epidermal cells, and the C-terminal amino acid sequence was necessary for its transactivation activity. Real-time PCR analysis revealed tissue-specific and drought stress-responsive expression of TabHLH49 in wheat. Furthermore, dual-luciferase assay showed that TabHLH49 can positively regulate the expression of WZY2 dehydrin. Transient expression of TabHLH49 and BSMV-mediated gene silencing also indicate that TabHLH49 positively regulates the expression of WZY2 dehydrin and improves drought stress resistance in wheat.

To explore the function of TabHLH49 in wheat, Agrobacterium GV3101 transformed with pCAMBIA1302:TabHLH49:GFP and pCAMBIA1302 empty vector (control) was injected into wheat leaves for transient gene expression assay. After 10 days of natural drought, the leaves of wheat overexpressing GFP showed obvious wilting and curling. However, wheat overexpressing TabHLH49:GFP showed better growth. Real-time PCR results showed that after drought stress, the expression of TabHLH49 and WZY2 was significantly increased in wheat leaves overexpressing TabHLH49:GFP compared with the control. The researchers also measured the malondialdehyde (MDA) content, relative water content and chlorophyll content in the CK and OE lines. After drought stress, the OE line showed significantly lower MDA content, higher relative water content, and higher chlorophyll content than the CK line.

Figure 1. Transient expression assay of TabHLH49. a Schematic representation of pCAMBIA1302 empty vector (control), pCAMBIA1302-TabHLH49:GFP (effector) and reporter constructs used in the transient expression system. b Transactivation of WZY2 (Pwzy2) promoter by TabHLH49 shown by dual-luciferase assay in tobacco leaves. Phenotype (c), TabHLH49 and WZY2 gene expression levels (d-e), relative water content (f), chlorophyll content (g) and malondialdehyde of wheat leaves injected with Agrobacterium GV3101 containing pCAMBIA1302 empty vector (CK) and pCAMBIA1302:TabHLH49:GFP (OE) under normal growth conditions or drought stress conditions for 10 days. (Liu H, et al. 2020)