Cloning And Functional Analysis Of DREB-Like Gene From Suaeda Salsa

Drought, high-salinity and low temperature are commonly adverse environmental conditions. These stresses were given to more attention during recent years. Plants have evolved to perceive different stress signals from their surroundings, to integrate them and to respond to these different stresses by modulating the expression of related genes in environmental stress conditions. Transcription factors play vital roles in stress signal transduction, gene expression modulation and phase transition during plant development. AP2/EREBPs are important transcription factors which related to plant development and physiology. It was investigated that AP2/EREBP transcription factors are able to improve tolerance to drought, high-salt and cold stresses in Arabidopsis and rice, of which, DREB might be involved in abiotic and biotic stress responses. Suaeda salsa grown in salt environment, has strong tolerance to salt and drought stress.Cloning and functional analysis of SsDREB transcription factor may help to understand the physiology in salt and drought stress, On the other hand, it can be transferred to other plants to improve plant abiotic stress resistance.In this paper, a 180 bp fragment was amplified from Suaeda salsa using its cDNA as the template and the conservative sequences of reported DREB protein genes as the primers PCR.A full-length cDNA with completed open reading frame of 364 amino acids was cloned using the strategy of RACE(rapid amplification of cDNA ends). This cDNA was designated as SsDREB, which contained 1493 bp with an untranslated region of 34 bp at 5'end and a polyA tail at the 3'end. The deduced amino acid sequence revealed that the protein contained a typical DREB-type AP2 DNA-binding domain consisting of 64 amino acids with valine (V) in the 14th position and leucine (L) in the 19th position.Real-time quantitative PCR analyses were performed to determine the expression profiles of the SsDREB gene. The results showed that SsDREB gene was induced by high-salt and drought, not by cold and ABA. The expression patterns of SsDREB gene in different organs by real-time quantitative PCR showed that SsDREB gene expressed strongly in leaf, weakly expressed in root and stem. The recombinant vectors pGADT7-Rec2-SsDREB was constructed and transformed into the yeast stains Y187, then the positive yeast transformants were selected using the SD/-His+10 mM 3-AT selective medium. The result testified that SsDREB contained a DNA-Binding domain and the SsDREB protein specifically bound to the DRE sequence.SsDREB was transformed into tobacco NC89 with Agrobacterium EHA105 containing the plant expression vectors pCAMBIA2301-SsDREB. The positive tobacco transformants were selected dusing MS medium containing kanamycin, PCR and RT-PCR.The results of stress tolerance experiments and physiological test showed that the expression of SsDREB could improve resistance to abiotic streses of transgenetic tobacco. Semi-quantitative RT-PCR analyses the regulation of downstream genes suggested that over-expression of SsDREB could enhance the expression of lipid transferase(ltp1)(X62395), Lea5(AFO53076),H+-ATPase B subunit(AF220611),H+-ATPase(X66737). Over-expression of SsDREB could enhance the expression of some stress-related genes and enhaneed the toleranee of transgenic lines.