Identification Of The Mechanism Of Transcriptional Regulation Of Stress-resistent Related Transcription Factor Gene, GmDREB3 From Soybean (Glycine Max L.)

The DREB (dehydration responsive element binding protein) is the stress-related transcription factor family. They exist in all kinds of plants, and regulate the expression of many stress-related genes. DREB transcription factors play a role in plant resistance to drought, hight salinity and low temperature. DREB genes current studies focused on functional analysis of DREB genes, little is known about their transcriptional of regulation.Functional studies have shown that overexpressing GmDREB3 can significantly improve the resistance of plants to drought. In order to analyze molecule mechanisms, we cloned promoter region of GmDREB3 gene from soybean genome. To identify key cis-elements in the promoter region controlling its expression, successive truncations were made in the promoter of GmDREB3 and put in front of the GUS reporter gene. These plant expression vectors were introduced to wheat callus under different stress conditions by particle bombardment. We analyzed the activities of different promoter regions by in vitro histochemistry staining and GUS fluorescence analysis. Our results suggested that promoter sequences between -705 and -1117 (relative to the translational start site) activated,while sequences between -1117 and -1464 decreased GUS gene expression response to dehydration and low-temperature stresses. Based on that, these two areas were analysed further. Our results showed that -705~-731bp contained a key MYB recognition motif and -1117~-1269 bp contain MYC key down-regulated motif. The bait vector was constructed using the regulation element, respectively. We used yeast one hybrid to screen for proteins that bind to the key elements, Using MYC-motif containing region as the bait, we identified a GmERF protein, while using MYB-motif containing region as bait, we identified a GmMYB protein. We verfied such protein-DNA interaction both in vivo and in vitro. GmMYB protein was capable of transcriptional activation in yeast, while GmERF was not. Analysis of the competitive binding of the GmDREB3 promoter, we find that GmERF protein binds to the GmDREB3 promoter with higher affinity than GmMYB. We determined the expression pattern of GmMYB and GmERF by RT-PCR. In addition, overexpression of GmMYB enhanced tolerance to drought and salt stress of transgenic Arabidopsis. While overexpression of GmERF did not significantly increase tolerance to drought and salt stress of transgenic Arabidopsis. In summary, low temperature treatment in soybean within 3h, GmMYB binds to the MYB element, activates DREB3, which then upstream activates resistance genes; 24h low temperature after treatment, GmERF protein binds to MYC and the ERF element, inhibits DREB3 expression, thereby inhibiting the expression of downstream genes. As a result, these motifs can activate gene expression in wheat callus responsing to stresses at appropriate levels due to interaction with two containing cis-regulatory elements.