Functional Identification And Promoter Separation Of Stress-resistent Related Transcription Factor Genes, GmDREB3, From Soybean (Glycine Max L.)

Plant growth and productivity are greatly affected by environmental stresses such as drought, high salinity, and low temperature. Expression of a variety of genes is induced by these stresses in various plants. DREB transcription factor plays a role in plant resistance to drought,hight salinity and low temperature. GmDREB3 from soybean is the member of soybean DREB transcription factor gene family and have been separated from soybean genome. Previous studies have shown that GmDREB3 could be induced by abiotic stress and improve the tolerance of transgenic tobacco and Arabidopsis plants in response to dehydration and low-temperature stresses. 15 transformated wheat lines were obtained by biolistic bombarding with GmDREB3 gene in order to analyse expression situations of GmDREB3 gene in wheat. The results of PCR analysis in T0,T1 generation transgenic plants showed that the transformated genes could be inherited to next generation stably. The results of identification of drought tolerance of some T1 generation transgenic lines showed that the level of drought tolerance of the transgenic lines, C1 and C2, were high than the wild type and GmDREB3 gene could improve transgenic wheat to drought tolerance. However, it is not clear that the transcriptional regulation mechanism of GmDREB3 in response to drought and cold stresses in plants. GmDREB3 gene promoter has been separated from soybean genome in order to analyze transcriptional regulation molecule mechanism of GmDREB3 gene. The GmDREB3 promoter, 1921 bp, has been separated in SiteFinding-PCR method which is a simple and efficient PCR method for chromosome walking. The sequence is abundant in A/T base and predicted to involve a lot of putative cis-element, such as low-temperature responsive element MYC,dehydration stress responsive element MYB,ABA responsive element ABRE as well as TATA-box in biological database. To identify the key promoter cis-element and promoter regulatory domain controlling gene regulation specificties, successive promoter truncations were made in the GmDREB3 gene promoter and attached to GUS reporter gene. Then, express these plant expression vectors in wheat callus in different stress-treatment conditions by particle bombardment and analyze these putative cis-elements function in vitro by histochemistry staining and GUS fluorescence analysis experiments. The results suggested that the promoters of between -285 and -1648 (relative to the translational start site) nucleotides activated GUS report gene expression in a number of cells of the wheat callus under drought and low-temperature stresses. But only the region between -285 and -1117 nucleotides can activate maximal GUS gene expression in wheat callus. Other region such as -1117 and -1648 decreased in activating abilities of GUS gene expression for the wheat callus in response to dehydration and low-temperature stresses. So, it is possible that there are some key up-regulated motif between -285 and -1117 and down-regulated motif between– 1464 and -1648. As a result, these motifs can activate gene expression in wheat callus responsing to stress in appropriate level due to interaction with up-regulated motif and down-regulated motif.