Functional Analysis On GmDREB Transcription Factor From Glycine Max (L.) Merrill

DREB (dehydration responsive element binding) was determined as a key factor which involved in many functions in plants, such as response to the stress of drought, low-temperature, or high-salt stress, regulating some physical metabolism in vivo. DREB was widely investigated and applied after the DRE core sequence being discovered by Yamaguchi-Shinozaki in 1994. There are more than 15 DREB transcription factors registered in GenBank at present. Couples of researchs indicated that these genes play crucial roles in response of abiotic stress.A new transcription factor (GmDREB, GenBank accession No.: AF514908) which involved in resistance to abiotic stress was cloned in our lab. Based on the previous research, the character, function and mechanisms of the gene was investigated through transform the gene to Arabidopsis thaliana.The results of Bioinformatics analysis indicated that there is one open reading frame of 525bp in the gene. The open reading frame codes a protein which was constituted by 174 amino, among them, there is a AP2/EREBP conserve region constituted by 58 amino. A nuclear location signal region and a transcriptional activation region locate on N-terminate and C-terminate respectively.In this study, the full length coding sequence of GmDREB was constructed to the pCAMBIA 1304 which included GFP and GUS regulated by CaMV35S promoter. The improved floral-dip method was employed to transform the wild type Arabidopsis. Transgenic homozygous lines which have genetic stability were obtained by resistant separation. The results of histochemical analysis of GUS indicated that the GUS gene was widely expressed in the whole planta after transforming the empty pCAMBIA 1304. The expression of GUS has tissue specificity after transforming the 35S:GmDREB-GFP-GUS into seedlings. The GUS was strong expressed in main roots, lateral roots, root hairs, hypocotyls and true leaves when the seedlings are 5d and 7d, whereas, they seldom expressed in cotyledons. The results of GFP fluorescence location analysis indicate that the GFP was widely expressed in the whole planta and whole cell after transforming the empty pCAMBIA 1304 vector. Similar to the expression of GUS, the GFP mainly expressed in roots, hypocotyls and located in cell nucleus and cytomembrane in the 35S:GmDREB-GFP-GUS transform seedlings.For further investigation on the functions and of GmDREB, the phenotype of wild type and transgenic planta was observed after drought and high-salt stress. Couples of physical index were determined after 0d, 6d and 15d treatment. The results of phenotype observation indicated the drought and high-salt stress can lead to reducing of the water content. The rate of reducing in transgenic lines was less than in wild type significantly. The drought and high-salt stress can lead to the rising of MDA content both in transgenic and wild type lines. The rate of rising in transgenic lines was less than in wild type. The content of soluble sugars and proline in transgenic lines are more than in wild type. These results indicated that the tolerance of transgenic lines is better than wild type, and the exogenous gene can improve the stress-resistance significantly.