Characteristic Analysis And Functional Identification Of DREB Transcription Factor Gene From Wheat (Triticum Aestivum L.)

AP2/EREBP transcription factors genes have been isolated from a wide variety of plants including Arabidopsis, wheat, maize, tobacco and rice, and regulate the expression of many genes in plant. In AP2/EREBP family, DREB transcription factors play important roles in response to abiotic stresses. The study is to investigate the new DREB genes, TaDREB1, TaDREB2, TaDREB3, TaDREB6, TaDREB9, W42 and TaDREB10 which were successively cloned from Triticum aestivum.(1) Analysis of expression pattern: To investigate the expression pattern of DREB genes responsive to different stresses, total RNA were extracted and purified from T. aestivum which subjected to various stress treatments (drought, high salt, low temperature and ABA) for different time. The results of RT-PCR analysis indicated that TaDREB3, TaDREB9, TaDREB10 and W42 could be induced by drought, high salt, low temperature and ABA, TaDREB6 could be induced by drought, but the expression mode was different. TaDREB3, TaDREB9, TaDREB10 and W42 may play important role in responsive to drought, high salt, low temperature and ABA stresses in wheat.(2) Subcellular localization assay: In order to detect the subcellular localization of DREB transcription factor proteins, TaDRF1, TaDREB3, TaDREB6, W42, TaDREB9 and TaDREB10 were fused to the 5'terminus of the coding region of green fluorescence protein (GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Then the fused plasmids were introduced into onion epidermal cells using a particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 25℃in the dark and localization of the fusion proteins was then detected using a confocal microscope equipped with appropriate filter. The subcellular localization assay indicated that TaDRF1, TaDREB3, W42, TaDREB6 can localize into the nuclei. However, TaDREB9 and TaDREB10 cannot localize into the nuclei.(3) Analysis of DNA-binding specificity and activity: To investigate the DNA-binding specificity in vivo and activity, we fused the entire encoding region of DREB genes into the yeast expression vector YepGAP without AD domain and transformed into two yeast strains carrying either the DRE or mutant DRE element. The result indicated that the yeast transformant with the DRE was able to grow on selective medium lacking His, Try and Ura in the presence of 3-AT, whereas transformant with mutant DRE was not able to grow under the same conditions. This indicated that W42, TaDRF1, TaDREB2, TaDREB3, TaDREB9 and TaDREB10 was able to bind to DRE element specifically in yeast cells and activated reporter gene expression.(4) Analysis of gene function: W42 was transformed into Arabidopsis thaliana by Agrobacterium infiltration method. T2 seeds of transformed Arabidopsis were surface-sterilized and planted on MS medium supplemented with 50 mg·mL-1 of kanamycin for selecting transgenic plants. The overexpression of W42 could improve plants the tolerance to drought and high salt.(5) Gene location: Using a nullisomic-tetrasomic series of T. aestivum. cv. Chinese Spring, the TaDREB6 gene was located on chromosome 3A by PCR using specific primers.The present study provides theoretical basis for us to further study the molecular mechanism of the DREB transcription factors in regulating the stress signal networks in plant, and good candidate gene resources for improving crop tolerance to adverse environmental conditions using gene engineering.