Cloning And Functional Analysis Of DREB Transcription Factor Genes From Populus Euphratica

Transcription factors play an important role in regulation of plant growth and physiological metabolism by regulation of gene expression at the transcription level. DREB transcription factors exist in all kinds of plants, such as Arabidopsis, maize, tomato,tobacco and rice et al., and regulate the expression of many functional genes in plant. In this paper,We researched systemically the basic characterization and biological function of two new transcription factor genes, PeDREB2a and Pe DREB2b,isolated from Populus euphratica. The results proved that two genes have the basal characterization of DREB transcription factor. The expression of these two genes in transgenic tabacco could improve tolerant to abiotic stresses, which is advantage for our to study the molecular mechanism of these two transcription factors regulating the signal network of response to environmental stresses in plant, and provide a new genes to improve crop tolerance to adverse environmental conditions.The main results of this study are as follows:1. A pair of degenerate primers was designed according to the conserved regions which encoding the DREB DNA binding domains in plant DREB genes. Two fragments were amplified from leaves of Populus euphratica using RT-PCR. Two novel genes encoding DREB transcription factors, PeDREB2a(EF597499) and PeDREB2b(EU195881) were isolated by RACE-PCR. Comparison of deduced amino acid sequences showed that they are typical DREB transcription factors.2. Semiquantitative RT-PCR showed that PeDREB was greatly induced by drought, high salinity, low tempreture , but not by ABA.3. The full-length DNA sequences of PeDREB2a and PeDREB2b were also amplified using genomic DNA as templates. The results indicated that the two genes have no introns.4. The recombinant vectors pBridge-PeDREB2a and pBridge-PeDREB2b were constructed and transformed into the yeast stains AH109,then the positive yeast transformants were selected using the SD/-Trp selective medium and the SD/-Trp/-His selective medium. The transcriptional activation of PeDREB2a and PeDREB2b proteins was confirmed by the yeast system, and itsβ-galactosidase activity was detected as 27.8U and 23.75U, respectively. The results of the colony-lift filters assay indicated that the yeast transformants of PeDREB2a and PeDREB2b showed a clear blue.5. The green fluorescent protein expression vectors p163-GFP-PeDREB2a,and p163-GFP- PeDREB2b were constructed and transformed into the epidermal cells of onion via particle bombardmental method. The results of instantaneous expression showed that PeDREB2a and PeDREB2b proteins all were localized in cell nucleus, which was consistent with the prediction of the subcellular localization.6. PeDREB2a and PeDREB2b were transformed into tobacco NC89 with Agrobacterium LBA4404 containing the plant expression vectors pCAMBIA2301-PeDREB2a and pCAMBIA2301- PeDREB2b, respectively. The positive tobacco transformants were selected using MS medium containing kanamycin, PCR and RT-PCR. Results of stress tolerence experiments and physiological test showed that the expression of PeDREB2a and PeDREB2b could improve resistance to abiotic stresses of transgenetic tobacco.