Cloning And Characterization Of The Gene Encoding DREB Protein In Turf Grass, Poa Pratensis

Plants are exposed to environmental stresses such as drought, high salt, and low temperature, which cause adverse effects on the growth and development of plants. These damages to plants caused by abiotic environmental stresses have been the important factors limited productivity of crops, improvement of ecological environment and sustained development. Therefore, it is a key and fundamental work for future agriculture to reveal the responsive mechanisms of plants to adverse environmental stresses and enhance the ability of crops to tolerate abiotic stresses, which could meet the food needs of increasing population in the world. To further study molecular mechanisms of plants to adverse environmental stresses and acquire efficient genes which are very useful to improve plant abiotic stresses tolerance, in this paper, a gene and two cDNA fragments were isolated by using RACE (Rapid Amplification of cDNA Ends) method.DREBs (dehydration responsive element binding protein) is a type of transcription factors related to abiotic stresses. These proteins are able to discern and bind the DRE cis- element in the promoter of some genes concerned with cold, drought and high-salt stresses, resulting in initiating the expression of these genes at transcriptional level. Therefore, it would be a good strategy to improve tolerance of various kinds of agriculturally important plants to abiotic stresses by DREB genes transfer. Using RACE methods, we obtained a cDNA clone encoding a new DRE-binding protein named PpDREB2 and two cDNA fragments in Poa pratensis. The PpDREB2 has a completed ORF, being 833 bp in length and encodes a deduced protein of 264 amino acid residues. It is shown from Protein Blast data that this protein belongs to a typical member of the AP2/EREBP transcription family with conserved NLS, AP2 DNA-binding domain and acidic region. Genomic DNA structure analysis of PpDREB2 indicated that there should be no intron in the genomic DNA of the PpDREB2 gene. The tissue organ-specific expression pattern of the PpDREB2 showed that its transcripts were detected abundantly in leaves and stems, and weakly in roots. Analysis of mRNA accumulation profiles of PpDREB2 showed that its expression is strongly induced by drought and salt treatment. However, Little changes in the amount of its mRNAs were observed during the process of ABA and cold treatment. It is concluded that the PpDREB2 would improve abiotic stress tolerance of plants through an ABA-independent pathway.The number of PpDREB2 homologous genes in Poa pratensis genome was estimated by DNA gel blot analysis. Nuclear DNA from Poa pratensis was digested with KpnI, SphI, and XbaI and hybridized by using the PpDREB2 cDNA as probe. The result indicated that the PpDREB2 is a three-copy gene.Two cDNA fragments encoding DREB protein are 598 bp and 296 bp in length respectively. There is a 176 bp overlap sequence between the two cDNA fragments.Besides, the plant transformation vector p3301-BI121-PpDREB2 was constructed in this experiment. The vector contains the genes encoding PpDREB2, gus and plant selection marker phosphinothricin acetyltrasferaese (PPT). In this vector, CaMV35S promoter controls the expression of PpDREB2. The p3301-BI121- PpDREB2 was transformed into rice via agrobacterium mediation and 15 regenerated positive rice plants have been obtained.All the results above would lay down the basis for further research on the tolerance of Poa pratensis to abiotic stresses and crop improvement by genetic engineer.