Cloning And Functional Analysis Of DREB Transcription Factors In Nictotiana Tabacum

Drought, high-salinity and freezing are the major environmental factors that influence the development, growth and quality of plant. Drought responsive element binding factors (DREB) are plant specific transcriptional factors, which play pivotal roles in plant resistance to abiotic stresses. In this study, new DREB transcription factors were cloned and functional analyzed from tobacco K326(Nicotiana tabacum) by using methods of biochemistry and molecular biology. This research results can help to further reveal regulation mechanisms of DREB family and provide a study foundation for improving tobacco comprehensive stress-tolerance by using transgenic technology in the future.Making use of Genome Walking, four DREB-Like transcription factor genes were cloned from tobacco ,and named as NtDREBI, NtDREB2, NtDREB3, NtDREB4, respectively. The sequences analysis indicated that they possessed typical structural characteristics of AP2/EREBP transcription factor family. Result of Phylogenic tree analysis showed that four genes attributed to DREB subfamily,and there were highly homologous for NtDREBI with BnDREB2-1(Brassica napus),as well as NtDREB2, NtDREB3, NtDREB4 with LeCBF1 (Lycopersicon esculentum), NtACRE111B(Nicotiana tabacum). The alignment in the ERF/AP2 domains found that the 11st amino acid residue was conservative Asp or Gly in all the AP2/EREBPs except NtDREB4.These four genes could be induced strongly by low temperature.Moreover, NtDREB4 also could be induced in some conditions such as drought, high salinity, ethylene.Yeast one-hybrid showed that NtDREBI could bind to DRE cis-element and activate the expression of downstream genes, NtDREB2 and NtDREB3 had capability of binding to DRE element but no activation function. However, NtDREB4 couldn't bind to DRE element.Alignment of deduced amino acid sequences of NtDREBI,BnDREB2-1, NtDREB1A and AtDREB1A found that the 148th amino acid residue in transcription regulation region had significant difference. Method of site-directed mutagenesis further indicated that the interaction between the 148th amino acid residue of DREB1A transcription factors and its neighboring residues play a crucial role for the transcription activation function.Using the construction PDI fusion expression system, we acquire the high-expressing soluble protein with the Ni-NTA purification method , further electrophoresis mobility shift assay(EMSA) result suggested that NtDREB2 and NtDREB3 had capability of binding to DRE element specifically whereas NtDREB4 couldn't bind to DRE and GCC box element. Due to the 11st amino acid residue difference in ERF/AP2 domain of NtDREB with other transcription factors, site-directed mutagenesis and yeast one-hybrid results further indicated that the 11st amino acid(G and D)located in loop structure of ERF/AP2 domain might be a crucial amino acid of DREB transcription factors binding to DRE cis-element and sustaining the stabilization of ERF/AP2 domain.