Isolation And Characterization Of Transcription Factor DREB/ERF Genes Related To Stress Tolerance In Triticum Aestivum L.

Plants have evolved to perceive different stress signals from their surroundings, to integrate them and to respond to these different stresses by modulating the expression of related genes. Transcription factors play vital roles in stress signal transduction, gene expression modulation and phase transition during plant development. The 145 AP2/EREBP transcription factors were classified into five groups: AP2 (14 genes), RAV (6 genes), DREB (also referred as CBF, 56 genes), ERF (also referred as EREBP, 65 genes) and others (4 genes) on the basis of number and similarity of the DNA-binding domain in Arabidopsis, of which, DREB and ERF might be involved in abiotic and biotic stress responses.DRE/CRT element is present in the promoters of multiple responsive to dehydration/cold-regulated (RDICOR) genes responding to both water deficit and low temperature, and DREB factors are involved in the regulation of abiotic stresses by DNA-binding activity with DRE/CRT element. ERE element (namely GCC-box) was in the promoter of pathogenesis-related (PR) genes shown to be the target for the ethylene signal transduction pathway. Preliminary analysis suggested that ERF transcription factors might be involved in the regulation of disease resistance responses by virtue of DNA-binding activity with ERE element.AP2/EREBP factors were given to more attention during recent years. It was investigated that DREB and ERF transcription factors were able to improve crops the tolerance to abiotic and biotic stresses. The intention of this paper is to isolate stress tolerance-related genes of transcription factors by Yeast one-hybrid system and hybridization in situ, and gain stress-tolerant wheat lines by transgenic technologies. It is of important significance to wheat stress-tolerant breeding and clarification of stress-tolerant molecular mechanism.Progresses of this research are following:1. Construction of cDNA library: Drought-induced and high-salt-induced wheat cDNA library with 3.6×10~6 pfu and 3.0×10~6 pfu were constructed using drought-tolerant cultivar Xiao Baimai and salt-tolerant cultivar Cha Dianhong as materials, respectively. The size of most inserts assembled about 0.7-2 Kb.2. Isolation of target genes: 10 DREB and 2 ERF transcription factor genes were isolated from drought-induced wheat cDNA library by hybridization in situ, 5'-RACE and RT-PCR. On the basis of similarity and structural characterization of amino acid sequence, 10 DREBs (DREB2-DREB11) were classified into 5 groups, first group: DREB3, DREB4 and DREB5; second group: W50, DREB6, DREB7 and DREB1; third group: DREB2 and DREB8; fourth group: DREB9 and DREB10; fifth group: DREB11; ERF1 and ERF2 were classified into different two groups. Except for third group DREB genes, other genes were new ones because they were not reported up to now.3. Analysis of gene structure: We investigated the configuration of 10 transcription factor genes(except for DREB2 and DREB8) and found that 9 genes (DREB3-7, DREB9-11 and ERF1) were all embedded in introns except for ERF2 gene. DREB4 cDNA corresponds to two genomic sequences with different size, 3.5 Kb and 1.2 Kb. Sequencing results showed that 1.2 Kb genomic sequence without intron corresponds to DREB4 cDNA sequence; 3.5 Kb genomic sequence was composed of 4 exons and 3 introns. There may be 3 different splicing ways to engender 3 cDNAs: DREB4A, DREB4B and DREB4C.4. Analysis of expression pattern: The results of Northern blot showed that DREB4 gene was induced by drought and high-salt in an ABA-dependent pathway, and cold also faintly affected the transcript of DREB4 gene. ERFl gene was highly induced by drought and cold, and faintly responds to exogenous ABA, however, ERF2 gene was highly induced by drought and high-salt, and faintly respond to exogenous ABA at early time. It was speculated that the transcript of ERFl and ERF2 might be dependent on ABA. ERFl and ERF2 genes were also highly induced by the inoculation of E. graminis f.sp. tritici. Therefore, some ERF genes were not only responsive to abiotic stress, but also involved in signal transduction induce by pathogen.5. Gene location: DREB2 and ERFl genes were assigned respectively to chromosomes 7A/7B/7D and 7A/7B by Southern blot analysis using nullisomic-tetrasomic series of T. aestivum L. cv. Chinese Spring as materials. ERF2 gene was located on chromosome 3D by PCR using specific primers. We firstly located AP2/EREBP transcription factors from monocotyledonous plants on specific chromosomes.6. Analysis of DNA-binding specificity and activation activity: Electrophoretic mobility shift assays (EMSA) revealed that DREB4 and DREB5 proteins could specifically bind to DRE m-acting element, furthermore, !C2C of DRE core sequence 'C2C3G4A5C might be more important than 3G4A5C in interaction with DREB4 and DREB5 proteins. To investigate the DNA-binding specificity in vivo and activity, we fused the entire encoding region of DREB4 and DREB5 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 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 DREB4 and DREB5 was able to bind to DRE element specifically in yeast cells and activated reporter gene expression. The |3-galactosidase activities of the survival clones were also analyzed and the result further confirmed the DNA-binding specificity and activation activity of DREB4 and DREB5 factors.7. Analysis of gene function: DREB4 and ERF 1/2 were respectively transformed into Aribidopsis thaliana and Nicotiana babacum by Agrobacterium infiltration method, and these three genes were also transformed into wheat by particle gun. The overexpression of DREB4, ERFl or ERF2 could improve plants the resistance to drought, high-salt or cold, for instance, the survival rate of transgenic A. thaliana overexpressing DREB4 gene enhanced 90%, 40% and 55% than control plants, respectively. Similarly, transgenic A. thaliana overexpressing ERFl and ERF2 gene also...