Genome-wide Analysis Of Target Genes Of Dehydration Responsive Element Binding Factor In Maize

Dehydration Responsive Element Binding protein (DREB) is one of the most important transcription factor which transducts abiotic stress signals at the transcriptional level. So far, researchers have cloned members of DREB family from multiple species, and validated their importance during the plant stress physiology.4 DREB genes have cloned from maize till now, which are DBF1, DBF1, ZmDREBIA and ZmDREB2A. And their transcription factor function at abiotic stress has been verified by experiments except DBF2. However, references to its regulatory pathways are so limited. In maize, rab17 is the only target gene of DREB verified by experiment. Identification of target genes of DREBs genome-widely is not only a fundamental step to elucidate the construction of regulatory networks, but also an important clue to understand the mechansim of plant response to enviroment.Studis show that DREBs activate their downstream genes expression through binding to a consensus sequence CCGAC at the regulatory region by their APETALA2 (AP2) domains. Additionly, transcription factors binding to the cis-acting element are often cooperate with other trans-acting factors. Therefore, with the completion of the maize genome sequencing project, it is theoretically feasible to discover the target genes of DREBs by looking for the DRE cis-elements and their conservation flanking sequences genome-widely through bioinformatics.In 2009, Wang et al. defined the DRE motif and its 200 bp length flanking sequences from identified DREB target genes as DRE frame sequence (DFS). Then by using HexDiff algorithm, they calculated the ratio of the frequency of hexamers in DFSs and nDFSs (which was contructed by DRE motif and random sequence on its both sides), and collect hexamsers with the ratio greater than 3.0 as the characters of DFSs. Finally, these datas were used to construct a DREBs target gene identification model by support vector machine (SVM).In this research,11472 full-length cDNAs were downloaded from maize full-length project of Starndford University. By using BLAST,8416 of them were mapped to sigle locuses on chromosomes of maize. Then search the DRE motifs in the region of 1000 bp ahead from the transcription initiation site. And though using the SVM trained by DREBs target gene identification model to classify those DRE motif with the 200 bp flanking sequences on both sides were DFSs or not, we got a total of 694 positive results. With Reference to gene expression profiles at low temperature, drought and high salt stress of maize, we finally identified 596 DREB target genes.In order to verify the prediction result, firstly we expressed and purefied DBF1, ZmDREB1A and ZmDREB2A by prokaryotic expression. And then randomly picked 17 genes, and use its DRE motifs and DREBs from mazie to do nitrocellulose filter membrane binding experiment. The result shows that DREBs can bind to these sequences in different levels, which greatly support our prediction. And the results obtained in this study provided the primary information that warranted further experimental investigation regarding the anti-abiotic stress regulatory network of DREBs in maize.