Cloning And Functional Analysis Of AP2/EREBP Transcription Factor In Malus Robusta R. And Oryza Sativa L.

Plant grows in an inconstant environment that frequently imposes constrains on growth and development. Drought, high-salinity and low temperature are commonly adverse environmental conditions. These stresses were given to more attention during recent years. 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 in environmental stress conditions. Transcription factors play vital roles in stress signal transduction, gene expression modulation and phase transition during plant development. AP2/EREBPs are important transcription factors which related to plant development and physiology. It was investigated that AP2/EREBP transcription factors are able to improve tolerance to drought, high-salt and cold stresses in Arabidopsis and rice, of which, DREB and ERF might be involved in abiotic and biotic stress responses. But there is less research in Malus robusta R. So it is important that this transcription factor can be isolated from Malus robusta Rehd. Using cis-element DRE and ERE as baits respectively, we isolated the transcription factor OsDREB1A and OsAP25 that specifically bind to the bait sequences in a cDNA library of Oryza sativa L. by yeast one-hybrid system and analyzed the structure and expression of OsDREB1A and OsAP25 genes. On the basis of similarity and structural characterization of amino acid sequence, except for OsDREB1A gene, OsAP25 gene was new one because it has not been reported up to now. The results of entire nucleotide sequence analysis revealed that OsAP25 contained a single open reading frame (ORF), encoding a predicted polypeptide of 345 amino acids with a molecular mass of 36.4kDa and a calculated pI of 5.54. OsAP25 also has the typical structure of ERF transcription factor. Two putative acidic domains in N-terminal and C-terminal regions were also detected. A putative nuclear localization signal located adjacent to C-terminal acid region. Furthermore, a putative mitogen-activated protein (MAP) kinase phosphorylation site was conserved in the C-terminal region of OsAP25 to involve in transcriptional active domain. The protein contained a typical DNA-binding domain consisting of 58 amino acids with alanine (A) in the 13^th position and aspartic acid (D) in the 18^th position, which was classified into ERF subgroup.Using two degenerate primers according to the sequences of DREB genes from Arabidopsis and rice. MrDREB2A gene encoding AP2/EREBP protein was cloned from Malus robusta R. by RACE. The results of DNA sequencing showed that there are five DREB homologous sequences. The deduced amino acid sequence revealed that the protein contained a typical DREB-type AP2 DNA-binding domain consisting of 58 amino acids with valine (V) in the 13^th position and leucine (L) in the 18^th position, which was classified into A-6 subgroup.RT-PCR analyses were performed to determine the expression profiles of the OsAP25 and the MrDREB2A genes. The results showed that OsAP25 gene was induced by drought and high-salt in an ABA-dependent pathway, and ethylene also faintly affected the transcript of OsAP25 gene. The MrDREB2A gene was highly induced by high-salt, cold, and drought and was strongly response to exogenous ABA. The expression patterns of OsAP25 and MrDREB2A genes in different organs and growth stages were shown. Under normal growth condition, OsAP25 expressed strongly in leaf and root and weakly expressed in shoot. MrDREB2A was expressed in leaf, faintly expressed in root and shoot. To sum up, the isolation of DREB and ERF transcription factors provide candidate genes for improving crop stress tolerances.