Identification And Characterization Of DREB Genes From Rice (Oryza Sativa L.)

Recently, DREB transcription factors have been identified in many plant species. It has been established that they specifically interact with DRE/CRT cis-acting elements and induce expression of multiple drought- or cold-responsive genes. Constitutive overexpression of DREB genes has been reported to trigger the DREB regulons and enhance freezing, drought, and salt tolerance in some of the transgenic plants. Due to its intrinsical nature and the potential importance to agriculture, considerable effort has been directing at isolating DREB genes and subsequently analyzing their functions.Five genes encoding AP2/EREBP containing proteins were cloned from rice (Oryza sativa L. cv Lansheng) by using PCR. The deduced amino acid sequences revealed that each protein contained a potential nuclear localization signal, a typical DREB type AP2 DNA-binding domain with valine (V) in the 14th position and glutamic acid (E) in the 19th position, and a possible acidic activation domain. CR350, possessing a conserved CEVR amino acid sequence between V14 and E19, was classified into A-1l subgroup and renamed as OsDREB1-1. CR102, CR103, CR223, and CR250, all possessing the same conserved SEIR amino acid sequence between V14 and E19, were classified into A-4 subgroup and renamed as 0sDREB4-l, OsDREB4-2, OsDREB4-3 and OsDREB4-4, respectively.Database search indicated that the five genes were single copy in the rice genome; both OsDREB4-1 and OsDREB4-3 were located on chromosome 2; both OsDREB4-4 and OsDREB1-1 were located on chromosome 4; OsDREB4-2 was located on chromosome 10.Northern and RT-PCR analyses were performed to determine the expression profiles of the genes. In three-week-old seedlings, expression of OsDREB4-2 and OsDREB4-3 was induced by dehydration (air dry) and high salt (250mM NaCl), but not by cold (4 ℃) or ABA treatment (20uM), whereas OsDREB4-1, OsDREB4-4, and OsDREB1-1 were expressed constitutively. Under normal growth condition, OsDREB4-1 was expressed strongly in stem and spike, weakly expressed in leaf, and undetectable in root and sheath; OsDREB4-2 was expressed in stem and leaf, faintly expressed in root and spike, and undetectable in sheath; OsDREB4-3 transcripts were higher in root, stem, and spike, lower in leaf, and undetectable in sheath; OsDREB4-4 was expressed uniformly in all the tissues tested; OsDREB1-1 transcripts were abundant in leaf, sheath, spike, as well as stem, but low in root.Since DREB1 transcription factors have been determined to interact specifically with DRE and the DNA-binding specificity of DREB4 transcription factors has not been examined, OsDREB4-2 and OsDREB4-3 were selected as typical DREB4 transcription factors, and their DNA-binding specificity was examined by using yeast one-hybrid assay. The experimental results indicated that both OsDREB4-2 and OsDREB4-3 specifically bound to DRE and activated expression of the dual reporter genes of HIS3 and LacZ.Taken together, these results imply that OsDREB4-2 and OsDREB4-3 could function as trans-acting factors in DREB/DRE regulated stress-responsive pathway, activate transcription of the DRE/CRT containing genes, and improve stress tolerance of plants. We are now generating OsDREB4-2 and OsDREB4-3 transgenic Arabidopsis plants. The precise function of OsDREB4-2 and OsDREB4-3, as well as the target genes upregulated by them will be further investigated.