Characteristic Analysis And Functional Determination Of ERF Transcription Factor Gene, TaEREB1/2 And TaERFL1/2 From Wheat (Triticum Aestivum L.)

AP2/EREBP is a family of plant-specific transcription factors containing the highly conserved AP2/EREBP DNA binding domain, which consists of about 60 to 70 amino acids, forming threeβ-sheets and anα-helix. The AP2/EREBP family regulates diverse process, including cell proliferation, plant development, secondary metabolism, biotic and abiotic stress responses, and responds to different phytohormones through directly interacting with GC-rich cis-element (eg.GC box, DRE element) in the promoter of their target genes. Previous studies have shown that the ERF subfamily has the 14th alanine and the 19th aspartic acid in theβ-sheet which are indispensable for binding to GCC box and DRE element, involved in abiotic and biotic stress signal transduction. The study is to investigate the new ERF genes, TaEREB1, TaERFL1, TaEREB2 and TaERFL2, which were successively cloned from Triticum aestivum.(1) Binding assay in vitro: The ERF/AP2 domains of TaEREB1, TaERFL1, TaEREB2 and TaERFL2 were fused to N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5 mM) was added to induce the expression of recombinators for 3-5 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled ERE or DRE sequence. The results of electrophoretic mobility shift assay (EMSA) indicated that TaEREB1, TaERFL1, TaEREB2 and TaERFL2 specifically bound to DRE (dehydration- responsive element) and ERE (ethylene-responsive element or GCC-box element) in vitro, which meant the four ERF genes may be involved not only in biotic stresses, but also in abiotic stress-induced signaling pathway. (2) Subcellular localization assay: In order to detect the subcellular localization of ERF transcription factor proteins, TaEREB1, TaERFL1, TaEREB2 and TaERFL2 were fused to the 5'terminus of the coding region of green fluorescence protein (GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Then the fused plasmids were introduced into onion epidermal cells using a particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22℃in the dark and localization of the fusion proteins was then detected using a confocal microscope equipped with appropriate filter. The subcellular localization assay indicated that TaEREB1, TaEREB2, TaERFL1 and TaERFL2 can localize into the nuclei.(3) Analysis of expression pattern: To investigate the expression pattern of TaEREB1/2 and TaERFL1/2 responsive to different stresses, total RNA were extracted and purified from T. aestivum which subjected to various stress treatments (eg. ABA, SA, MeJA, low temperature and high salinity) for different time. The results of RT-PCR analysis indicated that TaEREB1/2 and TaERFL1/2 could be induced by ABA, SA, MeJA, low temperature and high salinity, but the expression mode was different. TaEREB1/2 and TaERFL1/2 may play different role in different signaling pathway.(4) Achievement of transgenic Arabidopsis plants: The TaEREB1/2 and TaERFL1/2 were introduced into Arabidopsis through agrobacterium-mediated transformation, which is important to further investigate the gene functions in future.The present study provides theoretical basis for us to further study the molecular mechanism of the four ERF transcription factors in regulating the stress signal networks in plant, and good candidate gene resources for improving crop tolerance to adverse environmental conditions using gene engineering.