The Molecular Characterization And Biological Function Of Wheat Transcription Factor Gene TabHLH1

Classified into a superfamily with the conserved structural domain of basic-Helix-Loop-Helix, bHLH transcription factor members widely exist in plants and animals. In plant species, acting as the second anti-action transcription factors, bHLH transcription factor members play a critical role on regulation of plant growth and development, and on responses and tolerances of diverse abiotic stresses such as the deprivations of inorganic nutrients and stresses of drought, salinity, and low temperature. In this present study, a wheat bHLH transcription factor designated as TabHLH1 was identidied based on bioinformatic approach and was subjected to further analysis. We have systematically analyzed the molecular characterization and the expression properties of TabHLH1. Furthermore, transgenic tobacco plants with overexpression of TabHLH1 were generated via Agrobacterium-tumafeciens mediated method. The effects of TabHLH1 on regulation of plant responses and tolerances of diverse stresses such as low-Pi, low-N, drought, and salinity were investigated using the transgenic plants as the experimental materials. The main results are as follows.1. A transcription-derived fragment (TDF) responded to low-Pi stress was obtained by cDNA-AFLP technique. Sequencing result suggested that this TDF was functional classified into bHLH type transcription factors. The full-length cDNA corresponding to this TDF was identified using bioinformatic method and designated as TabHLH1. Based on the cDNA sequence, the open reading frame (ORF) of TabHLH1 was specifically amplified by RT-PCR using the cDNA to be transcribed from the transcripts derived from low-Pi treated wheat roots as the template.2. The full-length cDNA of TabHLH1 is 2208 bp, containing an open reading frame of 1443 bp and encoding a polypeptide of 480-aa. The translated protein by TabHLH1 has a molecular weight of 51.2 kD and an isoelectric point (pI) of 4.72. TabHLH1 harbors the conserved stuctural domain referred to bHLH (basic-Helix-Loop-Helix) that are generally identified in bHLH type transcription factors. In TabHLH1, the domain bHLH is located successively at zones of aa 326 to aa 338 (basic region), aa 339 to aa 353 (helix), aa 354 to aa 359 (loop), and aa 360 to aa 381 (helix). Therefore, TabHLH1 possesses the typical structural characterization of bHLH transcription factors.3. Based on homologous searches in GenBank, part of homologous counterparts sharing high similarities to TabHLH1 were identified and subjected to further phylogenetic analysis. The results implicate that TabHLH1 is homologous with OsPTF1, as well as those partners derived from Z. mays, T. aestivum, P. Edulis, S. bicolor, B. Distachyon and H. vulgare. Thus, TabHLH1 and its homologous conterparts are possibly generated from a close progenitor.4. using wheat cultivar Shinxin 828 as the materials, the expression patterns of TabHLH1 were detected. It was found that TabHLH1 responded to nutrient deprivations of phosphorus and nitrogen as well as abiotic stresses of drought and salinity, exhibiting up-regulated expression under conditions of above stresses. No respondings of TabHLH1 to deprivations of potassium and calcium, abiotic stress of low temperature, and exgenous abscisic acid (ABA) treatment were observed. These results suggest that TabHLH1 plays an important role on withstanding above diverse stresses.5. Using DNA recombinant technique, the binary expression plasmid fused the open reading frame (ORF) of TabHLH1 was constructed. The transgenic tobacco plants that integrated the TabHLH1 ORF were generated via Agrobacterium-tumafeciens mediated method. The transgenic tobacco plants with overexpression of TabHLH1 and the control plants with integration of empty binary vector were used for analysis of the ectopic gene function on tolerances of diverse stresses including deprivations of phosphorus and nitrogen, as well as abiotic stresses of drought and salinity. The results indicated that the transgenic plants behaved better growth behavior than the control plants.6. Under conditions of low-Pi stress, drought, and salt stress, the plant dry weights and related part of physiological parameters were assessed. Compared with the control plants, that TabHLH1 genetic transformed tobacco plants showed improved plant dry weights after above stress treatments. In addition, the transgenic plants had higher total P contents and more accumulative P amounts than the control plants under condition of Pi-deprivation. Under conditions of drought and salt stress, the transgenic plants behaved higher contents of leaf proline and soluble sugar than the control plants under conditions of drought and salinity. Taken together, ectopically expression of TabHLH1 was clearly confirmed to enhance significantly the plant capabilities on stresses of low-Pi, low-N, drought, and salinity. It is suggested that this wheat bHLH type transcription factor TabHLH1 has a potential value on evaluation, screening, and identification of wheat germplasm resources on tolerances of stresses of low-Pi, low-N, drought, and salinity. TabHLH1 is also useful for generation of plant novel germplasms with strong capabilities on tolerances of above diverse stresses in the future.