The Molecular Characterization Of Part Zinc Finger Protein Genes In Wheat (Triticum Aestivum L.) And Corresponding Function Analysis On Adaptations To Low-Pi Stress

As one of the indispensable macro-nutrients, phosphorus is acting as the components of nucleic acids, phospho-lipids, proteins, and part of enzymes in plants. Thus, phosphorus plays critical roles on regulation of diverse physiogical and biochemical processes, growth and development, and metabolic reactions in plants. Transcription factors, also refeered to trans-acting factors, are largely involved in reponding to the abiotic stresses such as deficiency of low-Pi and others. However, the transcriptional regulation mechanisms mediated by transcription factors have been largely elusive in wheat. In this study, thirty of Zinc-finger transcription factors belonging to WRKY type and five belonging to TFIIIA zinc finger type have been identified in wheat based on bioinformatic analysis. Of which, three genes responded largely to low-Pi stress were further transformed tobacco for identification of gene functions on adaptation to deficiency of Pi nutrition. The main results are as follows.1. Based on the conserved domains of WRKY and TFIIIA zinc-finger proteins in plants, the putative homologous sharing the conserved domians in wheat that released in GenBank database were identified. In total, thirty of transcription factors belonging to WRKY type and five belonging to TFIIIA zinc finger type have been obtained. RT-PCR analysis revealed 16 of the WRKY member and 5 of the TFIIIA member being specifically amplified.2. Among the WRKY members, most contain one of WRKY signature domain and one of zinc finger domain, besides WRKY5, WRKY11, and WRKY19 contain two of each domain. The cDNA length, open reading frame (ORF), and the translated polypeptides varied little among the tested WRKY members. According to the WRKY domain feature, the WRKY members could be classified into three groups referred to I, II, and III, with numbers of 3, 15, and 12, respectively. The five TFIIIA members all contain the conserved signature motif QALGGH, locating in the C2H2 zinc finger domain.3. Under low-Pi stress condition, the transcripts of TaWRKY12, TaWRKY20 and TaWRKY29 were gradually increased along with prolong of treatment. Among them, the expression of TaWRKY19 sustained high levels during the treatment, whereas the expression levels of TaWRKY20 and TaWRKY29 had obviously elevated expression levels after treatment of 3 h or 13 h. Along with the low-Pi stress progression, the expression of TaWRKY8 and TaWRKY15 droped largely and barely detected. The expression of TaWRKY3, TaWRKY4 and TaWRKY21 had similar levels at 3 h of treatment with the control, but decreased after that time point. TaWRKY4, TaWRKY17, and TaWRKY27 were down-regulated by low-Pi stress and sustained stable expression levels during the treatment period. The expression of TaWRKY25 was not changed in various Pi-supply conditions and shown to be constitutive.4. Under the various treatments of deficiencies of Pi, nitrogen, potassium, zinc, abscisic acid (ABA), drought (PEG), salinity (NaCl), and low temperature (4℃), all the sixteen members of the tested WRKY genes were shown to be regulated by several or all of the treatments. The five TFIIIA members were all up-regualted by low-Pi stress signaling. Of which, the member TaZFP2 was also shown to be up-regulated by low-N, and stresses of drought, and salinity. Two types of the expression pattern could be defined including up- and down- regulated in the gene responses. The responding degree to the treatments varied from intense to no obvious. Taken together, the members of WRKY and ZFP are widely involved in mediation of the abiotic stress signaling. The multi-responses of one member to various treatments suggest there are intricate transcriptional regulation networks to respond to distinct external signaling in which the members of WRKY and ZFP are involved.5. Using DNA recombinant technology and gene genetic transformation via Agrobacterium-tumefaciens mediated approach, the binary expression plasmids fused the open reading frame (ORF) of TaWRKY12, TaWRKY18 and TaZFP2 were constructed. Furthermore, the transgenic tobacco plant lines overexpressing above genes were generated. After low-Pi stress treatment, the transgenic plants with strong overexpression of TaWRKY12, TaWRKY18 and TaZFP2 showed much better phenotype fetures than the control plants, displaying much higher in the plant height, longer of the distance between joints, larger of leaf area. In the meantime, the transgenic plants had higher concentrations of Pi and total phosphorus, higher contents of soluble proteins and higher activities of acid phosphatases, and higher contents of chlorophyll a, chlorophyll b, and carotenoids than the control plants.6. The results in this study have implicated that TaWRKY12, TaWRKY18 and TaZFP2 are possibly acting as the critical components in the signal transduction pathways initiated by low-Pi stress. Up-regualted expression of aboves genes could induce the expression levels of part genes related to Pi acquisition and celular transportation, by which to improve the abilities of Pi uptake and Pi use efficiency in plants under low-Pi stress condition.