Molecular Characterization And Transcriptional Regulation Of Phosphate Transporter Gene TaPT2-1 In Wheat

Previously, a transcript-derived fragment (TDF) in leaves responding to the low-Pi stress was identified in our group based on cDNA-AFLP analysis. The TDF was used for identification of the molecular cheracterization and transcriptional regulation of the corresponding gene in this present study. The main results are as follows:1. BLAST search analysis suggests that the TDF, a transcript-derived fragment responding to the low-Pi stress, shares high similarities to the phosphate transporter (PT) genes identified in other plant species, including the chloroplast PT gene from Ricinus communis L. (GenBank accession number XM₀02527885) and the PT gene from Aarabidopsis thaliana (GenBank accession number AF515591). Based on search of the wheat full-length cDNA database, the full-length gene (clone number SET3_C22, and the GenBank accession number AK336079) coresponding to the TDF was identified. Owing to no report on this gene, it was referred to TaPT2-1.2. A specific primer pair was designed and the RT-PCR for amlification of the open reading frame (ORF) was successfully performed. It is found that the cDNA full-length of TaPT2-1 is 2075 bp, encoding a 568-aa polypeptide. Based on online analysis with a transmembrane prediction tool, TMprep, thirteen transmembrane domains were identified in TaPT2-1.3. Phylogenetic analysis displayed that TaPT2-1 shared much higher similarities to other four homologs from Arabidopsis thaliana, Solanum trberosum, Capsicum frutescens, and Solanum melongena. These results have implicated that all of them share a common ansestor along with the evolution process.4. Based on semi-quantitative RT-PCR analysis, the expression patterns of TaPT2-1 in leaves and roots under various Pi-supply condition were detected. It was ovserved that the transcripts of TaPT2-1 in leaves were significantly induced by the low-Pi stress. Whereas the expression of TaPHY2;1 in roots was shown to be constitutive, no obivious alteration detected with the changes of Pi-supply. Therefore, TaPT2-1 is possibly involved in the Pi recycling process in leaves under the Pi-stress condition.5. Using the genome walking approach, the promoter region of TaPT2-1 with a 1744 bp length was cloned from cv. Shixin828. The putative TaPT2-1 promoter contains the conserved boxes, TATA and CAAT, two playing important roles on gene transcriptional regulation. In addition, TaPT2-1 promoter also contains two regulatory elements, PIBS and Pho-like, being invoved in the responding to low-Pi stress signaling. Furthermore, some potential key regualtory elements, such as those involved in tissue-specific, defense response, auxin and salicylic acid responding, and signaling of light, sugar, and carbon, were also identified in TaPT2-1 promoter.6. The transgenic tobacco plants with the integrated TaPT2-1 promoter: GUS were generated. GUS histochemical staining analysis in the roots and leaves of the transgenic plants was performed. The results of GUS staining in roots and leaves under various Pi-supply conditions were in accordance with the TaPT2-1 transcripts detected based on RT-PCR analysis, showing a pattern to be induced expression in leaves. Also, the expression level of GUS was also induced by external treatment of salicylic acid.7. The expression level of reporter gene GUS under the control of TaPT2-1 promoter was much more induced in old leaves compared with those of novels. The distinct expression of low-Pi induced and leaf-predominantly expression of TaPT2-1 especially in old leaves suggest that this wheat phosphate transporter gene plays an critical role on cellelur transportation of Pi and improvement of Pi use efficiency in plants under low-Pi stress condition.