| Phosphorus (P) is required for plant growth and development. It is not only an important structural component of plant cell, also play an important role in regulation of cellular metabolism and the singnal pathways. Because of the low utilization of P, plants respond to phosphorus starvation through adaptive mechanisms involved in morphological, biochemical and molecular changes. The process of adaptation includs changing the morphology of the root system to achieve enhanced absorption of phosphorus. These changes are closely related to the specific regulation of gene expression under low phosphorus stress. Many genes involved in Pi-starvation responses have been cloned and their low Pi responses demonstrate the importance of transcriptional control in plants.In this study, a Ca2+binding protein, AtCBL1, was chosen for futher study the expression and fuction. The experimental results are shown as flollow:1、Expression analysis of AtCBLl geneAtCBLl gene was1,854bp (ORF642bp) encoding213amino acids. AtCBLl gene encoding the protein belongs to the CBL protein family. By real-time quantitative RT-PCR analysis of the gene expression patterns in low phosphorus stress, the results show that AtCBL1expression began to increase at3h of low phorphorous stress. At72h of low phorphorous stress, AtCBLl expression was still at a high level,suggesting that AtCBL1could be induced by low phosphorus stress.2、The assay of AtCBLl promoter activityApproximately1.2kb DNA fragment upstream of AtCBL1start codon was isolated from Arabidopsis genome and cloned into pBI101. The construction was introduced into Arabidopsis by the floral dip method. Histochemical staining of GUS activity revealed that AtCBL1promoter was weakly active in transgenic plants at normal level Pi conditions. At low Pi conditions, GUS activity was significantly induced in roots of transgenic plants.3、AtCBL1involved low Pi responseTo investigate the functions of AtCBLl in low Pi response, the transgenic Arabidopsis overexpressing AtCBLl and T-DNA insertion mutant cbll were obtained. Transgenic lines were selected by PCR identification. Phenotypic analysis of transgenic Arabidopsis indicated that AtCBL1overexpression promoted lateral root growth and development at either low or high level Pi conditions. And the seedlings of cbll mutant decreased number of lateral roots in normal Pi conditions and decreased sensation to low Pi stress showing longer primary root length in low Pi conditons.The expression of low Pi response genes in CBLl overexpressing plants and in cbll mutant had been analyzed. The results indicated that the genes coding members of Phtl family Pi transpoters, IPSl, RNS1were upregulated in CBL1overexpressing plants. And in cbll mutant, the expression of RNSl was downregulated. Whereas some other genes related to low Pi response, such as PHR1, PHO2, were not changed obviously.4、AtCBLl-AtCIPK17involved in Pi-starvation responseIn plants, CBLs interact with its target proteins, CIPKS, for signaling transduction. By yeast two-hybrid analysis, we found AtCBLl could interact with AtCIPK17in vitro. The transcriptional analysis also showed that AtCIPK17is involved in Pi starvation response. These data suggested that CBL1-CIPK17signaling is involved in Pi starvation response.5-. Identification of upstream regulators of AtCBLl geneBy bioinformatic analysis of AtCBL1promoter, some classic cis elements were found in, from which W-box, P1BS were related to Pi starvation response.Two W-box and three P1BS elements were found in AtCBLl promoter. To confirm the interaction between WRKY family transcription factors and the W-box elements, and the interaction between PHR1and the P1BS elements, EMSA and yease one-hybrid analysis were carried out. The results indicated that AtPHR1could interact with AtCBLl promoter showing PHR1could be the upstream regulator of AtCBLl gene. |
PDF Full Text Request