Functional Characterization Of High-affinity Pi Transporter Gene BnPpht1;4in Brassica Napus

Phosphorus (P) is an essential macronutrient for plant growth and development. It is a component of many important organic compounds in plants and participates in a variety of metabolic processes, such as carbohydrate metabolism, nitrogen metabolism, fat metabolism, etc. P is acquired by plants in the form of inorganic phosphate (Pi). The soluble Pi in soil is very low for plant absorption. The study of plant P absorption and utilization will uncover the molecular mechanism of Pi absorption and transport in higher plant. Cloning high affinity phosphorus transport protein gene and researching its function have important theoretical significance and potential of agricultural utilization value.In this study, from Pi starvation response genes in B. napus, one clone (cDNA) encoding high-affinity Pi transporters was identified and designated as BnPhtl;4,and study functions of the gene in low Pi conditions, the results for example:1. Isolation and characterization of BnPhtl;4geneBy designing a pair of specific primers, from the cDNA library of B. napus.one clone ORF is1605bp,and encoding a protein consist of534amino acids. Sequence analysis predicted that this gene encodes a protein shares95%identity with high-affinity Pi transporters AtPht1;4,so we name it BnPhl1,4.2. The subcellular localization of BnPhtl;4proteinTo determine the subcellular localization of BnPhtl;4protein, BnPht1;4-GFP fusion construct was generated and introduced into inner epidermal cells of onions by a helium biolistic device. Fluorescence of BnPhtl;4fused to GFP was confined to the periphery of cells, indicating that the BnPhtl;4protein was localized in the plasma membrane, the topology analysis of BnPhtl;4indicated that it as a integral membrane protein, consist of12membrane-spanning regions, separated into two groups by a large hydrophilic charged region between the sixth and the seventh transmembrane helices.3. The expression pattern analysis of BnPhtl;4To investigate the expression pattern of BnPht1;4in B. napus under normal and low Pi conditions, the total RNAs were isolated from B. napus roots, hypocotyls, cotyledons, stems, leaves, and flowers, and BnPhl1;4transcript was detected by qRT-PCR analysis. The results showed that BnPht1;4was induced under low Pi conditions. Under high level Pi conditions, BnPhl1;4transcript was high in stems and flowers. BnPht1;4transcript was significantly increased in B. napus roots under low Pi conditions, and was increased obviously in plant roots after growing in low Pi media for12h. 4. Isolated BnPhtl;4promoter and analysed activity under Pi deficiencyBy Genome Walker PCR, a1276bp fragment of BnPhl1;4gene was isolated from B. napus genome. By PlantCARE program, in BnPht1;4promoter, which included the WRKY type transcription factor binding element W-box, PHR1(MYB-CC type transcription factor) binding element P1BS, plant hormones responsive elements GARE, TGA,TCA and ABRE, and abiotic stresses responsive elements MBS and LTR. We introduced a transcriptional fusion between the BnPht1;4promoter and GUS gene into Arabidopsis by transformation. Histochemical staining of GUS activity revealed that the activity of BnPht1;4promoter was significantly detected in roots of transgenic Arabidopsis in low level of Pi when sugar in certain of concentration. This result indate that was induced by low Pi and dependent on exist of sugar.5. BnPhtl;4overexpression affects Arabidopsis root architectureBnPht1;4controlled by35S promoter was introduced into Arabidopsis, and analysed phenotype after selected transgenic lines by reverse-transcription PCR. On MS medium with high level (1mM) of Pi, the growth of35S:BnPht1;4seedlings was similar to wild type plant and no obvious difference was found in plant size, fresh weight, primary root length and lateral root number between wild type and transgenic plants. On MS medium with low Pi (50μM Pi), the fresh weight of transgenic plants was higher than that of wild type plants, and the primary-root length of35S:BnPht1;4transgenic plants was obviously longer than that of wild type plants. Although lateral root density on35S:BnPht1;4transgenic plants is less than that on wild type plants, the lateral root number on35S:BnPht1;4transgenic plants is more than that on wild type plants. On the other hand, Pi content in roots of35S:BnPht1;4Arabidopsis was also higher than that of in wild type.6. Transcriptional regulation of BnPhtl;4by PHR1in low Pi responseBecause there are two P1BS (GNATATNC) elements were found in BnPht1;4promoter sequence, which PHR1as a transcription factor could bind. To investigate the necessity of P1BS elements for BnPht1;4induction in low Pi conditions, the P1BS elements in BnPht1;4promoter were respectively deleted by primer-based mutagenesis resulting in BnPht1;4PMJ:GUS, BnPht1;4PM2:GUS and BnPht1;4PMM:GUS mutation constructs. The constructs were introduced into Arabidopsis by transformation. Histochemical staining of GUS activity revealed that the induction of BnPhl1;4PMl, BnPht1;4PM2by low Pi was decreased in root of Arabidopsis compared to that of BnPhl1;4P. However, the GUS activity was not absolutely impaired in BnPht1:4PMM:GUS transgenic plants, which suggested that BnPhl1;4could be regulated by other regulators than PHR1in low Pi responses. 7. Yeast one-hybrid and EMSA indacted that WRKY75can interacte with BnPht1;4promoterIn Arabidopsis, a member of WRKY family transcription factor, WRKY75also is the key regulator in low Pi response, which was functionally redundant with PHRl in regulation of the low Pi response genes (Rubio et al.,2001; Devaiah et al.,2007). In BnPhtl;4promoter, two W-box motifs, the WRKY family transcription factor binding elements, were found. Through building a carrier and synthetic probe, by yeast one-hybrid and EMSA indacted that WRKY75can interacte with BnPht1;4promoter.