BnPHR1介导的油菜磷饥饿应答分子调控机制研究

Phosphorus is one of the essential nutrient elements for plant development. In our previous work, from the Pi starvation induced genes of Brassica napus, one clone was identified as homologous gene of AtPHR1 and consequently designated as BnPHR1, which encodes a novel MYB transcription activator in B. napus. Available evidences suggest that auxin plays an important role in mediating the Pi starvation effects on root system architecture of plants. Although no data directly approve the crosstalk between PHR1 and auxin signaling in Pi starvation responses so far, it has been reported that AtSIZ1, the upstream regulator of AtPHR1, is involved in auxin signaling. Potentially, there is a conjunction between PHR1 regulation and auxin signaling in Pi starvation response. Our data showed that BnPHR1 was regulated by auxin in roots. The BnPHR1 promoter activity was significantly decreased in roots of Arabidopsis arf19 mutant. The data suggested that the AUXIN RESPONSE FACTOR may be the switch of BnPHR1 on transcriptional level. In this project, the expression of BnPHR1 will be analyzed in roots of B. napus treated with exogenous auxin and auxin transport inhibitor. The activity of BnPHR1 promoter and mutated BnPHR1 promoter in transgenic B. napus will be analyzed. The BnPHR1 promoter and mutated BnPHR1 promoter will be introduced into arf7, arf19, arf7arf19 mutants and the promoter activity will be assayed. The interaction between ARF19 and BnPHR1 promoter will be investigated by protein-DNA interacting assay in vitro and in vivo to showing the molecular mechanism of transcriptional regulation of BnPHR1 by AUXIN RESPONSE FACTOR. Our previous data also showed that the overexpression of BnPHR1 in both Arabidopsis and B. napus remarkably enhanced the expression of the Pi starvation induced genes. Additionally, our data suggested that BnPHR1, as crucial regulator in Pi starvation, directly activates those genes encoding high affinity Pi transporters which promote the uptake of Pi for plant growth. In this project, to investigate the functions of BnPHR1 in Pi starvation response, the phenotype of BnPHR1 over-expressing and RNAi transgenic B. napus in different level of Pi conditions was assayed.To investigate the regulating mechanism of BnPT2 by BnPHR1 in Pi starvation response, the expression of BnPT2 in BnPHR1 over-expressing and RNAi transgenic B. napus was analyzed. The interaction between BnPHR1 and BnPT2 promoter in vivo was investigated by ChIP assay. The activity of BnPT2 promoter and mutated BnPT2 promoter in transgenic B. napus will be analyzed. On the other hand, the expression of the Pi-starvation induced genes, which had been identified in B. napus, will be analyzed in BnPHR1 over-expressing and RNAi transgenic B. napus, by which more genes regulated by BnPHR1 in Pi starvation will be identified.Based on the above data, the molecular mechanism of Pi starvation response modulated by BnPHR1 will be uncovered.

本课题组鉴定出BnPHR1基因，前期研究表明其在转录水平受到生长素信号调控并在磷饥饿应答中具有重要功能。本项目拟通过外源生长素和生长素极性运输抑制剂处理油菜分析BnPHR1表达，在油菜中分析生长素应答元件突变后的BnPHR1启动子活性变化，在拟南芥arf7，arf19，arf7arf19中分析BnPHR1启动子活性变化，通过蛋白质-DNA结合实验分析生长素应答因子与BnPHR1启动子的相互作用等揭示生长素信号对BnPHR1调控的分子机制；通过BnPHR1过量表达及RNAi转基因油菜表型分析揭示BnPHR1在磷饥饿应答中的功能；通过BnPHR1转基因油菜中BnPT2表达、BnPHR1与BnPT2启动子互作分析揭示BnPHR1对BnPT2调控的分子机制。通过上述研究阐明BnPHR1介导的油菜磷饥饿应答分子调控机制，获得磷素利用效率提高的转基因油菜株系，为油菜磷素高效利用遗传改良提供科学数据。

本课题研究鉴定并克隆了BnPHR1基因，研究分析发现BnPHR1基因在转录水平受到生长素信号调控并在磷饥饿应答中发挥重要作用。通过外源生长素和生长素极性运输抑制剂处理油菜分析发现BnPHR1表达与生长素呈正相关性。生长素和生长素运输抑制剂处理也显示BnPHR1活性与生长素信号正相关。在拟南芥arf7，arf19单突变体，arf7arf19双突变体中BnPHR1启动子活性显著降低，通过酵母单杂交，EMSA和ChIP实验分析证实ARF7，ARF19能与PHR1启动子相互作用。BnPHR1过量表达转基因拟南芥、phr1突变体、BnPHR1过量表达转基因油菜及RNAi转基因油菜表型分析表明BnPHR1在植物磷饥饿应答中发挥重要功能。鉴定并克隆了BnPHR1下游靶基因BnPT2，进一步分析了BnPT2表达特性、通过酵母单杂交，EMSA实验表明BnPHR1能与BnPT2启动子互作。BnPHR1和BnPT2的过量表达都能提高植物从外部环境获取磷素的能力。上述研究阐明Auxin-PHR1-PT2等转运蛋白油菜磷饥饿应答分子调控路径，获得磷素利用效率提高的转基因油菜株系，为油菜磷素高效利用遗传改良提供科学数据。