茉莉酸（JA）调控油菜低磷应答的分子机制研究

Phosphorus is one of the essential nutrient elements for plant development. Our previous work indicated that JA is involved and could be a positive regulator in plant low-Pi response. In high-Pi conditions, JA could induce the activity of BnPht1;4 promoter in transgenic Arabidopsis. However, in wrky75 mutant the induction of BnPht1;4 promoter activity by JA was decreased obviously. So the regulation of low-Pi response genes by JA could depend on low-Pi regulators such as WRKY75. On the other hand, in low-Pi response, WRKY75 was modified by phosphorylation with western blot analysis. Based on these data, JA could be the signal for activating the regulators such as WRKY75 by phosphorylation in low-Pi response. The phosphorylated regulators could activate the expression of low-Pi response genes such as Pht1;4. In this project, B. napus will be treated with JA and JA inhibitor in low- and high-Pi conditions for investigating the effects of JA signal on the growth and development of B.napus. The BnPht1;4P:GUS and BnIPS1P:GUS transgenic plants will be treated with JA and JA inhibitor in low- and high-Pi conditions for investigating the effects of JA signal on the promoter activity of low-Pi response genes. With BnWRKY75 overexpressing transgenic plants, phosphorylation of BnWRKY75 in low-Pi response and JA treatment will be confirmed by Western blot and by LC-MS/MS analysis. By stable isotope metabolic labeling-based quantitative phosphoproteomics, the other phospho-regulators in low-Pi response and JA treatment will be identified. The specific phospho-antibody will prepared and the phospho-regulators in low-Pi response will be proved by Western blot analysis. The phospho-site mutation of the regulators will be carried out and mutated transgenic plants will obtained. The phenotype of transgenic plants in low-Pi conditions will be investigated. In low-Pi conditions, the expression of low-Pi response genes in transgenic plants will be analyzed. Based on the above data, the molecular mechanism of JA involved in low-Pi response in B. napus will be uncovered which will be useful in genetic modification of B. napus for improving Pi absorption and usage in the future.

磷是植物生长所需的必需元素之一。研究并揭示植物（作物）低磷适应分子调控机制有着重要理论和实践意义。本课题前期研究表明JA参与植物低磷应答并可能在其中扮演正调控角色。低磷应答中JA信号可能通过磷酸化修饰激活WRKY75等转录调控因子进而调控下游应答基因。本课题拟通过油菜生长发育表型分析及低磷应答基因表达分析说明JA作为正调控信号参与油菜低磷应答。BnWRKY75磷酸化修饰分析说明JA对低磷应答基因调控是通过BnWRKY75等转录调控因子磷酸化修饰来实现的。通过14N和15N代谢标记定量磷酸化蛋白质组学方法分析鉴定低磷及JA处理条件下其它的磷酸化转录调控因子。利用磷酸化位点特异性抗体验证磷酸化转录调控因子。BnWRKY75等转录调控因子磷酸化位点突变后分析转基因油菜表型变化及低磷应答基因表达变化。最终阐明JA信号在低磷应答中的分子调控机制，为油菜磷素高效利用遗传改良提供科学数据。

磷(Phosphorus，P)是植物生长所需的必需元素之一，在植物中具有多种功能。植物所利用的磷主要吸收形式是无机磷酸盐(phosphate, Pi)。磷营养缺乏是作物生长发育的限制因素，也是农业面对的主要问题之一。磷在植物中扮演功能的复杂性，使研究植物磷素吸收，运输及低磷应答分子调控等成为一项艰巨的工作。油菜是我国重要的油料作物，其在生长发育中需要较多的磷，油菜油脂合成和脂肪代谢同样与磷有关。实践证明油料作物生长发育需要较多的磷。研究并揭示油菜低磷适应分子调控机制，对于油菜磷肥的合理施用，以及磷营养性状的遗传改良，促进油菜可持续发展及降低环境污染都有着重要意义。本课题研究发现低磷应答中JA在油菜根和地上部分均扮演重要调控角色。不同浓度JA及JA信号抑制剂处理后发现JA信号在低磷胁迫应答中影响油菜根的发育及地上部分花青素的积累。RT-PCR分析结果表明JA及JA信号抑制剂处理后低磷应答基因表达变化。这些结果显示JA是油菜低磷胁迫应答正调控信号。低磷应答中JA信号可以通过磷酸化修饰WRKY75、PHR1转录调控因子进而调控下游应答基因。JA及低磷处理BnWRKY75过量表达转基因油菜后通过抗体及质谱分析鉴定出BnWRKY75蛋白磷酸化位点。低磷条件下JA对低磷应答基因调控可以通过BnWRKY75转录调控因子特定氨基酸磷酸化修饰来实现的。MYB-CC家族转录因子PHR1是植物低磷应答最重要调控因子。JA对低磷应答基因调控也可以通过BnPHR1磷酸化修饰来实现的。通过BnWRKY75和BnPHR1磷酸化位点突变转基因油菜株系，进一步分析了转基因油菜中低磷胁迫应答基因表达变化。结果显示BnWRKY75与BnPHR1磷酸化修饰模式存在显著不同。这些研究数据为最终阐明JA信号在低磷应答中的分子调控机制，为油菜磷素高效利用遗传改良提供科学数据。