转录因子BnWRKY47s调控油菜硼胁迫反应的分子机制

When plants suffer from boron stresses, the homeostasis of uptake and transport of boron in plants is regulated by genes. However, the regulation mechanism that these NIPs and BORs genes respond to boron stresses is unknown. We found two BnWRKY47s genes (BnaA09WRKY47 and BnaCnnWRKY47) in Brassica napus were up-regulated under under boron deficiency condition, which was also induced in boron deficiency confirmed by RT-qPCR analysis and the complementary experiments that overexpressing AtWRKY47 in mutant wrky. At the same time, we also found that the promoters of NIPs and BORs genes contain the W-box combined specially by WRKY. Based on these results, we put forward the hypothesis of the molecular mechanism that BnWRKY47s regulate the responses to Brassica napus to boron stresses, and design the research contents and its route map for the project. Firstly, we will create transgenic lines with overexpression or knockdown of BnWRKY47s, and then study the expression and function of BnWRKY47s in the resistance to boron stresses. Secondly, with yeast one/two-hybrid, EMSA, CHIP and other techniques, the interaction of BnWRKY47s with NIPs, BORs, and the genes identified from yeast two-hybrid experiments will be investigated in both in vitro and in vivo. The aim of the project is to unveil the molecular regulation mechanism or pathway of BnWRKY47s resisting boron stresses in Brassica napus.

植物遭受硼胁迫时硼吸收转运和分配的自稳态平衡由NIPs和BORs基因调控，然而这些基因响应硼胁迫的分子调控机制尚未有报道。我们研究发现2个甘蓝型油菜BnWRKY47s基因（BnaA09WRKY47和BnaCnnWRKY47）在缺硼胁迫的基因表达谱中均显著上升表达，并在RT-qPCR验证和转基因互补试验中发现WRKY47同时响应缺硼和硼毒胁迫，还发现NIPs和BORs启动子含有WRKY专一识别的W-box元件。在此基础上，本项目研究提出BnWRKY47s调控抗硼胁迫分子机制的假设，制定合理的研究内容和技术方案：在油菜中超表达或敲除BnWRKY47s基因，获得转基因纯合株系，研究其在油菜抗硼胁迫中的表达和生物学功能；同时，利用酵母单/双杂、EMSA、CHIP等技术研究BnWRKY47s与NIPs、BORs等基因互作，细胞亚细胞定位等，揭示BnWRKY47s介导油菜响应硼胁迫的分子调控机制途径。

植物遭受硼胁迫时硼吸收转运和分配的自稳态平衡由NIPs和BORs基因调控，然而这些基因响应硼胁迫的分子调控机制尚未有报道。我们研究发现2个甘蓝型油菜BnWRKY47s 基因（BnaA09WRKY47和BnaCnnWRKY47）在缺硼胁迫的基因表达谱中均显著上升表达，并发现NIPs和BORs启动子含有WRKY专一识别的W-box元件。在此基础上，本项目研究首先在油菜中超表达或敲除BnWRKY47s基因；在获得转基因纯合株系的基础上，研究其在油菜抗硼胁迫中的表达和生物学功能；同时，利用酵母单、EMSA等技术研究BnWRKY47s与NIPs、BORs等基因互作，细胞亚细胞定位等。其目的在于揭示BnWRKY47s介导油菜响应硼胁迫的分子调控机制途径。在研究中，发现BnaA9.WRKY47、BnaA1.WRKY53和 BnaCn.WRKY57基因响应缺硼胁迫，在根和地上部受缺硼诱导显著上升表达，而AtWRKY47基因响应硼过量胁迫，在根和地上部受硼过量毒害诱导显著下调表达，亚细胞定位显示它们位于细胞核；BnaA9.WRKY47 敲除的转基因株系对缺硼敏感，而超表达株系对缺硼表现抗性，进一步利用酵母单杂、EMSA、原位杂交和烟草瞬时表达等技术证明BnaA9.WRKY47与BnaA3.NIP5;1互作，正向调控BnaA3.NIP5;1基因的表达，促进硼的吸收累积而表现抗缺硼胁迫的生物学功能。相反，AtWRKY47 的超表达材料对硼毒敏感，而突变体wrky47表现对硼毒的抗性，wrky47-1 突变体中地上部的硼浓度显著低于野生型，表明AtWRKY47 在高硼胁迫反应中发挥了负调控的生物学作用。另外，我们研究发现，BnaA9.WRKY47不仅响应缺硼胁迫，也参与油菜缺氮反应，调控体内氮的自稳态平衡。综上所述，本研究揭示BnaA9.WRKY47 受缺硼诱导上调，进而激活硼高效基因BnaA3.NIP5;1的表达而表现抗缺硼的正向调控机制，其途径为：“-B → BnaA9.WRKY47→ BnaA3.NIP5;1 →B uptake → B-deficient tolerance”；而AtWRKY47抵抗过量硼的毒害是一条负向的调控机制，其调控途径涉及“++B—┤AtWRKY47 —┤ BOR1&BOR2 →B transport → excess-B tolerance”。