UGT73C6与ZFP36互作在低磷诱导丛枝菌根形成中的作用机制

Arbuscular mycorrhizal fungi play a key role in stabilizing ecosystem. One of the most important function of AM is the source of soil available phosphate (Pi). High Pi inhibits AM formation while low Pi increases AM formation, in which the phytohormone abscisic acid (ABA) plays a vital role, however, the underlying mechanism remains unclear. CCaMK, a Ca2+/calmodulin dependent protein kinase, has been identified as a key role in the symbiosis of arbuscular mycorrhizal fungi with plants root. However, the study of its upstream factors remains almostly unknown. Our group found that a transcription factor zinc finger protein ZFP36 could directly target to CCaMK by yeast one hybrid, chromatin immunoprecipitation combined with high-throughtput sequencing (ChIP-seq), immunoprecipitation gel kinase activity assay. According to the mycorrhizal incubation assay and gene expression analysis, we confirmed ZFP36 participates in AM symbiosis formation under low Pi and confers a positive role in Pi absorption. Based on these, this project is intended to further clarify the molecular mechanism by which ZFP36 regulates CCaMK via Electrophoretic Mobility Shift Assay (EMSA), protoplast transient transformation method, and transient luciferase assay in tobacco; And to further verify the interaction between ZFP36 and UDP-glycosyltransferase UGT73C6 by yeast two-hybrid assay, bimolecular fluorescence complement (BiFC), co-immunoprecipitation (Co-IP), glutathione pull-down (GST pull-down). Additionally, by combining mycorrhizal infect rate analysis and non-invasive microelectrode ion flux measurements (MIFE) in mutants, we would elucidate the function of the interactor in mycorrhizal symbiont formation and mycorrhiza-regulated phosphorus absorption in rice. All above, this research will not only help to understand how low Pi increase AM formation, but also have an important significance for the enhancement of the crop stress tolerance and plants nutrient absorption by means of molecular biology combined with mycorrhizal technology.

丛枝菌根（AM）是维持生态系统稳定的重要因子，其显著功能是活化土壤有效磷。土壤有效磷低时有利于AM的形成而高磷抑制，脱落酸ABA在其中发挥重要作用。钙/钙调素依赖型蛋白激酶CCaMK是植物形成AM的关键蛋白，也是ABA信号系统重要组分，其上游调控因子的研究较少报道。本研究前期通过酵母单杂交、染色质免疫共沉淀结合高通量测序（ChIP-seq）、免疫沉淀凝胶激酶活性分析等技术，发现ZFP36直接靶定CCaMK启动子区。基于此，本项目拟采用EMSA、原生质体体系等技术进一步澄清ZFP36调控CCaMK分子机理；通过酵母双杂交、BiFC、Co-IP、GST pull-down验证ZFP36与UGT73C6互作，并利用突变体功能研究，阐明该互作体在调控丛枝菌根形成方面的功能。综上，本研究不仅有助于人们了解低磷如何促进AM形成，而且对利用分子生物学手段结合AM菌剂技术提高菌根生态效益具有重要指导意义。

丛枝菌根（Arbuscular mycorrhiza, AM）作为绿色生物有机肥，具有广阔的应用前景，尤其在低磷环境有利于菌根共生体系的形成。前期研究表明，低磷促进植物激素脱落酸（abscisic acid, ABA）的积累，ABA会活化AM形成的关键蛋白CCaMK，从而促进AM形成。然而，AM形成过程中，低磷如何调控CCaMK来决定菌根形成？CCaMK上游调控信号还不是很清楚。本项目主要研究内容包括三点：1. ABA及不同浓度磷酸盐处理下，ZFP36/UGT73C6基因表达；2. ABA信号系统中，UGT73C6与ZFP36互作验证及UGT73C6与ZFP36糖基化修饰；3. ZFP36调控CCaMK活性对菌根介导的磷素营养的贡献。得出结果如下：1. 不同浓度磷酸盐处理下，表现出低浓度磷酸盐LP促进ABA积累，高浓度磷酸盐HP抑制ABA积累；ABA/不同浓度磷酸盐影响UGT73C6基因表达；2. 低浓度ABA促进丛枝菌根的形成并增加侵染率、高浓度ABA抑制菌根形成并降低丛枝菌根侵染率；3. 低磷促进、高磷抑制菌根侵染依赖ZFP36；4. UGT73C6与ZFP36在体内外互作；5. ZFP36通过直接靶标CCaMK启动子区而影响菌根形成，并影响菌根介导的磷素营养。综上，UGT73C6通过直接相互作用于ZFP36而影响ZFP36结合CCaMK，从而影响菌根形成。此外，在本项目资助下，课题组还揭示了一种RNA结合蛋白OsDIP1在ABA介导的抗氧化防护中的作用；阐明了DNAJ蛋白作为分子伴侣稳定ZFP36来调控ABA信号；揭示了ABA信号系统中，ZFP36互作系统。本项目研究结果，将有利于阐明菌根形成的关键信号通路，并为菌根途径磷素营养提供参考。