ANAC032调控拟南芥应答镁毒害的分子机制研究

世界分布较广的蛇纹石土壤中存在过量的镁，抑制植物生长发育。ABA信号途径参与调控植物对镁毒害响应，但其分子机制尚不清楚。前期我们鉴定出一个ABI5互作蛋白ANAC032。与野生型相比，abi5和anac032突变体对镁毒害敏感性提高，而过表达ANAC032或ABI5拟南芥植株表现出相反的表型。ANAC032表达受镁毒害诱导。在此基础上，本项目拟通过生理生化和代谢组分析等手段，分析ANAC032或ABI5过表达和突变体植株在镁毒害条件下，根和叶中矿质元素及代谢物含量变化；采用转录组学和ChIP-seq方法鉴定ANAC032参与调控矿质元素累积、根系发育和镁毒害响应的下游靶基因，并通过ChIP-qPCR、Y1H、EMSA和瞬时表达等实验进一步确认；同时采用遗传学和分子生物学方法，分析ANAC032与ABI5互作在调控响应镁毒害中的生物学功能。本研究使我们更深入理解植物应答镁毒害的分子调控网络。

In serpentine soils, which are widely distributed in the world, there are serious magnesium (Mg) toxicity, which inhibits plant growth and development. ABA signaling pathway is involved in the regulation of plant response to Mg toxicity. However, the molecular mechanisms underlying ABA-mediated Mg toxicity response remain largely unclear. We identified a ABI5-interacting protein SNAC-A type transcriptional factor ANAC032 using yeast two-hybrid (Y2H) assay. Our previous study showed that overexpression of ANAC032 (OX32) or ABI5 (ABI5OX) improves Mg toxicity tolerance in Arabidopsis compared with Col-0 plants; while loss of function anac032 or abi5 mutants showed the opposite phenotypes. Quantitative real time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of ANAC032 is induced by Mg toxicity. This project will investigate the contents and distribution of mineral elements and metabolic profiling in roots and leaves of Col-0, OX32, anac032, ABI5OX and abi5 plants under normal or Mg toxicity conditions using physiological and metabonomics analysis; identify the downstreamed target genes of ANAC032 that involved in modulating the uptake, transport and accumulation of mineral elements, root system development and stress responses to Mg toxicity using transcriptome combination with chromatin immunoprecipitation (ChIP)-seq analysis; and further confirm these downstreamed target genes using yeast one-hybrid (Y1H), ChIP-qPCR, electrophoretic mobility shift assay (EMSA) and transient dual-luciferase reporter assay. Using genetic and molecular biological methods, we will further clarify the biological function of ANAC032-ABI5 interaction and the molecular regulatory pathway of ANAC032 in responses to Mg toxicity in plants. This study is helpful for better understanding of the molecular regulatory network of plants in response to the mineral element toxicity.