miR164-NAC通路调控拟南芥适应缺铁胁迫的机制研究

Iron (Fe) is essential for all living organisms as the cofactors for many important enzymatic reactions. Fe-deficiency has become one of the major environmental factors limiting crop productivity worldwide. Limited Fe availability in soil reduces the iron content of plant-derived food and therefore, Fe deficiency anemia becomes the most common and widespread nutritional disorder for humans. One effective way to overcome these problems is to understand the genetic mechanisms of iron homeostasis in plants. Plants have evolved two response mechanisms, reduction (strategy I) and chelation (strategy II) to cope with Fe-deficient environments. A few evidences showed that the NAC family could be associated with Fe homeostasis in strategy II plants. However, the mechanisms by which NAC family members linked to Fe homeostasis have not been well characterized in strategy I plants. miRNAs have attracted attention because of their important roles in regulating gene expression through post-transcriptional gene silencing, translational inhibition, or heterochromatin modification. Plant miRNAs are involved in various developmental processes. Recent findings have revealed the important roles of miRNAs in plant adaption to nutrient deficiency. However, the functional significance of miRNAs in Fe-deficient responses in plants remains to be explored. In our pre-experiment, we found that miR164 might be related to Fe homeostasis in Arabidopsis. In the present project, we plan to demonstrate the important roles of miRNA164-NACs module in regulating Fe homeostasis in Arabidopsis through the technogies of RNomics, plant nutrition, molecular biology and biochemistry. Our research will significantly contribute to the understanding of the cooperation between transcriptional and post-transcriptional regulatory pathways, and provide valuable information for breeding abitotic-stress-resistance cultivar.

土壤缺铁直接影响人类的铁营养状况，因此研究植物适应缺铁胁迫的生理及分子机制具有重要的理论与现实意义。我们已有的结果表明：缺铁影响miR164的表达；其靶基因只有NAC1/NAC5的表达量在缺铁条件下与miR164的丰度呈负相关关系；mir164b基因敲除突变株、35S::NAC5转基因植株在缺铁条件下具有更长的主根，暗示miR164能够通过NAC1/NAC5参与植物适应铁胁迫反应。因此本申请在现有结果的基础上，拟深入研究miR164与NAC1/NAC5在植物耐受缺铁胁迫中的作用，阐明miR164及NAC1/NAC5参与植物适应缺铁胁迫的分子路径。通过对这种复杂调控网络的分析，有助于我们理解转录因子介导的转录水平调控及其miRNA介导的转录后水平调控在植物适应缺铁胁迫中的作用。由于miRNA在进化上的保守性，这种双重调控机制可以借鉴到其它植物，为培育抗逆作物品种打下坚实的分子生物学基础。

铁是动植物所必需的微量营养元素。土壤缺铁严重降低了作物籽粒中铁含量，直接影响人体的铁营养状况。因此深入研究植物适应缺铁胁迫的生理及其分子机制具有重要的理论与现实意义。为探究miRNA在植物适应铁胁迫的分子机制，本实验室前期对缺铁处理2天的成熟期拟南芥构建的小RNA文库进行深度测序，发现miR164能够响应铁胁迫。miR164是一类在物种间高度保守的miRNA，拟南芥miR164的靶基因是6个NAC转录因子。NAC转录因子在机理Ⅱ植物（禾本科单子叶植物）适应铁胁迫中的作用已很明确，但在机理Ⅰ植物（双子叶植物及非禾本科单子叶植物）中的作用机制还不清楚。本课题利用小RNA技术、分子生物学等技术手段，深入研究了miR164-NAC5通路在机理Ⅰ植物适应铁胁迫中的功能。发现miR164家族受缺铁胁迫变化显著，其中根系中miR164a和miR164b受缺铁胁迫显著下调表达，而在叶片中受缺铁胁迫上调表达；为了研究miR164在拟南芥适应缺铁胁迫中的作用，检测了miR164b突变体对缺铁胁迫的响应情况，证实mir164b突变体对缺铁胁迫具有更强的耐受性；NAC5为miR164的靶基因，过量表达NAC5提高植物对铁胁迫的耐受性；利用ChIP-seq、ChIP-qPCR、EMSA、双荧光素酶等实验，证实NAC5直接调控NFYA8的表达；35S::NFYA8转基因植株的主根长度、侧根数量、铁还原酶活性、铁含量以及IRT1和FRO2的mRNA丰度均高于野生型这些结果表明证实“miR164-NAC5-NFYA8”调控通路在维持机理I植物拟南芥铁吸收稳态中起重要作用。