microRNA408在水稻响应镉胁迫过程中的调控机制

Cadmium (Cd） is able to be accumulated in rice seeds, which severely threatens food safety. MicroRNA (miRNA) is a small RNA molecular that regulates gene expression at post-transcriptional level in eukaryote. Artificial regulation of miRNA expression nowadays is an efficient approach to improve crop yield and tolerance against adverse stresses. It has been shown that over-expression of miR408 can enhance the photosynthesis and thereby increase seeds yield in rice, however, whether miR408 is involved in other physiological processes is largely unknown, interfering the wide application of miR408 in crop improvements. Our preliminary data show that miR408 can be highly induced whereas its target gene Uclacyanin 7 (UCL7) is significantly repressed by a Cd treatment. Moreover, over-expression of miR408 in transgenic rice is more sensitive to Cd stress than the wild-type. These results suggest an involvement of miR408 and target UCL7 in response to Cd stress. In this project, we aim to pursue the biological significance of miR408 and UCL7 in Cd environment in detail and investigate the miR408 upstream modulators. We furthermore attempt to illustrate a regulatory module that is constituted by miR529-Squamosa promoter-binding protein-like (SPL) transcription factor-miR408-UCL7, which plays a critical role in tolerance to Cd stress. These findings will not only provide molecular insights into comprehensively understanding the functions of miRNAs, but also set a theory foundation for improvement of yield and quality by artificial control of miRNAs in rice.

重金属镉可在稻米中大量积累，严重影响食品安全。miRNA是转录后水平调控基因表达的重要分子，人工调控其表达水平是作物品种改良的一种有效手段。研究表明，过量表达miR408可以促进水稻光合作用并增加籽粒产量，但miR408是否参与水稻其他生物学过程尚不清楚，阻碍了其在水稻育种中的广泛应用。前期研究我们发现：镉诱导miR408积累而抑制靶基因UCL7表达；过量表达miR408的水稻对镉更加敏感，暗示miR408及靶基因参与了水稻镉胁迫应答过程。本研究以此为出发点，将详细阐述水稻miR408和靶基因响应镉胁迫的生物学功能；同时，还将揭示miR408上游调控因子，即深入解析miR529通过调控SPL转录因子，从而影响miR408和靶基因UCL7的表达，参与调控水稻镉耐受性调控模块的分子机制。研究成果不仅可为解析miRNA的生物学功能提供启示，而且可为利用其提高稻米品质和产量提供参考。

镉（Cadmium，Cd）作为一种危害性极强的环境重金属污染物，高浓度Cd会严重干扰水稻的生长发育以及食品安全。microRNAs(miRNAs)作为响应环境胁迫的重要调节因子，水稻miR408调节Cd胁迫响应。本项目利用定量PCR，GUS染色，酵母单杂，转录组测序以及降解组测序试验，不仅发现miR408-3p参与Cd胁迫，其互补链miR408-5p同样参与Cd胁迫过程。进一步研究发现miR408-3p主要是通过靶向UCLs家族成员中的UCL7调节Cd的胁迫过程，miR408-5p则对Cd胁迫更为敏感，其靶基因是生长素早期相应蛋白基因家族成员IAA30，该基因同样响应Cd胁迫。再利用已知的ChIP-seq数据库以及酵母单杂，发现miR156家族成员可通过调控靶基因SPL14调节miR408的表达。另外，本项目发现miR408通过对靶基因的调控，不仅影响Cd转运蛋白基因OsHma3，OsNarmp5和OsLct1的表达，对激素相关基因的表达也有不同程度的影响。项目的完成，获得了低Cd积累的遗传育种材料ucl7 和 miR408过表达材料。本项目首次解析了成熟miR408的双链，miR408-3p和miR408-5p在水稻调控Cd胁迫中的生物学功能。本研究的发现不仅有助于我们更好地理解miR408调节Cd胁迫的上下游调控网络，也为在OsUCLs和OsHma基因家族中挖掘更多的低Cd相关基因提供了有效参考。阐明miR408的调控机制，不仅可以深入挖掘水稻UCLs基因与下游基因Cd转运基因之间密切相关的调控机制，进一步研究下游调控机制提供参考，也为利用CRISPR/Cas9基因敲除技术以获得天然优质水稻突变体提供了有利参考。