水稻AGO4家族蛋白的功能分化

小RNA是真核生物基因表达的重要调控分子，在植物生长发育和抵御环境胁迫过程中扮演着重要角色。作为小RNA效应复合物的核心组分，ARGONAUTE（AGO）蛋白通过装载小RNA来介导靶基因调控。目前，水稻小RNA的鉴定和功能研究较多，但AGO作用机制研究很少，制约了小RNA在水稻育种中的利用。申请人前期发现了水稻24-nt miRNA进入AGO4s沉默复合物介导DNA甲基化。本项目在此工作基础上，着重阐述水稻四个AGO4家族蛋白作用方式和生物学功能的异同，并力求回答三个重要问题：（1）不同AGO4蛋白如何实现不同小RNA的装载和分拣？（2）AGO4家族蛋白是否具有mRNA切割活性并怎样识别DNA甲基化位点？（3）AGO4家族蛋白不同成员间如何实现其功能分化？ 本研究将为解析水稻AGO蛋白与小RNA互作的分子机制提供直接基础，也可为通过表观遗传手段，改良水稻重要农艺性状提供启示。

Small RNAs (sRNAs) are important regulators for gene expression, and have been demonstrated to be extensively involved in development and adaption to biotic and abiotic stresses in rice. When controlling gene expressions, sRNAs must be loaded into ARGONAUTE (AGO) effector complexes and then take effects via the mode of mRNA cleavage, protein degradation or DNA methylation. Most studies so far are focused on characterization and functional exploration of sRNAs in rice, however, molecular investigation of AGO complexes are pretty limited, hindering the wide application of sRNAs in rice breeding. We previously identified a class of long microRNAs (lmiRNAs), which can direct DNA methylation for target regulation through associations with four AGO4 clade proteins in rice. In this project, we will determine the differences among these AGO4 proteins in their functions and action modes, and thereby attempt to answer three critical questions: (1) how do AGO4 proteins recruit different sRNAs in rice? (2) whether AGO4s have mRNA slicer activity and how AGO4s recognize different DNA methylation loci? (3) what specific roles do the different AGO4s have in rice physiology? Our results will not only provide insights into understanding of molecular interactions between sRNAs and their silencing complexes in rice, but also set a theoretic foundation for developing epigenetic approaches for crop improvement.